bluuebunny/crossref_metadata_2025 huggingface dataset:
Rhodopsin–lipid interactions
Authors: Hong Keelung , Chen Yong Shiau , Hubbell Wayne L.
Published: May 2005

Purified, lipid‐free rhodopsin has been incorporated into bilayers of natural and synthetic phosphatidyl cholines and a natural digalactosyl diglyceride. Successful incorporation and high regenerability of rhodopsin appears to depend on the nature of the hydrocarbon chains and not specifically on the type of polar head group of the lipids.

Electron paramagnetic resonance spectra of recombinant membranes containing small amounts of spin labeled phosphatidyl cholines reveals that rhodopsin interacts directly with the hydrocarbon chains of the phospholipids in the bilayer, resulting in a dramatically decreased motional freedom of the chains.

In studies of the recombinant membranes using freeze–fracture electron microscopy, the presence of rhodopsin is detected as discrete particles in the plane of fracture. The particle distribution is dependent on the nature of the lipid hydrocarbon chains and whether or not the rhodopsin has been photolyzed.

From these studies, it is concluded that the rhodopsin‐bilayer structure is stabilized primarily by direct interaction of the hydrocarbon chains with the protein molecule, and that structural parameters in this model system are subject to modulation by visible light.
Adenylate cyclase and cyclic AMP phosphodiesterase of normal and rous sarcoma virus transformed chicken embryo fibroblasts
Authors: Russell Tom R. , Anderson Wayne B.
Published: May 2005

Cyclic AMP appears to regulate cell growth. Cyclic AMP levels are high in normal chicken embryo fibroblasts and drop to very low levels when the cells are transformed by the Bryan high‐titer strain of Rous sarcoma virus. Cells infected with a temperature‐sensitive mutant of the virus have normal levels of cyclic AMP at the nonpermissive (nontransforming temperature), but when the cells are shifted to the permissive (transforming) temperature the cyclic AMP levels rapidly fall to values that are found in transformed cells. Studies on the adenylate cyclase and cyclic AMP phosphodiesterase in normal and transformed chicken embryo fibroblasts have shown that the adenylate cyclase is greatly decreased in the transformed cells whereas the phosphodiesterase is increased. The decrease in adenylate cylcase activity is the result of an increase in the Km of the substrate and a loss of a magnesium ion activator site. The increase in phosphodiesterase activity is the result of an increase in total phosphodiesterase activity and a decrease in the negative cooperativity of plasma membrane bound phosphodiesterase. Thus the fall in cyclic AMP levels that occurs on transformation can be correlated with changes in the activity of adenylate cyclase and cyclic AMP phosphodiesterase.
Red cell aggregation by macromolecules: Roles of surface adsorption and electrostatic repulsion
Authors: Chien Shu , Jan Kung‐Ming
Published: May 2005

Aggregation of normal and neuraminidase‐treated human red blood cells (RBC) by dextrans of various molecular sizes and concentrations was quantified by microscopic counting and light reflectometry. The influences of variations in the ionic strength and the cationic valency of the dextran solution on RBC aggregation were also investigated. The data on RBC aggregation were correlated with measurements of the zeta potential by cell electrophoresis and the intercellular distance in the rouleaux by electron microscopy. With the use of the classical equations and newly developed knowledge in colloid chemistry, the electrostatic repulsive force between adjacent cell surfaces (Fe) was calculated from the experimental data. The macromolecular bridging force causing RBC aggregation (Fb) has also been derived as a function of dextran concentration. Other forces that cause RBC disaggregation are the mechanical shearing force (Fs) and the RBC membrane bending force (Fm). The net force for RBC aggregation is equal to Fb – Fe – Fm – Fs. This model of aggregation involving force balance at the surface can explain known experimental results on factors influencing cell aggregation. It is proposed that such force balance between cell surfaces may be applicable in other cell or particulate systems and that it may be of fundamental importance in many physiological functions.
The surface chemistry of the erythrocyte and thrombocyte membrane
Authors: Seaman G. V. F.
Published: May 2005

