COMPOSITIONS AND METHODS FOR REGULATING CELLULAR SIGNAL TRANSDUCING SYSTEMS

Abstract: Disclosed is a method of regulating the metabolic pathway of inositol in a cell. This method includes the introduction into said cell of a molecule which is then allowed to couple a membrane-bound receptor, activatable by an external signal, to an intracellular phosphodiesterase of the type responsible for generating second messenger substance, IP3. This molecule is selected from the group consisting of a purified A-protein, and active fragments, active analogs, and active fusion products thereof. Also disclosed are methods of detecting an A-protein and methods of inhibiting the growth and proliferation of cancer cells using anti-A-protein antibodies.

Description: COMPOSITIONS AND METHODS FOR REGULATING CELLULAR SIGNAL TRANSDUCING SYSTEMS CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of U.S. Patent Application Serial No. 802,370, filed December 4, 1991, which is a continuation of U.S. Patent Application Serial No. 170,737, filed March 21, 1988 (now U.S. Patent No. 5,100,661). BACKGROUND OF THE INVENTION Fundamental to cells is the response to stimulation of cell surface receptors by external signals. While there are a number of different receptors embedded within the plasma membrane, and a variety of such external signals, e.g., hormones, blood and growth factors, neurotransmitters, and radiation of a specific wavelength, there are a limited number of internal signals or second messengers employed within the cell. A second messenger is one that activates an appropriate cellular response to a specific external signal. It becomes activated when the receptor stimulated by an external signal excites an internal molecule, e.g., an enzyme, which in turn stimulates the production of a second messenger substance. An early signal transduction system identified in the art was the beta adrenergic receptor-adenylate cyclase pathway. This system employs the second messenger cyclic adenosine monophosphate (cAMP), a derivative of adenosine triphosphate (ATP) . Its mechanism of action is now understood to proceed as follows: the external signal-receptor complex interacts with a guanine nucleotide binding protein called a G-protein. G-protein activates adenylate cyclase, which in its activated form can catalyze the production of the second messenger, cAMP, from ATP. cAMP, in turn, causes cellular activity, e.g., protein synthesis, secretion, cytos eletal movement, constituting a cellular response. G-proteins are a class of regulatory proteins which bind guanosine di- and triphosphate nucleotides, i.e., GDP and GTP, respectively. The family of G-proteins serves as peripherally membrane-bound signal transducing polypeptides, coupling activation of cell surface receptors to the regulation of intracellular effectors. These proteins can activate the enzymatic abilities of adenylate cyclase or a phosphodiesterase while binding GTP. Examples of known and probable G-proteins include G s and Gi, which are responsible for the regulation of adenylate cyclase; transducin, which activates a cGMP-specific phosphodiesterase in the retina; ADP-ribosylation factor (ARF) in the liver (Kahn et al. -L. Biol. Chem. 25_9_:6228-6234, 1984); and P21, the product of the ras protooncogene. (For a review, see Whitman et al., Phosphoinositides and Receptor Mechanisms, copyright 1986 by Alan R. Liss, Inc., pp. 197-217). A second signal transduction system serves as a basis for cellular signalling by mitogenic growth factors such as growth hormone (GH), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and radiation of specific wavelengths. This system involves various intermediates in the inositol metabolic pathway. It employs calcium ions and a combination of second messengers ultimately derived from phosphatidylinositol (PI), which is a minor plasma membrane constituent. In this system an external signal, such as light in the photoreceptor cell, activates a receptor, e.g., rhodopsin, which then, by means heretofore unknown, stimulates the catalytic activity of phospholipase C. A key event with regard to the second messenger function is the hydrolysis of an inositol derivative, phosphatidylinositol 4,5-biphosphate (PIP2), a minor membrane constituent, by phospholipase C (PL-C) to yield inositol-1,4,5- triphosphate (IP3) and diacylglycerol (DG) . Both of these reaction products act as second messengers in at least two different systems: DG controls ion currents through the membrane by regulating membrane permeability to various ions and the activity of protein kinase C; while IP3 regulates the concentration of intracellular Ca +2 which in turn affects many cellular processes, e.g., cell division and proliferation. Because of the intimate involvement of the inositol metabolic pathway in this second messenger system, it is understood that failure in the pathway mediates the development of a number of disease states. For example, there is now a large body of evidence supporting the concept that the secondary effects of diabetes, i.e., vascular degeneration and slowed nerve conduction, are the result of stepped-up sorbitol production that results from a failure of the inositol metabolic pathway. The effect of chronically high blood sugar levels on the inositol pathway is to retard inositol metabolism. This may be the result of the inactivity of a regulatory G-type protein due to the glycosylation of nuclear elements, e.g., genes or regulatory proteins, or be the result of the direct glycosylation of the G-type regulatory protein. Retinitis pigmentosa, a disease of the eye characterized by toxic levels of unmetabolized GTP in photoreceptors, may result from a failure of GTP hydrolysis due to absent or reduced levels of GTPase activity of a G-protein, or by the inability of a mutated G-protein to bind or mediate hydrolysis of GTP. Current evidence suggests that at least some types of human cancer, or uncontrolled cell proliferation, are the result of a mutation in a regulatory enzyme of the inositol system. The suspected mutation is understood to prevent the hydrolysis of GTP, the inactivating step for the entire inositol metabolic pathway, including systems initiated by growth hormone (GH) . If the inositol system is unable to shut off, the result is uncontrolled cell division, or malignancy. Alternatively, the malignant state could result from the hyperproduction of IP3 caused by the overproduction, or faulty production, of an enzyme controlling the inositol pathway. Disease states characterized by the lack of cell division, i.e., the lack of proliferation, can also be the result of a failure in the inositol-related signal transduction system to increase intracellular Ca +2 levels, or to respond to GH or other growth factors. Accordingly, the elucidation of the regulatory mechanism involved in the inositol-related signal transduction system will provide a better understanding of the disease states which result from its dysfunction, and can lead to the development of preventative and/or compensatory measures. More specifically, there exists a need for methods of treating disease states resulting from the dysfunction of this system, and for methods of regulating inositol metabolism in cultured cells and cells of higher organisms. Therefore, it is an object of this invention to provide compositions of matter that can link cell membrane receptors with the inositol-related signal transducing system, thus enabling the manipulation of the inositol metabolic pathway to compensate for disease states resulting from its dysfunction. It is also an object of the invention to provide a method of regulating the metabolic pathway in a cell. Another object is to provide a method of controlling the secondary effects of disease states such as diabetes which may result from a dysfunction of the metabolic pathway. Yet another object of this invention is to provide a method of detecting the presence of A-protein in a mammalian subject and in biological sample. Still another object of the invention is to provide a method of controlling the growth and proliferation of cancerous cells. These and other objects of the invention will be apparent from the description, drawing, and claims which follow. SUMMARY OF THE INVENTION It has now been discovered that intracellular molecules, called A-proteins, are responsible for the regulation of the inositol-related signal transducing system. In this system, A-proteins function by activating PL-C to generate the second messengers IP3 and DG. Upon stimulation of a membrane bound receptor, an A-protein binds with GTP to form an intermediate which functions to activate PL-C. When the GTP of the intermediate is hydrolyzed to GDP, PL-C activation terminates. A-proteins are accordingly important G-type signal transducing molecules critical to proper functioning of the cellular inositol metabolic pathway. This knowledge has been exploited to develop methods for regulating metabolic pathways that include the involvement of a cell membrane receptor responsive to, or activatable by a hormone, enzyme, growth factor, radiation of a particular wavelength, or a neurotrans itter. In some aspects of the invention, novel bioactive compositions are provided including an A-protein, which stimulate, inhibit, or normalize cellular metabolism, in particular the inositol pathway. For example, the inositol metabolic pathway in a cell can be sensitized by introducing a signal transducing molecule into that cell, e.g., by means of a synthetic lipid vesicle or liposome. The inositol pathway may be stimulated by the introduction of preactivated A-protein, e.g., an A-protein-GTP conjugate, preferably comprising a non-hydrolyzable GTP analog, e.g., a commercially available material such as GTP-gamma-S or GMPPNP. The molecule useful in these methods of the invention has the ability to functionally couple an activated membrane-bound receptor to a phosphodiesterase, which then becomes enzymatically active. The phosphodiesterase is the enzyme responsible for- generating a second messenger that causes a cellular response. The signal transducing molecules useful in the foregoing methods of the present invention are isolated and purified A-proteins, active fragments of an A-protein, active A-protein analogs, and active fusion products and derivatives thereof. Incubation of these materials with non-hydrolyzable GTP analogs, or other complexes of the two, provide inositol metabolism stimulants. The term "A-protein" was originally used to describe rod photoreceptor protein of approximately 20 kilodaltons molecular weight. Improved extraction and separation methods combined with preliminary sequence data on the separated forms indicates that the entity referred to previously as A-protein may consist of at least two structurally and functionally related proteins; one membrane-bound and one soluble. On this basis, the terminology used reflects the presumed is vivo state: A m , membrane bound (19 kD); and A s , soluble (20 kD) . These A-proteins include the amino acid sequences set forth in the Sequence Listing as SEQ ID NO:l (A m ) and SEQ ID NO:2 (A s ). Further characterizations of the A-protein related molecules, for the practice of the invention, are that they comprise a single polypeptide chain with a significantly hydrophobic region. These molecules also have the ability to bind and hydrolyze guanosine nucleotides adenosine and guanosine triphosphate, and have the ability to activate phospholipase C and other phospholipases in the presence of GTP. Native A-proteins may be recovered from known and available cells, e.g., photoreceptor cells of the eye, and many other cell types. Native A-proteins can be obtained in purities greater than 80% from vertebrate photoreceptors and other types of cells using the methods disclosed below. The stability of A-protein in aqueous suspension is enhanced by the addition of nonionic detergents. The membrane-bound receptor which activates the signal transducing molecule may be one which is responsive to mitogenic signals such as hormones, growth factors, radiation of a particular wavelength, or neurotransmitters. Additionally, the metabolic pathway of inositol in a cell, in further accord with the invention, can be inactivated or inhibited by the introduction to that cell of an antibody which binds a molecule such as an A-protein, or an active fragment, analog, or fusion product thereof. The antibody may be a monoclonal antibody, and may be administered via a liposome. A similar effect can be achieved using an enzymatically non-functional A-protein analog which competes with a native A-protein with the effect of reducing the level of inositol metabolism. Such a construct retains the ability of an A-protein to interact with a membrane-bound receptor or with GTP, but has diminished ability to activate PL-C. The present invention provides a method of stimulating the proliferative abilities of a cell. This method comprises the step of introducing into the cell a signal transducing molecule as characterized above conjugated with a non-hydrolyzable analog of GTP. Conversely, cell proliferation can be inhibited by introducing an antibody which recognizes, binds, and inactivates the signal transducing molecule, as characterized above, or by introducing a non-functional A-protein analog. Further, the present invention provides methods for controlling secondary effects of diabetes, including vascular degeneration and slowed nerve conduction, for reducing the intracellular concentration of GTP, and for regulating the level of calcium ions in a cell. These methods comprise introducing into the cells of a subject a signal transducing molecule as characterized above. The invention also provides novel compositions of matter, useful for stimulating the inositol metabolic pathway in a cell, and for promoting cell proliferation, consisting of an A-protein, an active fragment, analog, or fusion product thereof, coupled to a non-hydrolyzable GTP analog, e.g., guanosine-5'-0-[3 thiotriphosphate] or β-γ-imidoguanosine 5' triphosphate. Other aspects of the invention include antibodies reactive with an A-protein such as a monoclonal antibody that binds to a particular epitope on A m or A s . Such an antibody is useful in a method of detecting the presence of an A-protein in a mammalian subject. This method includes obtaining a biological sample from the subject, such a blood serum, lacrimal secretions, or tissue, treating the sample with the antibody, thereby forming an antibody-A-protein conjugate with any A-protein in that sample, and then detecting the presence of the conjugate. Detection may be accomplished by utilizing an anti-A-protein antibody that has a marker adhered thereto. Useful markers include enzymes, fluorescent dyes, and radionuclides. Alternatively, the conjugate may be isolated by the standard biochemical methods used to obtain immunoprecipitates, such as centrifugation. This invention also includes a method for detecting an A-protein in a biological sample. In this procedure a first antibody which binds to a first epitope on an A-protein is adhered to a solid support. The first antibody is contacted with the biological sample to be tested. A labeled second antibody which binds a second epitope on an A-protein is then added to the solid support. This second antibody further comprises a marker. Any unbound antibody is removed, and the presence or absence of the marker is then detected, its presence being indicative of the presence of A-protein in the sample. The antibodies of the present invention may also be used to inhibit the growth and proliferation of cancer cells. In addition, these antibodies may also be used to image (in vivo and in vitro) a cell, tissue, or organ that has an A-protein present on its surface. This requires the administration of an antibody specific for a membrane-bound form of A-protein (A m ) conjugated to a detectable marker to the cell, tissue, or organ in a pharmacologically acceptable vehicle. The presence or absence of the marker on the cell, tissue, or organ is then determined, its detection or presence being indicative of the presence of A-protein. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other objects of this invention, the various features thereof, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings, in which: FIG. 1 is a schematic representation of the known inositol lipid metabolic pathway that yields second messengers IP3 and DG, supplemented to show the function of A-proteins; FIG. 2 is a bar graph depicting the results of reconstitution experiments in which rod outer segment (ROS) membranes, prelabeled with 3 H-myoinositol, were reconstituted with an A-protein and PL-C, and exposed to light (L) or dark (D) . The release of radioactivity due to the degradation of PIP2 is measured in the presence and in the absence of A-protein; FIG. 3 is a bar graph depicting the results of substrate specificity experiments in which ^H- PIP2 is incorporated into stripped ROS membranes which are then reconstituted with A-protein and PL-C, and subjected to light (L) and to dark (D) conditions; FIG. 4 is a graphic representation of the effect of anti-A-protein mAb-containing liposomes on the proliferation of cancer cells; FIG. 5 demonstrates the chromatography of soluble A-protein: FIG. 5A is an optical scan of the s form of A-protein purified on a high pressure liquid chromatography gel filtration column; FIG. 5B is an optical scan of the HPLC-purified A s rechromatographed on a DEAE-cellulose anion exchange column with a linear salt gradient (dashed line) to confirm the effectiveness of the purification procedure; FIG. 6 is a photographic representation of the purification of the A m and A s forms of the A-protein by SDS-PAGE. The gel was stained with silver. Lane 1 is purified A m ; lane 2 is purified A s ; and lane 3 shows molecular weight standards; FIG. 7 is a diagrammatic comparison of the amino-terminal sequence of A-protein with ARF and ras p 21 proteins. The amino acid sequence analysis was performed on A m and A s purified by ultrafiltration and size exclusion chromatography or Western blotting onto nitrocellulose; FIG. 8 is a graphic representation of the ATPase activity of purified A m and A s combined (1:2) and reconstituted with 32p-ATP and washed ROS membranes and incubated in light or dark conditions; FIG. 9 is a graphic representation of the binding of GTP-analog GMP-PNP by soluble and membrane-bound forms of A-protein in the presence of adenosine nucleotides; and FIG. 10 is a graphic representation of the ATPase activity of A s and A m in presence of GTPγS. DESCRIPTION OF THE INVENTION The proteins responsible for the regulation of the inositol-related signal transducing system have now been discovered. These new signal transducing molecules, collectively named A-protein, have the ability to hydrolyze ATP and GTP, and therefore constitute an ATPase and GTPase. They also has the ability to activate phospholipases including phospholipase C, phospholipase D, and possibly also phospholipase A2. FIG. 1 shows the inositol-lipid metabolic pathways which provide these second messengers. As illustrated, A-protein is directly responsible for the activation of PL-C which catalyzes the production of second messengers IP3 and DG from PIP2/ which is an inositol and lipid derivative. Upon activation of a receptor, A-protein present on or about the interior of the cell membrane binds with GTP. This complex activates PL-C, which in turn acts on PIP2 to yield second messengers IP3 and DG. The stimulating activity of the A-protein-GTP complex ceases upon hydrolysis of GTP which is then released as GDP. A-protein present within an animal cell is inactive in the absence of receptor stimulation, but addition of excess A-protein increases sensitivity to receptor stimulation. Accordingly, a cell which expresses a mutant form of A-pfotein with reduced activity may have its metabolism corrected by the introduction of A-protein or an expressible gene that encodes it. Persistent activation of the inositol pathway can be provided by introducing into a cell a conjugate of A-protein and a non-hydrolyzable analog of GTP. This bypasses the necessity of receptor activation and results in persistent stimulation. Introduction of an A-protein-non-hydrolyzable GTP analog conjugate can temporarily "transform" a cell, inducing cell proliferation for a limited time. A cell's inositol metabolism may be inhibited by the introduction of a nonfunctional A-protein analog which competes with the native form upon stimulation of a receptor. Thus, a truncated form or analog of an A-protein which retains the ability to bind GTP and/or to couple with a receptor, but lacks the ability to activate PL-C inhibits inositol metabolism when introduced into a cell. Antibodies against A-proteins bind and inactivate them, thus also inhibiting inositol metabolism and reducing or terminating the effect of receptor stimulation. Intracellular administration of such a non-functional analog or antibody can inhibit cell mitosis. A-proteins have been isolated from mammalian (bovine) and amphibian (frog) rod outer segments (ROS) by extraction, centrifugation, chromatography and other protein purification techniques known to those skilled in the art. See U.S. Patent Application Serial No. 170,737, the disclosure of which is herein incorporated by reference. Other proteins with similar or identical physical and functional characteristics as the A-proteins have been isolated from various other tissues from vertebrates and invertebrates. These findings indicate that the structure of the A-proteins has been conserved through evolution. The present invention is based on the recognition that the A-proteins have a universal regulatory role in cells which employ inositol-type metabolism. A-proteins are quite labile in aqueous solution, but can be significantly stabilized if disposed in aqueous solutions containing a nonionic surfactant. Theyt have a molecular weight in the range of 20 to 21 kD, as inferred by comparison to molecular