The electrokinetic procedures used for investigation of the nature and distribution of cellular surface charge groups are outlined. The application of ion binding studies, the action of enzymes, and the effects of specific functional group reagents are illustrated by discussion of the results obtained for erythrocytes and thrombocytes. The human erythrocyte has been shown to be a macropolyanion possessing about 107 charged groups per cell of which more than 60% are the carboxyl group of N‐acetylneuraminic acids; some of the remaining groups at least are the α‐carboxyl groups of protein‐bound amino acids. The electrokinetic constitution of the human blood platelet is more complex and includes sialic acid carboxyl groups and phosphate groups and at least one other species of unidentified anionogenic groups as well as amino groups. Lastly the effects of neutral and charged polymer adsorption on the electrokinetic properties of blood cells are briefly considered.
The salt‐extractable fraction of dynein from sea urchin sperm flagella: An analysis by gel electrophoresis and by adenosine triphosphatase activity
Authors: Kincaid Jr. Harvey L. , Gibbons Barbara H. , Gibbons I. R.
Published: May 2005

Previous work has shown that the dynein from axonemes of sea urchin sperm consists of two distinct fractions which differ substantially in their extractability by salt. Upon gel electrophoresis of whole demembranated axonemes solubilized with sodium dodecyl sulfate, the dynein fraction shows two closely spaced bands with apparent molecular weights of 520,000 and 460,000; the proteins in these bands are termed the A and B components of the dynein. Similar electrophoresis of the soluble fraction obtained by extracting the axonemes with 0.5 M NaCl shows a single prominent band containing approximately half of the A component of the dynein (A1 component). The residue of extracted axonemes contain the other half of the A component of the dynein (A2 component) and all the B component. Densitometry of the bands indicates that the A1, A2 and B components of the dynein are present in approximately equal molar quantity. Electron microscopic studies show that the A1 component of the dynein constitutes the outer arms on the doublet tubules. Assay of ATPase activity in 0.05 M KCl and l mM ATP indicates about 65% of the total ATPase activity becomes soluble when the A1 component of the dynein is extracted with salt.
Three‐dimensional structures at 5.5 Å resolution and regulatory processes in aspartate transcarbamylase from E. coli
Authors: Lipscomb W. N. , Evans D. R. , Edwards B. F. P. , Warren S. G. , Pastra‐Landis S. , Wiley D. C.
Published: May 2005

The three‐dimensional structure of the multisubunit allosteric enzyme, aspartate transcarbamylase, has been determined to 5.5 Å resolution. An unusual feature of the molecule is a large central aqueous cavity 50 Å × 50 Å × 25 Å, into which the active sites face. Access to the central cavity and the active site region is provided by six equivalent channels of 15 Å diameter.

A complex C6R4, composed of catalytic trimers C3 and of regulatory dimers R2, has been isolated upon treatment of aspartate transcarbamylase (ATCase, C6R6) by mercurials. The specific catalytic activity of C6R4 is essentially the same as that of ATCase, about 70% of that of the catalytic trimers at 30 mM aspartate and saturating carbamyl phosphate. Allosteric interactions are reduced in C6R4 as compared with those in ATCase. In the homotropic interactions the Hill coefficient is reduced from approximately 3.3 to 2.1 at pH 8.3, while the heterotropic interactions of both cytidine triphosphate (CTP) and adenosine triphosphate (ATP) are reduced substantially but not abolished at pH 8.3. Thus, the allosteric transitions involved in the regulatory mechanisms do not require the intact structure C6R6. Also, this regulation is not simply the control of access of substrates or products to or from the large central aqueous cavity in the ATCase molecule.

Comparison of electron density maps at 5.5 Å resolution for ATCase and for the complex of ATCase with CTP shows substantial similarities throughout the three‐dimensional electron density maps. Significant differences are seen, however, in the region of the regulatory dimers R2 where CTP adds, and near the active sites in the catalytic trimers C3.
A regulator protein for the length determination of bacteriophage lambda tail
Authors: Katsura Isao , Kühl Peter W.
Published: May 2005

Mutants in gene U of phage λ produce polytails. Those polytails have a tail fiber and a basal part like normal tails, but their major tubular part is longer than that of normal tails and shows a wide length distribution.

We established the morphogenetic pathway of the λ tail and found that U gene product (pU) acts throughout the assembly of the major tail protein (pV). Polytails in U− lysate are activated by pU in vitro and form long‐tailed phage which are infectious to a small extent.