weight standards during electrophoretic separations. Preferred methods of isolating the native protein are disclosed in detail below. Good purification results have been achieved using filters with molecular weight cutoffs in the range of 10 kD and 30 kD. A-proteins, various truncated or mutein analogs thereof, and fused proteins comprising an A-protein and other protein domains can be produced by various synthetic and biosynthetic means. For example, an appropriate host cell such as a microorganism, yeast, or eucaryotic cell culture can be genetically engineered to express an A-protein, or a portion or analog thereof. This may be accomplished by now well established recombinant DNA technologies known to those skilled in the art. The recombinant procedure may include the isolation or synthesis of a gene encoding an A-protein, a portion, or analog thereof, and the integration of that gene into a plasmid. The amino acid sequences of the A-proteins may be established readily given this disclosure. The Sequence Listing sets forth the N-terminus amino acid sequence of two forms of A-protein (A s and A m ) as SEQ ID NO:l and SEQ ID NO:2. Gene synthesis from synthetic oligonucleotides and known mutagenesis techniques provide the technologies to prepare an array of analogs, truncated A-protein forms, and fused proteins comprising A-protein or a domain thereof. Production of such materials further may include the transformation of an appropriate host cell with a vector harboring the recombinant DNA, culturing that transformed host cell, and isolation of the expressed protein. Given the availability of A-protein-rich samples producible as disclosed herein, the recombinant production of the native form and various analogs thereof is well within the current skill in the art. Alternatively at least portions of the protein may be produced synthetically by chemically biasing amino acids in the correct sequence. A-proteins are also useful as antigens to produce antibodies useful to depress a cellular mechanism which relies on the enzymatic action of A-proteins. Antibodies may be part of a polyclonal antisera, or an active portion thereof, raised against an A-protein, and shown to react with an A-protein or with its analogs and fragments. However, the antibody is preferably a monoclonal antibody produced by methods known per se. The antibody preferably is selected so as not to cross-react with the cellular components. This antibody can be of any class and subclass as determined by the Ouchterlony double diffusion test. Alternatively, an antibody which recognizes an A-protein can be synthesized by biosynthetic or recombinant means, either in whole or in part. In addition, the antibody can be linked to other functional molecules such as toxins, dyes, enzymes, or radioactive markers. Such linked antibodies may be useful for detecting an A-protein and imaging a cell, organ, or tissue having an A-protein on its surface. The antibody-marker complex may be prepared by chemical linkage or by recombinant DNA techniques if the marker is proteinous. For example, the antibodies may be labelled with a reagent which enables the monitoring of the antibody after its administration to a patient. The label may be, for example, a radioisotope such as 125- or 99m-- C/ both of which may be imaged extracorporeally by radiation detection means such a gamma scintillation camera. Alternatively, the antibody may be labelled with a non-radioactive, paramagnetic contrast agent capable of detection in MRI systems. In such systems, a strong magnetic field is used to align the nuclear spin vectors of the atoms in a patient's body. The field is then disturbed and an image of the patient is read as the nuclei return to their equilibrium alignments. In the present invention, antibodies can be linked to paramagnetic contrast agents such as gadolinium, cobalt, nickel, manganese or iron complexes, to form conjugate diagnostic reagents that are imaged extracorporeally with an MRI system. Such a labelled antibody may be administered to a patient via arterial or venous injection. Alternatively a non-hydrolyzable derivative of the antibody may be administered by mouth. An anti-A-protein monoclonal antibody can be obtained from a hybridoma cell line formed upon the fusion of a mouse myeloma cell with a spleen cell of a mouse previously immunized with an A-protein purified, for example, from bovine ROS. The immunogen alternatively can be a derivative of A-protein, or an analog or portion thereof, produced in vitro according to known mechanical or manual procedures of peptide synthesis. Alternatively, the immunogen (A-protein) can be synthesized by biosynthetic means using recombinant DNA technologies known to those skilled in the art. The mice whose spleen cells are chosen for fusion are preferably from a genetically defined lineage such as Balb/c. The myeloma cells used in the fusion are from a mammalian, antibody-producing cell line, but most preferably are from a mouse cell line such as, e.g., NS-1. The monoclonal antibody can be obtained from ascites fluid of mice injected with the fusion product. The antibody so produced is specific for an A-protein, and therefore is particularly useful in regulating mechanisms which involve A-proteins. For example, the antibody will be useful in inhibiting the metabolism of PIP2 to second messengers DG and IP3, as shown in FIG. 1. In the studies described in the exemplification below, eighteen anti-A-protein monoclonal antibody-producing hybridomas have been injected into mice in order to produce more antibodies. In this frequently used method the hybridomas are injected into the peritoneum of an immunosup ressed mouse. The hybridoma cells normally reproduce to form a tumor which secretes the antibody of interest in a pure form; in this case, antibodies against A-protein. Ordinarily, animals used for producing antibodies in this way are sacrificed after a defined period, and the antibody is harvested from the ascites fluid. Under normal circumstances, mice survive for two to four weeks after infusion. Of the four different hybridomas injected into 40 mice, all caused an apparent immune response in the host animal. The animals all died prematurely with significantly enlarged spleens and lymph nodes as well as generalized edema. Post-mortem examination revealed no tumors in any of the mice. This result is significant in two ways. First, the antibodies appear, to have prevented the growth of the very tumor cells producing them. This observation signals the effectiveness of the antibodies in checking the growth of cancer cells. The second aspect of this procedure, the apparent autoimmune response elicited, was unusual in that the animals were already immunosuppressed prior to treatment and yet developed a significant immune response. The inference is that the antibodies induce a generalized toxic immune response. The antigenicity of the hybridoma itself is not suspect due to the common usage of this technique without the autoimmune response when similar hybridoma types are injecte . In cancer, a failure of the immune system can be one component in the establishment of tumor cells. A treatment that both checks cancerous growth and recruits a directed autoimmune response to the tumor is thus effective in causing regression of neoplastic tissues. Since anti-A-protein antibodies will affect normal tissues as well as neoplastic growth, it is necessary to ensure that the antibodies are directed to the target cells. This becomes particularly important if the antibodies attract an immune response from the host. The better localized the response, the more effective it may be. in addition, the antibodies may cause related autoimmune problems if they are generalized in the blood stream. One way to specifically deliver agents to cancer tissue is to package the antibodies in carriers such as synthetic lipid vesicles, or liposomes, that are coated with a different antibody which specifically binds to cancer cells. In this case, the liposome fuses with the outer membrane of the cancer cell and the contents are disgorged into the cell. Thus, A-proteins or antibodies thereto, conjugates, or analogs thereof may be administered, via the use of a protective and directive vehicle such as liposomes, to a subject afflicted, for example with cancer, diabetes, retinitis pigmentosa, etc., or to a cell culture to stimulate or depress inositol metabolism. Liposomes contacting a cell membrane can deposit their contents into the cell via endocytosis. Liposomes useful for this purpose can be prepared by any number of methods (e.g., Bangham et al. (1965) _∑_. Mol. Biol. 11:238-252; Deamer and Bangham (1976) Biochem. Biophys. Acta, 4_4_3_:629-634; and Ghalayini and Anderson Q3iochem. Biophys. Res. Comm. (1984) 421:503-506). Briefly, these methods include mixing the material to be entrapped or incorporated, e.g., A-protein, an A-protein non hydrolyzable GTP analog conjugate, or anti-A-protein antibodies, with the appropriate lipids, e.g., ROS membrane-extracted, in a buffer, and sonicating the mixture. Another possibility is the implantation of porous plastic discs, preloaded with anti-A-protein antibody, an A-protein, an A-protein conjugate, or analog thereof, into the site of the lesion. This technology is already used for site-specific drug delivery and recent advances in the technology now make it possible to use large macromolecules in combination with the discs. Any other localized delivery system, e.g., microcapsules or polymeric agents, could similarly be used. From the foregoing, it will be apparent that compositions of the types described above have a number of utilities, both in vitro and in vivo. The introduction of the native or of active analog forms of A-proteins into cells having an overabundance of GTP, or expressing a defective form of a native A-protein, can reduce intracellular GTP concentration and restore or improve inositol metabolism. Activated A-protein conjugates can stimulate inositol metabolism, leading to cell replication. Thus, introduction of such conjugates into the cells of a transformed or non-immortal cell culture can produce a pulse of replication. Non-functional A-protein conjugates and antibodies to A-protein can depress inositol metabolism, and thus mitosis, in, for example, malignant cells. The following examples further disclose the nature of the invention, without limiting the scope thereof. EXAMPLE 1 Purification Of Soluble (A^) And Membrane-Bound (Am) A-Protein A-proteins were isolated from the retinas of cow eyes essentially as described by Schmidt et al. (J. Biol. Chem. (1987) 2£2:14333-14336) . Bovine (cow or calf) eyes were obtained from a local abattoir within 2 hours of killing. Bovine eyes were kept on ice in the dark for 30 to 60 minutes. Retinas were dissected out and placed in buffer A (130 mM NaCl, 20 mM Tris-HCl, pH 7.0; 1 ml per calf retina or 2 ml per cow retina). Gentle, repeated inversions of the container liberated large numbers of ROS into the buffer. The mixture was poured through a Buchler funnel to remove the retinas. The filtrate was allowed to settle in a conical-bottomed tube on ice for 5 minutes, allowing gross particulate matter to settle out of the ROS suspension. The supernatant is found, by means of microscopic examination on a hemocytometer, to consist of greater than 95% ROS. The ROS were disrupted with shear created by repeatedly drawing the suspension into the syringe and forcing it out against the wall of the container. To separate particulate and aqueous fractions, the suspension was centrifuged at 10,000 to 12,000 x g for 20 minutes at 4°C. The pellet containing ROS membranes was washed once with a volume of buffer A equal to that of the removed supernatant. The resulting pellet was resuspended in 3 to 6 ml of buffer T (0.05%) Tween 20/80 (1:1) in double-distilled water) and spun at 15,000 x g for 45 minutes. The above manipulations were carried out under dim red light. Both A-protein solutions (the supernatants containing soluble and membrane-bound A-protein, respectively) were filtered through Centricon 30 microconcentrators (molecular weight cut-off of 30 kD, Amicon Corporation), with centrifugation at 5,000 x g in a refrigerated centrifuge. The ultrafiltrates were then concentrated and dialyzed in Centricon 10's (molecular weight cut-off of 10 kD) at 5,000 x g. The retentates contained proteins of 10 kD to 30 kD, with average concentrations of 100 to 200 μg/ml for soluble A-protein (A s ) and 30 to 100 μg/ml for membrane-bound (A m ), reduced to a volume of 0.5 to 1 ml. The purification of A s results in a 320-fold enrichment and A m is purified 20-fold. If the soluble A-protein solution was to be kept overnight before concentration and use, buffer A with 0.05% Tween 20/80 (1:1) was added 1:5 to minimize aggregation of proteins in the concentrated solution. The A-proteins, purified by ultrafiltration as described above were further purified for sequence analysis by HPLC on a Bio-Sil SEC-125 column in buffer A (A s ) or buffer T (A m ) . Elution was isocratic. Pooled peaks were concentrated, dialyzed against water and lyophylized prior to analysis. For the purpose of confirming the purity of soluble A-protein used in experiments, A s , purified by ultrafiltration as described was run on a HPLC size exclusion column (TSK-2000, Bio-Rad) in buffer A (FIG. 5A), and then rechromatographed in 0.1 M potassium phosphate buffer on a DEAE anion-exchange column. Protein was eluted with a 0 to 200 mM NaCl gradient (FIG. 5B) . The estimation of molecular weights of A m and A s were made from the relative mobility of each on SDS gels (see EXAMPLE 3 and FIG. 6) and a calibrated gel filtration column. The agreement of weight determination between the native forms from the column and the denatured forms on gels indicates that A-protein exists in vivo as a monomer. The purification of A s as described above results in preparations of greater than 95% purity. Any protein contaminant of the purified A s preparations has been shown not to interact with guanosine or adenosine nucleotides under any of the conditions tested. Since the extractions are sequential, A m is purified to essential homogeneity by the procedure described with no detectable contaminants, as shown in the SDS gel described below in EXAMPLE 9 and pictured in FIG. 6, lane 2. Once purified, the stability of the A-proteins differs markedly in aqueous solution. A m is metastable in the purified state and retains most of its functional properties for several days at 4°C. A can also withstand freezing and thawing in detergent without losing more than 15 to 20% of its original activity. In contrast, A s is labile under a variety of conditions and no satisfactory methods of treatment have been found to prevent greater than 80% activity loss over a 48 hour period at 4°C. The soluble A-protein is markedly thermo- and cryolabile. Purified A s loses activity rapidly at room temperature (half life = 2 hrs) and freezing results in loss of virtually all activity, presumably due to denaturation and/or aggregation. The purified soluble form aggregates readily in the absence of detergent treatment and will precipitate overnight in the refrigerator under those conditions. EXAMPLE 2 Gel Electrophoresis Polyacrylamide gel electrophoresis was performed according to a modification of the methods of O'Farrell (_I. Biol. Chem. (1975) _25JL:4007) in the presence of 0.1% SDS in a pore gradient gel (10 to 20%). Samples were applied in a sample buffer of 0.33 mM DTT, 7% SDS, 17% glycerol and 0.5 M Tris-HCl, pH 6.8, and run to equilibrium. Samples were not heat-denatured, in order to avoid the appearance of additional bands caused by the formation of stable polymers. Proteins were visualized with the Bio-Rad silver stain kit. Gels were calibrated using pre-stained molecular weight standards from Bio-Rad (range 17 to 94 kD) . As shown in FIG. 6, "A-protein" includes A m , a membrane bound form having a molecular weight of about 19 kD and A s , a soluble form having a molecular weight of about 20 kD. The soluble protein resolves into two closely spaced bands on gels. The membrane-bound form migrates ' as a single band. EXAMPLE 3 Protein Sequencing Sequencing was performed on an automated pulse liquid phase system (477A Protein Sequencer, 120A Analyzer, Applied Biosystems) using Edman degradation. Derived amino acids are separated by reverse-phase chromatography and read by an on-line analyzer. The primary amino acid sequences of the N-terminal region of A s and A m determined by automated microsequencing are shown in FIG. 7 and in the Sequence Listing as SEQ ID NO:l and NO:2, respectively. The sequence of both forms of A-protein are identical to the extent that they have been analyzed except for the Met residue in position 1 and the presence of carbohydrate on the membrane-bound form. Average amounts degraded and number of analyses were 348 pmol (n=8) and 632 pmol (n=4) for A m and A s , respectively. Most runs consisted of at least fifteen cycles. The first two residues, Met-Gly, are conserved among GTP binding proteins. Repeated analysis of both A s and A m demonstrates some ambiguity concerning the N-terminal methionine. Methionine was present in all samples that were not immobilized on nitrocellulose although the average proportion of total protein with methionine in the first position is 6.9% and 37% for A m and A s respectively. The remainder of both proteins analyzed have a glycine as the N-terminal amino acid indicating a post-translational removal of the methionine, possibly by an endogenous aminopeptidase. FIG. 7 also shows a comparison of the initial amino acid sequences of As, bovine retinal ARF (brARF) protein, bovine adrenal ARF (baARF) protein, and the H, K, AND N ras ρ21 proteins. Samples of A m blotted on nitrocellulose and analyzed had virtually no methionine on the N-terminus. Analysis of these samples could not proceed beyond the first residue, perhaps indicating the attachment of a blocking group such as a fatty acid to the exposed Gly. Such a modification of the amino terminal residue could serve to enhance protein-membrane interaction. This is further supported by the fact that the concentration of amino acids liberated by hydrolysis distal to Gly-2 during sequencing drops an average of 89% and 58% for A m and A s , respectively indicating the possible presence of a blocking ligand at that position. EXAMPLE 4 Reconstitution Experiments A. Preparation of Components A-proteins were purified as described in EXAMPLE 1. A cytosolic fraction containing PL-C activity was prepared by extracting soluble proteins from ruptured ROS in buffer A (100 mM NaCl, 20 mM Tris, pH 7.0, 1 mM MgCl2) • This solution was washed 3 times to remove all of the A-protein with buffer B (10 mM Tris, pH 7.0, 0.1 mM EGTA) and concentrated on Centricon 30 ultrafiltration instruments. Stripped ROS membranes (containing rhodopsin as the receptor) were prepared by washing the membranes 3 times in buffer A and 3 times in water, containing 0.01% polyoxyethylene 23 lauryl ether (Brij 35 nonionic detergent, Sigma Chemical Co.). The stripped - membranes were resuspended in buffer B (rhodopsin concentration = 100 mM) prior to use. B. ROS Membrane Labelling Prior to stripping, ROS membranes were ruptured in buffer A (1 ml/retina) containing 1 mM ATP and 200 ml (50 μCi) of myo-[2- 3 H(N)]-inositol (250 μCi/ml) (New England Nuclear, Boston, MA) was added. This mixture was allowed to incubate at room temperature for 3-5 hours. This resulted in significant uptake of radiolabel by the membranes as PI, phosphatidylinositol 4-phosphate (PIP), and (PIP 2 ). C. Experimental Procedure The stripped, radiolabelled ROS membranes (50 μl) were recombined with 30 μl of A-protein (5-8 μg) solution and the cytosolic PL-C-containing fraction in buffer B which contained 1 mM GTP, 1 mM ATP, and 0.01% nonionic surfactant. The final volume of each sample was 200 μl. In addition to these samples, control samples containing either no A-protein, no PL-C, or none of either were prepared to check the PL-C solution and ROS membranes for background activity. Samples were either exposed to a bright 10 msec xenon flash (Nikon) (delivering 1.8 x 10 3 μWcm ~2 sec~l) sufficient to bleach greater than 70% of the rhodopsin present in each sample or kept in the dark as control. Both dark and light samples were simultaneously quenched with 200 μl of ice cold 15% trichloroacetic acid immediately following the light flash (within 10 seconds) . Following quench, the samples were kept on ice for 30 minutes. Samples were spun down in a microcentrifuge for 5 minutes and 100 μl of supernatant was aliquoted for liquid scintillation counting. D. Results FIG. 2 shows the radioactivity, represented as counts per minute (cpm), recovered in the aqueous phase of ROS membrane, prelabelled with tritiated myoinositol, and reconstituted with purified A-protein and the phospholipase C ROS fraction. Bars in FIG. 2 marked "L" represent samples exposed to a xenon flash; samples represented by "D" were kept dark. The bars on the right in the drawing are for identical samples without the A-protein and hence depict background. In this experiment, only the samples containing A-protein showed a specific release of radioactivity sufficient to indicate that A-protein is capable of activating PL-C in the presence of bleached rhodopsin. The high levels of activation seen in the "dark" A-protein samples may be due to stray light activation of "dark" rhodopsin combined with an excess of A-protein. Comparison of light and dark samples reveals the light-dependent component of ' the reaction. EXAMPLE 5 Substrate Specificity Tests A. Extraction of ROS Lipids Two milliliters of ROS suspension were prepared from 10 bovine retinas, as described in EXAMPLE 1, above, as previously described. The suspension was mixed with a five-fold excess of chloroform-methanol (2:1) and allowed to stand on ice for 1 hour. The mixture was spun briefly and the lower phase was removed with a pipette. This lower phase (-2 ml) was removed and washed once with 3 milliliters of chloroform-methanol-0.2 N HCl (3:47:48). The lower phase was removed and used as described. This solution contained - 1 μM lipid/ml. B. Preparation of Liposomes Liposomes were prepared according to the method of Ghalayini and Anderson Q3iochem. Biophys. Res. Cornm. (1984) 414:503-506). Purified A-protein and A-protein-free PL-C fractions were prepared as described previously. 