If the formation of the basal part of the tail is blocked, pV (the major tail protein) remains unassembled as long as pU is present in the cell. However, we found that part of pV assembles into giant polytubes of several microns in length in lysates of a double mutant U− · H− in which both the basal part and pU are absent.

pV in purified tails can be dissociated into disks (about 10S) or smaller units (about 2.5S) in vitro under extreme conditions. The disks form polytubes efficiently under physiological conditions, but the smaller units do not form polytubes efficiently. The smaller units have in vitro complementation activity with V− lysate. In vitro complementation activities with V−, U−, and Z− lysates are detected in the dialyzed extracts of SDS gel electrophoresis of purified tails. The molecular weights of the polypeptide chains containing those activities are estimated to be 31,000, 14,000 and 20,000 daltons, respectively.
The transformation of τ particles into t4 heads. II. Transformations of the surface lattice an related observations on form determination
Authors: Aebi U. , Bijlenga R. , v. d. Broek J. , v. d. Broek R. , Eiserling F. , Kellenberger C. , Kellenberger E. , Mesyanzhinov V. , Müller L. , Showe M. , Smith R. , Steven A.
Published: May 2005

In part I of this paper (1) we give evidence that the P23‐capsoid of τ‐particles is transformed in situ into the P23*‐capsid of normal phage. Using the polymorphism of phage T4, we have chosen polyheads as representative of P23 assemblies and giant phages as representative of P23* assemblies in order to study their surface crystals by optical filtration of micrographs. We found for polyheads a lattice constant of 112 Å with the typical hexameric, ringlike capsomer and for the giants a lattice constant of 124 Å with quite a different capsomer morphology, of the type (6+1). From the stoichiometry of the proteins composing the normal capsid we conclude that the protomer is a single P23* molecule and that the minor capsid‐proteins must be in singular positions on the surface lattice or on the polyhedral head (center of capsomers, vertices, or basal part).

We extrapolate the findings on the giant head to the normal head and give a geometric model which is consistent with 1,100 molecules of P23* per capsid.

We discuss the part of form inheritance contributed by P23 and the other formgiving gene products and give evidence that morphologic characters are the result of pairs of a reaction chain of interacting gene products. The example we give is the giant head produced by a ts mutant in gene 24 at 36°C.
The mechanism of microtubule assembly in vitro
Authors: Kirschner Marc W. , Williams Robley C.
Published: May 2005

The mechanism of microtubule polymerization and depolymerization has been studied with protein purified from extracts of porcine brain. Under polymerizing conditions characteristic microtubules composed of parallel protofilaments are observed in the electron microscope. Under depolymerizing conditions three forms are observed: double rings of outside diameter 49 nm, spirals, and 7‐nm globular subunits. Under the same conditions two boundaries are observed in the analytical ultracentrifuge at 6S and 36S, whether depolymerization is accomplished by cooling to 0°C, by addition of 1 mM CaCl2 at 25°C, or by removal of GTP. On polymerization all of the 36S and most of the 6S is converted to a fast‐sedimenting form which the electron microscope reveals to be microtubules.

The depolymerization mixture may be fractionated by gel chromatography into two fractions, one consisting solely of 6S and the other mostly 36S. Neither fraction regenerates the original equilibrium mixture. The 36S form may be reversibly dissociated into 6S subunits by addition of NaCl. From these and other considerations we have postulated that microtubule protein is composed of two different types of tubulin, both of which participate in polymerization. Studies are reported showing that colchicine does not dissociate microtubule rings but blocks polymerization by interfering with their proper lateral association into a protofilament array within microtubules. The role of GTP in polymerization is also discussed. Electron micrographic evidence is presented suggesting the conversion of protofilaments directly into rings and spirals, and a pathway for microtubule assembly is proposed.
Structural studies of spherical viruses
Authors: Harrison Stephen C. , Jack Anthony , Goodenough Daniel , Sefton Bartholomew M.
Published: May 2005

Structural studies of tomato bushy stunt virus and Sindbis virus are discussed in terms of the information they provide about specificity and control in virus and membrane assembly.
Undelivered summary remarks for the 1974 squaw valley meeting on assembly mechanisms
Authors: Wood W. B.
Published: May 2005