3 H-PIP2 (L-alρha-[myo-inositol- 2- 3 H(N)]) phosphatidylinositol; New England Nuclear, Boston, MA) was incorporated into stripped ROS membranes, prepared as described above, by means of liposomes. 3 H-PIP2 (0.5 μCi) was dried under N2 after being mixed with lipids extracted from ROS. Buffer B was added to the test tube containing the dried H-PIP2/lipid residue and the contents of the tube were sonicated for 10 minutes in a sonicating water bath (Branson Ultrasonic Cleaner, Shelton, CT.). This resulted in a fine suspension (clear) of liposomes containing H-PIP2- This suspension was combined with an equal volume of stripped ROS membranes suspended in buffer B (rhodopsin concentration = 500 μM) and allowed to stand for 10 to 24 hours at 0°C. C. Experimental Procedure 3 H-PIP2~labeled ROS membranes (100 μl) were combined with purified A-proteins (5 to 10 mg), and A-protein-free PL-C fraction in a buffer. The buffer contained 20 mM Tris, pH 7.0, 1 mM ATP, 0.25 mM GTP and 0.05 mM GMPPNP (β-γ-imidoguanosine 5'-triphosphate) (Sigma Chemical Co., St. Louis, MO), a non-hydrolyzable GTP analog. Each sample had a final volume of 300 μl. Control samples were made up as above except that either the A-proteins or PL-C or both were deleted. Identical samples, including control samples, were either kept dark or exposed to room light for 30 minutes. Aliquots of each sample (100 μl) were removed at 1, 10, and 30 minutes of the incubation and quenched with an equal volume of ice-cold 15% trichloroacetic acid. Quenched samples were kept on ice for 30 minutes and then spun for 5 minutes in a microcentrifuge. 100 ml of the supernatant of these samples was assayed for radioactivity by liquid scintillation counting. The results are shown in FIG. 3, which graphically shows counts per minute of released radioactivity for samples subjected to light (L) and for samples that remained dark (D), with background response being subtracted. The samples containing activated A-protein and PL-C were the only ones to demonstrate significant levels of PIP2 hydrolysis. According to the measured data, at 30 minutes, the A-protein-containing "light" sample showed 24% of the available radioactive PIP2 had been hydrolyzed. This activation of PIP2 hydrolysis is direct evidence of PL-C activation by A-protein, since PIP2 has been shown to be the specific and only substrate of PL-C. The greater release of radioactivity in the light-exposed sample compared to the "dark" sample is a demonstration that this process is receptor mediated. EXAMPLE 6 Production of Monoclonal Antibody to A-Protein Balb/c mice (6-8 weeks old; The Jackson Laboratory, Bar Harbor, ME) were immunized with four injections of A-protein. The injections were performed one week apart and 1 mg A-protein (either As, Am, or both) is injected on each occasion. The first three injections were given intraperitoneally. and the fourth intravenously. A-protein was injected with complete Freunds adjuvant on the first occasion, incomplete adjuvant on the second and third occasions, and without adjuvant on the last occasion. Serum withdrawn prior to the last injection showed prominent binding to purified A-protein using a solid phase microtiter plate enzyme-linked immunoassay. The mouse with the best immune response was sacrificed three days after the last injection. Hybridomas were produced by fusion of spleen cells from the sacrificed mouse with NS-1 (P3NS-1/1-Ag4-1) myeloma cells (American Type Culture Collection, Rockville, MD; Accession No. TIB18) . In the present example, the method of Nadakavukaren (Differentiation 22:209-202, (1984)) was employed to perform the fusions. Resultant clones were tested for binding to A-protein. Subcloning by serial dilution was carried out on one clone. The most productive subclones were injected into the peritoneal cavity of Balb/c mice to produce ascites fluid containing monoclonal antibody. The ascites fluid which is obtained is centrifuged, tested for activity, and then stored at -70° C until required. The resulting 18 anti-A-protein antibodies were screened for antibody isotype by the Ouchterlony double diffusion test in agar plates against anti- IgM, anti-IgG, anti-IgGl, anti-IgG2a, anti-IgG2b, and anti-IgG3 antibodies (Cappell) . The results are shown in TABLE 1 "n.d." - not determined EXAMPLE 7 Treatment of Cancer Cells With Anti-A-Protein Antibodies A. Methodology Spent medium from flask cultures of monoclonal antibody cells was spun down and the cell-free supernatant, containing IgG in solution, was concentrated by centrifugation in Centriprep 30's (ultrafiltration devices with a molecular weight cut-off 30 kD) . The IgG was purified from the retentate using Protein-A columns. The purified IgG was dialyzed out of the acidic eluting buffer into pH 7.4 buffer (10 mM KC1, 10 mM Tris-HCl, pH 7.7). Rat mammary adenocarcinoma (RBA) cells (ATCC No. CRL 1747) grown to confluency in flasks of MEM (10% fetal bovine serum, 12% penicillin- streptomycin, 0.1% fungamycin; incubated at 5% CO2) were plated out at low density into 2 mm culture dishes. These cells were used because they contain an activated H-ras oncogene. Liposomes were used to introduce the anti-A-protein antibodies into the cells by fusion. These liposomes were prepared by drying down 100 ml negatively charged lipid (L-α-phosphatidylcholine, dicetyl phosphate, and cholesterol, in a molar ratio of 63:18:9) in a glass tube, and adding 1 ml of IgG solution (or 1 ml pH 7.4 buffer, in the case of "empty" control liposomes) before sonicating for at least 2 hours. Liposome solutions were sterile- filtered immediately before using 0.45 mM cellulose acetate filters to create a population of uniformly sized liposomes. After 8 days, the medium was removed, the cells washed once with PBS and 100 ml of liposomes (or in the case of controls, 100 ml sterile PBS) added. The dishes were agitated manually for 3 minutes at room temperature to maximize cell-liposome contact. Fresh medium was then added and the cells incubated at 37°C. Cells were counted under a light microscope, and growth was monitored in specific, premarked areas of the dish. General observations were made of the overall growth in the dishes, also. These experiments were done in duplicate. B . Results The results are shown in FIG. 4. On day 2, the population of control cells had usually more than doubled. The population of cells treated with "empty" liposomes had increased by 34%, but the cells treated with immunoliposomes containing IgG has been reduced to 66% of the original cell count. On day 5, the control cell population was 400% of the original, the cells treated by empty liposomes had increased to 225% of the original population, whereas the cells treated by the anti-A-protein IgG had further decreased to 38% of the original population. These results indicate that antibodies raised against ROS A-protein not only prevent growth of adenocarcinoma cells, but decrease it significantly. In addition, these results demonstrate that not only is A-protein involved in the process of cell division, but that an activating mutation or overproduction of an A-protein can cause certain types of cancer (in this case, ras-related transformation), and that monoclonal antibodies raised against it can inhibit the growth of cancer cells. EXAMPLE 8 Effect of Anti-A-Protein Monoclonal Antibodies On Animal Subjects A. Methodology Balb/c mice (1 or 9 weeks old, both sexes) were immunosuppressed on arrival by injection of 0.2 ml Pristane. 12 days later, hybridoma cells (raised initially in Balb/c mice, then grown in flask cultures) which had been spun down into phosphate buffered saline (PBS) were administered by intraperitoneal injection (4 x 10 6 cells per 1 week old mouse) . Four different anti-A-protein IgG producing hybridomas (3F5H11, 4C5F3, 3A7G6 and 7E4F9) were used. 4C5F3 was also introduced to 9-week-old (adult) mice, to see how much, if any, difference the age of the subject makes. Controls were not injected. Over the next 2 to 10 days, the mice were sacrificed using either CO2 gas or Nembutal, and the eyes immediately removed by severing the optic nerve. The eyes were fixed in Karnovsky's solution, embedded, sectioned to 3 mm thickness, and stained with haemotoxylin and eosin. Slides were photographed at 40 x objective with Kodachrome, Kodak Technical Pan film (b/w) and Kodak Ektar 125 film (color) . Autopsies were conducted on mice sacrificed on days 9 and 10 after removal of eyes and a brief external evaluation. Fluid removed from the peritoneal cavity was frozen whole. Both juvenile and mature controls were used in parallel to treated subjects. Mice were injected as groups of 4 to 6, and sacrificed at intervals of 2, 4, 7, 8, 9, and 10 days. B. Results 1. Autopsies All trial injections of myeloma hybridomas into mice were run in multiples and in parallel with controls. Propositus and control mice were all immunosuppressed with pristane prior to treatment. Mice from the second study were enucleated and the retinas were sectioned to examine the rod outer segments for damage. Generally, the mice from both studies failed to grow or maintain significant tumor tissue. Upon post-mortem examination, most mice had few or no hybridoma cells in evidence in the peritoneal cavity. There was some fluid in the peritoneum of most experimental mice which often contained significant red cell infiltration. There was little or no hemolysis in fluid specimens that were spun down, as determined by inspection. All of the injected mice displayed generalized toxic immune reactions, as inferred from enlargement of abdominal lymph modes and enlargement of the spleens, ranging from mild to acute. The retinas of mice injected with hybridomas were uniformly damaged at the level of the rod outer segments beginning as early as day 2. This damage appeared to involved disruption of the outer segment membranes and occasionally extended partially into the inner segments. Damage was completely confined to the photoreceptor cells. Disruption was occasionally severe enough to cause retinal detachment. Several mice showed the presence of small numbers of hybridoma tumors or detectable levels of myeloma-like cells in the ascites fluid. Descriptions of these individuals are as follows: One mouse containing 3F5H11 cells died overnight between days 8 and 9, so it was processed on day nine. To avoid another loss overnight the remaining juvenile mouse containing 4CSF3 was sacrificed. Both of these mice exhibited swelling of the abdomen, and were not very active prior to sacrifice. Autopsies revealed that their peritoneal cavities contained fluid; almost 4 milliliters was recovered from the 4C5F3 mouse. The 3F5H11 mouse showed a large immune response, in that the lymph nodes, not visible normally, were swollen and very evident. The 4C5F3 mouse exhibited less of an immune reaction than the 3F5H11 mouse that died overnight. Some tumor deposits were visible on the peritoneal wall, and food was found in the gastrointestinal tract: this is the more usual reaction to injection of monoclonal antibody cells to the peritoneal cavity of mice; normally the cells grow in the ascites fluid as tumors. It is a common means of propagating monoclonal antibody cell lines. The remaining mice left in each group were sacrificed on day 10. The 7E4F9 mouse had a swollen abdomen, and almost 4 milliliters of fluid was removed from its cavity. There was an immune reaction, and the lymph nodes were swollen to visibility. The 3A7G6 mouse exhibited a more normal reaction, in that its abdomen was not swollen, food was present in the tract, little fluid was found in the cavity (1 milliliter), the lymph nodes were not swollen, and small tumors were present. The two 4C5F3 mature mice remaining displayed differing reactions. One had a large immune reaction, almost 5 milliliters of fluid was found in the cavity, no food was found in the tract, and the lymph nodes were swollen. The other had no immune reaction, nor was there fluid in the cavity. All the immunosuppressed controls were also sacrificed on day 10. These looked normal, and autopsy revealed no tumors or abnormalities. 2. Microscopic Examination of Sections of Retina 3 mm sections were used for slide preparation. The results of microscopic examination of these sections are summarized in TABLE 2, below. TABLE 2 Observations A normal retina, showing intact photoreceptors Normal; intact photoreceptors Normal; intact photoreceptors I I Early damage manifested as apparent damage to membranes : at proximal end of ROS Progressive damage: gross disruption of ROS membranes, extending from proximal end i Complete involvement of ROS in degenerative process: virtually no normal ROS visible. This view is of a retinal evagination, which probably occurred due to a retinal detachment. Morphology of both ROS and RIS is grossly abnormal TABLE 2 (continued) Observations Complete retinal detachment, probably due to apical disruption of ROS Moderate damage to proximal end of ROS I Progressive damage to ROS, spreading from proximal to apical region 10 Area Centralis Mature 4C5F3 Extensive ROS disruption at proximal end o 11 Area Centralis Mature 4C5F3 Severe, progressive damage extending from proximal to apical region of ROS. The beginnings of a retinal detachment 12 Area Centralis Mature 4C5F3 10 Severe, progressive damage to ROS, with evidence of vacuolar disruption of ROS TABLE 2 ontinued) Days After Observations Damage visible at proximal end of ROS, with some RIS disruption 8 Damage to ROS extending from proximal to central region of ROS, with some RIS disruption I *>. 10 Damage similar to that seen after 8 days I : 4 Early damage to proximal end of ROS 10 Moderate damage extending from proximal to apical region of ROS. Disruption is confined to central area of retina -46- These results show a definite progression over time; disruption of outer segments seems to spread from the periphery inward toward the middle of the retina. This indicates that the principle site of action of the monoclonal antibody is at the end of the ROS that is undergoing active membrane renewal. This is consistent with the idea that A-proteins are important in regulation of phospholipid (phosphatidylinositol) metabolism. The monoclonal antibodies that give the most striking effect were 3F5H11 (which also gave the best results in the immunoliposome test on cancer cells) and 4C5F3. 3A7G6 produced good, although not uniform, effects. 7E4F9 produced moderate results. The autopsies show an abnormal reaction of many of the mice to the introduction of monoclonal antibody producing (hybridoma) cells into the peritoneal cavity. Hybridoma cell lines can routinely be successfully grown in ascites fluid as tumors; this does not seem a possibility with hybridoma cell lines producing antibody directed against bovine ROS A-proteins. The lack of tumor establishment and/or growth appears to derive from the anticancer effects of the monoclonal antibodies. These studies demonstrate that large immune reactions occurred in mice injected with the hybridoma cells, and that virtually no tumor cells escaped destruction. The evidence presented here points to a connection between A-proteins and cancer-associated retinopathy (CAR), a phenomenon observed clinically in humans; a patient exhibiting retinal degeneration of unknown origin is later -47- diagnosed with cancer (see Thirkill et al. (1987) Arch. Ophthalmol. 105:372-375) . The immune system of the body produces antibodies against an antigen secreted by the tumor. This antigen is immunologically indistinguishable from A-proteins, found abundantly in the outer segments of retinal photoreceptors. The antibodies cross-react with native A-proteins in rods and cause retinal damage to appear before the cancer is detectable. These experiments demonstrate the development of CAR by direct introduction of anti-A-protein monoclonal antibodies into mice. The elaboration of an A-protein-like antigen by human neoplasms is an anticipated result of the involvement of this regulatory enzyme in the formation of cancerous tissues. The tumors secreting this antigen are probably formed due to overproduction of the enzyme, creating a runaway growth cycle within the cancerous cells. The retinal degeneration seen in CAR patients generally precedes a diagnosis of cancer by weeks or usually months, indicating the presence of this antigenic marker in the bloodstream at early stages of the disease process. This is a critical time for therapeutic intervention. The monoclonal antibodies that have been produced against A-protein are likely to be highly specific for the tumor antigen and can be used as a diagnostic tool for screening or early detection purposes. In addition, there is some indication that the same antigen is involved in other cancer- associated autoimmune disease, including that of the central nervous system. -48- The results demonstrate evidence of the ability of the monoclonal antibodies to suppress or, in some cases, reverse the establishment and growth of cancerous tissue. The use of hybridomas to establish antibody-producing tumors in mice is usually so dependable that it is a standard method of monoclonal antibody production. Yet in the overwhelming majority of the mice to which these tumor-producing cells were administered, there is not only a complete lack of tumor growth, but there is virtually no evidence of myeloma cells in the peritoneum after a brief exposure to the monoclonal antibodies being produced. Evidence that significant levels of antibody are initially generated by growing hybridoma tumors comes from the postmortem examination of the retinas of mice injected with hybridoma cells. The damage, in some cases extensive, of the rod outer segments of these animals can only be produced by significant serum titers of antibody in circulation. The most straightforward synopsis is that tumors were formed as a consequence of injection of the hybridoma into the peritoneal cavity. These tumors secreted increasing amounts of antibody with anti-proliferative properties which, in turn, suppressed tumor cell growth. Either the antibody itself, or the significant immune reaction they elicited, resulted in the actual disappearance of tumor cells. Almost all animals sacrificed on days 8 to 10 of the study were found to be free of tumors or hybridoma cells. -49- EXAMPLE 9 Inhibition of Phospholipase C Activation Purified mAb to A-protein (100 to 200 μg) was preincubated with purified A-protein (50 to 100 μg) for 30 minutes prior to the experiment. Stripped ROS, partially purified phospholipase C, 3 H-PIP2 vesicles (0.1 μCi; 0.022 μg PIP2/sample), GTPγS (final concentration is 100 μmol/sample), and the mAb/A-protein mixture (about 10 μg mAb; about 5 μg A-protein/sample) were combined with buffer (10 mM Tris-HCl, 10 mM KCL, pH 7.4) to a final volume of 300 μl/sample. A sample without antibody was used as control. All samples were made up and assayed in triplicate. The samples were incubated at room temperature for 30 minutes in room light and quenched with 300 μl of 15% trichloroacetic acid (TCA) . Following quench, samples were placed on ice for 30 min and then spun down in a microcentrifuge. The supernatant was removed, and soluble inositol phosphates (primarily IP3) are assayed by direct counting following separation on formate-agarose affinity columns. The results are shown in TABLE 3 below. -50- TABLE 3 INHIBITION OF IP3 FORMATION (PIP2 HYDROLYSIS) BY A-PROTEIN ACTIVATION OF PL-C NORMALIZED CPM'S FOR VARYING MOLAR RATIOS OF A m :mAb 1.5:1 2:1 In these studies a value of 1.00 is indicative of no inhibition (compared to controls) of IP3 formation (PIP2 hydrolysis) by A-protein activation of PL-C. A value of 0.65, for example, is equivalent to approximately 35% inhibition due to antibody inactivation of A-protein. These studies demonstrate that the monoclonal antibodies listed in TABLE 3 are specific for A m , and can, at a molar ratio of about two parts A-protein to one part antibody, inhibit IP3 formation. -51- EXAMPLE 11 Assays A. GTPase and ATPase Assays 5 The rate of hydrolysis of GTP or ATP by both soluble and membrane-bound A-protein was assayed in a total volume of 200 μl. 1.0 to 10.0 μg A m , 5 x 10 ~5 M GTP or ATP, 67 to 335 nM [γ" 32 P] GTP or 88.8 to 10 177.5 nM [γ" 32 P] ATP (2.8 Ci/mmol) and 20 μl of stripped ROS membranes (in the case of A m ) were mixed in buffer J (20 mM Tris-HCl, pH 7.0, 0.1 mM EDTA) . The effect of light on the hydrolysis of GTP or ATP was investigated by means of duplicate incubations in 15 the dark. Samples were incubated at room temperature for 5 minutes and quenched with 200 μl ice-cold quench buffer (50 nM KH2PO4, 6% Norite A, 10% TCA) . The samples were kept on ice for 30 minutes and spun down for 5 minutes in a microcentrifuge. 50 μl 20 aliquots of each supernatant were placed in a vial with 5 ml scintillation fluid and assayed for radioactivity. The hydrolysis of [γ~ 32 p]-GTP was measured in the presence and absence of photolyzed rhodopsin. 25 The GTPase activity of A m was enhanced in the presence of the activated receptor. In contrast, no effect on the hydrolysis rate was observed when unphotolyzed rhodopsin was added to the incubation in 30 the dark. In the absence of rhodopsin or the presence of unbleached rhodopsin, A m had negligible GTPase activity. -52- On a mol/mol basis, the rate of GTP hydrolysis by A m (0.458 GTP sec ~ l/A m ) is comparable to that of transducin (0.512 GTP sec~-/Tα) when both are measured at submaximal velocity in the presence of photoactivated rhodopsin. The GTPase rates for A m and transducin are additive when the two purified proteins are present in approximately equimolar concentrations. Under all experimental conditions tested, the rate of GTP hydrolysis by purified A s was insensitive to the presence of bleached or unbleached rhodopsin. The apparent Michaelis constant was determined for A s and A m by measuring the rate of GTP hydrolysis over a thousand-fold range of substrate concentrations. K m and V max were determined by construction of double reciprocal plots and regression analysis. Data are presented as mean ± S.D. The results are given in TABLE 4. -53- TABLE 4 Values For GTPase I ATPase In) - f n :A s 2.06 (±2.46) x 10 _6 M (4) 1.50 (±1.47) x 10- 4 M (3) Am 2.39 (±1.87) x 10--M (3) 1.44 (±0.56) x 10 _5 M (3 ) v max !A s N - D« 0.56 (±0.09) pmol/mg A-protein min-- • »m 16.4 nmol/ingAπ-iin -1 2.90 (±0.16) pmol/mg A-protein min~- In the presence of rhodopsin the K m values for A m and A s are similar indicating similar affinities for GTP. A m and A s were found to have ATPase activity that was not receptor coupled. The K m values for both A m and A s ATPases are given in TABLE 4, above. Comparison of the rate constants of A m indicates that its affinity for ATP is approximately an order of magnitude greater than that of A s . The relative K m values for GTPase and ATPase activity of both A and A s indicate that GTP is the preferred substrate for binding and hydrolysis. The addition of rhodopsin to incubations did not enhance the rate of ATP hydrolysis of either pro¬ tein (FIG. 8). The ATPase rate declines slightly in the presence of the activated receptor. -54- B. GTP Binding Assays Assay of the binding of the GTP-analogs Gpρ(NH)p and GTPγS (New England Nuclear) by both A m and A s was performed according to the methods of Northup et al. (J_. Biol. Chem. (1982) 257:11416-11423). Binding was carried out in a total volume of 100 μl of solution containing 5 to 10 μg purified A-protein, 15.3 μM 3 H-Gpp(NH)p (lOμ Ci) or 1.32 μM GTPγS 35 (1 μCi) and buffer (100 mM NaCl, 0.1 mM EDTA, 20 mM Tris-HCl, pH 7.0). The samples were vortexed and incubated at 25 β C for 30 minutes, quenched with 200 μl ice-cold buffer (0.5 M NaCl, 0.1 M Tris-HCl, 0.1% Tween 80), and kept on ice for 30 minutes. The samples were placed onto nitrocellulose filters that had been previously washed with 2 ml of the same buffer. The filters were rinsed 5 times with 2 ml ice-cold buffer and assayed for radioactivity. A m bound both GTPγS and Gpp(NH)p spontaneously during a brief incubation at room temperature. The results are shown in TABLE 5 below. TABLE 5 form of Gpp(NH)p GTPγs A-protein mol GTP analog/mol protein A m 0.57 + 0.12 0.93 A. 0.29 ± 0.08 0.47 -55- This process apparently required no cofactors. A m bound less than stoichiometric amounts of each GTP analog under the experimental conditions used (TABLE 5) . A s bound significantly less of these analogues on a mol/mol basis after a similar incubation in the absence of cofactors. GTPγS was more readily bound than Gpp(NH)p by both A m and A s . C. ATP/GTP Competition The effect of adenosine nucleotides on the binding of Gpp(NH)p to a mixture of A m and A s was examined because of the ability of the A-proteins to bind and hydrolyze ATP. Purified A m and A s were mixed (1:2), preincubated with ATP or ADP, and assayed for Gpp(NH)p binding after a brief incubation by the rapid filtration method. As shown in FIG. 9, ATP was an effective inhibitor of binding at all concentrations tested. ADP was inhibitory in a concentration-dependent manner at higher concentrations. In contrast to the effect of adenosine nucleotides on the binding of Gpρ(NH)p, they had negligible effects on the hydrolysis of GTP by A-protein. However, at micromolar concentrations, GTPγS was found to significantly inhibit the hydrolysis of ATP during the course of a one hour incubation (FIG. 10). GTP has a similar but less pronounced effect on ATP hydrolysis, indicating that both nucleotides compete for the same or closely related binding sites on A-protein. These results also support the finding that GTP is a more effective competitor than ATP for A-protein binding. -56- D. Protein Assays Protein concentrations were determined according to Bradford (Anal. Biochem. (1976) 22:248), using the Bio-Rad Microassay (Coomassie Brilliant Blue G-250) . Bovine serum albumin was used as a standard. On the basis of protein assays performed on the purified species, the complement of A m relative to A s (A m /A s ) is 0.47. The ratio of the amounts of the separated soluble species recovered from high pressure columns (as estimated by optical density at 280 nm) and slab gels (as estimated by densitometry) is approximately unity. This indicates that all forms of A-protein are extracted in equivalent amounts from rods if the two soluble forms are the products of separate genes. If not, the membrane-bound form would thus be present at half the concentration of the soluble. E. Assay for A-Protein The monoclonal antibodies produced against A-protein were used to create an assay for the detection of that antigen in the serum of humans. The procedure employs an enzyme-linked immunosorbent assay (ELISA) . More specifically, the type of ELISA being used in this context is of the "sandwich" variety. This assay requires two different antibodies that are specific for A-protein, but which each recognize and -57- bind to separate epitopes on the protein. One of the antibodies serves as the "capture" agent and is used unmodified to coat the bottom of a chamber in a standard 96-well microtiter plate. The antibody is then removed from the well and a blocking agent (1% bovine serum albumin (BSA)) is then placed in the chamber to block non-specific binding sites. Serum from the propositus is then incubated in the prepared well for from 1 to 12 hours, and then removed. The well is washed with a detergent solution, and the second antibody in solution is added to the well. The second antibody used in this procedure is physically linked to an enzyme; in this case the enzyme used is horseradish peroxidase (HRP). Following this incubation, the second antibody is removed and the well is washed once more. The final step consists of adding an appropriate colorimetric substrate. The specificities of these antibodies for different epitopes in A-protein were predetermined by performing the ELISA using various combinations of capture and conjugated enzymes with purified antigen (A-protein) to determine possible overlap of recognition sites. The amount of color development is directly proportional to the amount of enzyme-linked antibody bound in the well, which is proportional to the amount of antigen bound in the well by the "capture" antibody. The results are quantitated by a spectrophotometric determination of the amount of color produced over a predetermined period of time (typically 30 to 120 minutes) . -58- The ELISA described above has been used to test human serum from normal healthy volunteers, cancer patients, and individuals with diagnosed bacterial infections, for the presence of the A-protein antigen. The normal population was used to determine the threshold of positivity in this assay. The sensitivity of the assay extends at least to the single ng/ml range as determined by the construction of standard sensitivity curves using known amounts of purified antigen. The patient population tested in preliminary use of this assay yielded the following qualitative results. A positive reaction in the test was obtained from patients diagnosed with lung, lymphoma, stomach, colon, rectal, and breast cancer when compared to normal subjects. A positive reaction in the test was also obtained from individuals with a diagnosed bacterial infection, when compared to normal subjects, including both intestinal and venereal sites. The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present examples are therefore to be considered in all aspects as illustrative and not restrictive, the scope of the invention is indicated by the claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. -59- SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Schmidt, Geoffrey A. (ii) TITLE OF INVENTION: Compositions And Methods For Regulating Cellular Signal Transducing Systems (iii) NUMBER OF SEQUENCES: 5 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Lahive & Cockfield (B) STREET: 60 State Street (C) CITY: Boston (D) STATE: Massachusetts (E) COUNTRY: U.S.A. (F) ZIP: 02109 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.5 inch, 720kb storage (B) COMPUTER: IBM XT (C) OPERATING SYSTEM: DOS 3.30 (D) SOFTWARE: Word Perfect 5.0 (vi) CURRENT APPLICATION DATA: (B) FILING DATE: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) APPLICATION NUMBER: (D) FILING DATE: (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear -60- (ii) MOLECULE TYPE: protein (vii) FEATURE: (A) NAME: Membrane-bound A-protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: Met Gly Gin lie Phe Val Lys Lys Leu Asn Val 5 10 Lys Ala Asn His Gly 15 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vii) FEATURE: (A) NAME: Soluble A-protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Gly Gin lie Phe Val Lys Lys Leu Asn Val Lys 5 10 Ala Asn His Gly 15 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal fragment -61- (ix) FEATURE: (A) NAME: bovine retinal ARF (B) LOCATION: 1 to 10 (x) PUBLICATION INFORMATION: (A) AUTHORS: Price, S. Nightingale, M. Tsai, S. Williamson, K. λdamik, R. Chen, H. Moss, J. Vaughn, M. (B) TITLE: Guanine Nucleotide-Binding Proteins That Enhance Choleragen ADP-Ribosyl Transferase Activity: Nucleotide And Deduced Amino Acid Sequence Of An ADP Ribosylation Factor (C) JOURNAL: Proc. Natl. Acad. Sci. (USA) (D) VOLUME: 85 (F) PAGES: 5488-5491 (G) DATE: 1988 (K) RELEVANT RESIDUES IN SEQ ID NO:3: FROM 1 TO 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Met Gly Asn Val Phe Glu Lys Leu Phe Lys 5 10 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids -62- (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: polypeptide (v) FRAGMENT TYPE: N-terminal fragment (ix) FEATURE: (A) NAME: bovine adrenal ARF (x) PUBLICATION INFORMATION: (A) AUTHORS: Sewell, J. ahn, R. (B) TITLE: Sequences of the Bovine and Yeast ADP Ribosylation Factor and Comparison to Other GTP-Binding Proteins (C) JOURNAL: Proc. Natl. Acad. Sci. (USA) (D) VOLUME: 85 (F) PAGES: 4620-4624 (G) DATE: 1988 (10 RELEVANT RESIDUES IN SEQ ID NO:4:FROM 1 TO 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Gly Asn lie Phe Ala Asn Leu Phe Lys 5 10 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal fragment (ix) FEATURE: (A) NAME: £-£ p21 (x) PUBLICATION INFORMATION: -63- (A) AUTHORS: Barbacid, M. (B) TITLE: ras Genes (C) JOURNAL: Ann. Rev. Biochem. (D) VOLUME: 56 (F) PAGES: 779-827 (G) DATE: 1987 (K) RELEVANT RESIDUES IN SEQ ID NO:5:FROM 1 TO 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Met Thr Glu Tyr Lys Leu Val Val Val Gly 5 10

Claims: -64- What is claimed is: 1. A method of regulating the metabolic pathway of inositol in a cell comprising the steps of: (a) introducing into said cell a molecule selected from the group consisting of a purified A-protein, and active fragments, active analogs, and active fusion products thereof; and (b) allowing said molecule to couple a membrane-bound receptor, which is activated by an external signal, to an intracellular phosphodiesterase of the type responsible for generating second messenger substance, IP3- 2. The method of claim 1 wherein said introducing step comprises introducing into said cell an A-protein which is characterized by: (i) having a molecular weight of about 19 to 20 kilodaltons; (ii) activating phospholipase C in the presence of GTP; (iii) binding and hydrolyzing guanosine nucleotides; and (iv) binding and hydrolyzing adenosine and nucleotides. -65- 3. The method of claim 1 wherein said introducing step comprises introducing a protein including an amino acid sequence selected from the group consisting of SEQ ID NO:l and SEQ ID NO:2. 4. The method of claim 1 wherein said introducing step comprises introducing into said cell a molecule further characterized by coupling to a receptor that is responsive to one of the group consisting of hormones, growth factors, radiation of a particular wavelength, and neurotransmitters. 5. The method of claim 4 wherein said introducing step comprises introducing into said cell a molecule further characterized by coupling to a receptor that is responsive to a growth factor. 6. The method of claim 1 wherein said introducing step comprises contacting said cell with a liposome containing said molecule. -66- 7. A method of stimulating inositol metabolism in a cell comprising the steps of: (a) providing a protein conjugate formed of: (i) a molecule selected from the group consisting of a purified A-protein, an active analog, active fragment, or active fusion product thereof, said molecule coupling a cellular membrane-bound receptor activatable by an external signal to an intracellular phosphodiesterase of the type responsible for generating IP3; coupled to (ii) a non-hydrolyzable analog of GTP; and (b) introducing into said cell an effective amount of said protein conjugate such that said conjugate activates a phospholipase contained therein. 8. The method of claim 7 wherein said providing step comprises providing a protein conjugate including a non-hydrolyzable analog of GTP selected from the group consisting of guanosine-5'-0-[3- thiotriphosphate] and β-γ-imidoguanosine 5'-triphosphate. -67- 9. The method of claim 7 wherein said said providing step comprises providing a protein conjugate characterized by: (i) having the ability to activate phospholipase C; (ii) having a molecular weight of about 19 to 20 kilodaltons; and 10 (iii) having an amino acid sequence selected from the group consisting of SEQ ID NO:l and SEQ ID NO:2. 15 10. An isolated and purified composition of matter useful in stimulating inositol metabolism in a cell, said composition being a conjugate comprising: (a) a nonhydrolyzable analog of GTP; 20 coupled to (b) a molecule selected from the group consisting of a purified A-protein, or an active fragment, active analog, or active 25 fusion product thereof, said molecule coupling a cellular, membrane-bound receptor activatable by an external signal to an intracellular phosphodiesterase of the type responsible for generating IP3. 30 11. The composition of claim 10 wherein said non-hydrolyzable GTP analog is selected from the group consisting of guanosine-5'-0-[3-thio- triphosphate] and β-γ-imidoguanosine 5' triphosphate. 35 -68- 12. The composition of claim 10 wherein said molecule is further characterized by: (i) activating the phosphodiesterase 5 phospholipase C in the presence of GTP; and (ii) having a molecular weight of about 19 to 20 kilodaltons; and 10 13. The composition of claim 10 wherein said A-protein includes an amino acid sequence selected from the group consisting of the sequences set forth as SEQ ID NO:l and SEQ ID NO:2. 15 14. A liposome containing the composition of claim 10. 15. A method of controlling the secondary effects of diabetes, including vascular degeneration and slowed 20 nerve conduction, in a subject, said method comprising the steps of: (a) providing a molecule selected from the group consisting of a purified A-protein, 25 active analogs, active fragments, and active fusion products thereof, said molecule coupling a cellular membrane-bound receptor, activatable by an external signal, to an intracellular phosphodiesterase of the type 30 responsible for generating IP3; and (b) administering said molecule in a physiologically acceptable carrier to the circulatory system of said subject. 35 -69- 16. The method of claim 15 wherein said providing step comprises providing an A-protein including an amino acid sequence selected from the group consisting of SEQ ID NO:l and SEQ ID NO:2. 17. The method of claim 15 wherein said providing step further comprises providing an A-protein characterized by: (i) having a molecular weight of about 19 to 20 kilodaltons; (ii) activating phospholipase C in the presence of GTP; (iii) binds and hydrolyzes guanosine nucleotides; and (iv) binds and hydrolyzes adenosine and nucleotides. 18. The method of claim 15 wherein said providing step further comprises providing said molecule in a liposome. -70- 19. A method of reducing the concentration of GTP in a cell comprising the steps of: (a) providing a molecule selected from the group consisting of a purified A-protein, active analogs, active fragments, and active fusion products thereof, said molecule having the ability to couple a cellular, membrane-bound receptor activatable by an external signal to phospholipase C; and (b) introducing said molecule, in a physiologically acceptable carrier, into said cell. 20. The method of claim 19 wherein said providing step comprises providing a purified A-protein including an amino acid sequence selected from the group consisting of SEQ ID NO:l and SEQ ID NO:2. 21. An antibody which is reactive with an A-protein selected from the group consisting of soluble A-protein (A s ), membrane-bound A-protein (A ), and fragments thereof. 22. The antibody of claim 21 which is a monoclonal antibody. -71- 23. A method of detecting the presence of an A-protein in a mammalian subject, said A-protein coupling a cellular membrane-bound receptor activatable by an external signal to an intracellular phosphodiesterase of the type responsible for generating IP3, said method comprising the steps of: (a) obtaining a biological sample from said subject; (b) treating said sample with an antibody which binds to said A-protein, thereby forming an antibody-A-protein conjugate; and (c) detecting the presence or absence of said conjugate, the presence of said conjugate being indicative of the presence of said A-protein in said sample. -72- 24. A method for detecting an A-protein in a biological sample, said A-protein coupling a cellular membrane-bound receptor activatable by an external signal to an intracellular phosphodiesterase of the type responsible for generating IP3, said method comprising the steps of: (a) adhering a first antibody to a solid support, said first antibody binding to a first epitope on said A-protein; (b) contacting said adhered first antibody with said biological sample to be tested; (c) adding a second antibody to said solid support, said second antibody binding a second epitope on said A-protein and further comprising a marker; (d) removing any unbound antibody; and (e) detecting the presence or absence of said marker, the presence of said marker being indicative of the presence of said A-protein in said sample. 25. The method of claim 24 wherein said adding step (c) further comprises adding a second antibody having a marker selected from the group consisting of enzymes, fluorescent dyes, and radioisotopes. -73- 26. A method of regulating a metabolic pathway in a cell comprising the step of introducing into said cell a molecule which couples a membrane-bound receptor, activatable by an external signal, to an intracellular phosphodiesterase, thereby generating a second messenger substance, said molecule being selected from the group consisting of a purified A-protein, active fragments, active analogs, and active fusion products thereof. 0 27. The method of claim 26 wherein said introducing step comprises introducing into said cell a molecule which couples a membrane-bound receptor to phospholipase C. 15 28. A method of stimulating metabolism in a cell comprising the step of introducing into said cell an effective amount of a protein conjugate to activate a phosphodiesterase contained therein, said protein 20 conjugate including: (i) a non-hydrolyzable guanosine nucleotide analog; coupled to 25 (ii) a molecule selected from the group consisting of a purified A-protein, active analogs, active fragments, and active fusion products thereof. 30 29. The method of claim 28 wherein said introducing step comprises introducing into said cell a protein conjugate including a non-hydrolyzable GTP analog. -74- 30. A method of regulating the level of calcium ions in a cell comprising the step of introducing into said cell a molecule coupling a membrane-bound receptor, activated by an external signal, to an intracellular phosphodiesterase, thereby generating second messenger substance, IP3, and binding and hydrolyzing adenosine and guanosine nucleotides. 31. The method of claim 30 wherein said molecule is further characterized by: (i) having a molecular weight of about 20 to 21 kilodaltons; (ϋ) activatating phospholipase C in the presence of GTP. 32. The method of claim 30 wherein said introducing step comprises introducing into said cell a protein including an amino acid sequence selected from the group consisting of SEQ ID NO:l and SEQ ID NO:2. 33. The method of claim 30 wherein said introducing step further comprises contacting said cell with a liposome containing said molecule. -75- 34. A method of inhibiting the growth and proliferation of cancer cells comprising the steps of: (a) providing an antibody which binds specifically to an A-protein; and (b) administering said antibody in a pharmacologically acceptable vehicle to said cancer cells. 35. The method of claim 34 wherein said providing step comprises providing a monoclonal antibody specific for an A-protein. 36. A method of imaging a cell tissue or organ that expresses an A-protein on its surface, said method comprising the steps of: (a) providing an antibody which binds a membrane-bound form of A-protein (A m ) conjugated to a detectable marker; (b) administering said antibody-conjugate marker in a pharmacologically acceptable vehicle to said cells, tissue, or organ; and (c) determining the presence or absence of said marker, on said cell, tissue, or organ the presence of said marker being indicative of the presence of A m on said cell, tissue, or organ.

Inventors: SCHMIDT GEOFFREY J, SCHMIDT, GEOFFREY, J.
Applicants: SCHEPENS EYE RES INST, THE SCHEPENS EYE RESEARCH INSTITUTE
Application year: 1992
Main classification: G01N33
IPC codes: A61K38, A61K39, A61K47, A61K49, A61P3, A61P35, C07K7, C07K14, C07K16, C07K19, C12N9, C12N15, C12P21, C12R1, G01N33