The status of research on macromolecular assembly is similar in several respects to that of research on macromolecular synthesis in the late 1950's. The work of that era can teach us some lessons, but it also has left us with some preconceptions that may be misleading us in our attempts to understand assembly mechanisms.
The biochemistry of an acetylcholine receptor
Authors: Raftery M. A. , Vandlen R. , Michaelson D. , Bode J. , Moody T. , Chao Y. , Reed K. , Deutsch J. , Duguid J.
Published: May 2005

The acetylcholine receptor from Torpedo californica electroplax has been studied at three levels of molecular organization: receptor‐rich membrane fragments, solubilized and purified receptor, and reconstituted receptor in phospholipid vesicles. The binding of cholinergic ligands to the membrane‐bound and the solubilized material is not cooperative, and the number of ligand sites is less than the number of toxin sites. In addition, the purified macromolecule contains the molecular features necessary for ion‐translocation during postsynaptic depolarization, since a chemically excitable membrane can be formed from purified receptor and Torpedo phospholipids.
Reconstitution of mycoplasma membranes
Authors: Razin Shmuel
Published: May 2005

Detergent‐solubilized proteins and lipids of mycoplasma membranes reassemble spontaneously into membranous structures on the removal or dilution of the detergent in the presence of divalent cations. The cations seem to function by neutralizing the negatively charged groups on membrane lipids and proteins which interfere by electrostatic repulsion with membrane reassembly. Moreover, salt bridges formed by the divalent cation between acidic groups on membrane proteins and lipids seem to play an important role in the reconstituted membrane stability. Electron transport activity, as measured by the transport of electrons from NADH to oxygen, has been demonstrated in reconstituted Acholeplasma laidlawii membranes. However, restoration of active transport of sugars or ions has not been achieved so far. The conditions for obtaining properly sealed vesicles, which are obligatory for demonstrating transport activity, are still rather poorly defined. The reassembled membranous structures cannot be distinguished from the native membranes in chemical composition, density, and thin sections. However, probe techniques, x‐ray diffraction, and freeze‐fracturing electron microscopy indicate that the proteins are organized differently in the reassembled membranes, though the lipid bilayer is restored. The results obtained so far leave little hope for successfully reconstituting the molecular organization of membranes as complex as those of mycoplasmas by a single‐step reassembly of detergent‐solubilized membrane components. The prospects appear brighter with membranes having only a few protein species, such as the outer membrane of gram‐negative bacteria. In spite of the failure to reconstitute fully active mycoplasma membranes, the reassembly procedure was found valuable in studying the interactions of detergent‐solubilized membrane proteins with lipids, the effects of a hydrophobic environment on hydrophilic enzymes, and the production of “hybrid” membranes having selected membrane components.
Biochemical characterization of the golgi complex of mammalian cells
Authors: Fleischer Becca , Zambrano Fernando , Fleischer Sidney
Published: May 2005

We have developed methods for the isolation of Golgi apparatus from a number of mammalian tissues. The Golgi is distinct both chemically and enzymatically from the other membranes of the cell. For both liver and kidney, galactosyltransferase has been found to be a useful marker enzyme for Golgi membranes. This enzyme is involved in the modification of glycoproteins during secretion. In addition to lipoproteins and glycoproteins, the Golgi apparatus of liver is involved in the secretion of albumin, a simple protein. It does not, however, take part in the synthesis of sphingomyelin, lecithin, or triglycerides which are present in the secreted lipoproteins. These lipids appear to be synthesized predominantly by the endoplasmic reticulum. In kidney, which is rich in glycolipids, 3′‐phosphoadenosine 5′‐phosphosulfate, an enzyme which converts cerebroside to sulfatide, is localized predominantly in the Golgi apparatus. Thus, Golgi functions to modify glycolipids as well as mucopolysaccharides and proteins. Sulfatide constitutes a significant fraction of the total lipid of both Golgi and plasma membranes of kidney. When 35S‐sulfate is injected into rats, it is incorporated first into the sulfatides of the Golgi apparatus and later appears in the sulfatides of the plasma membrane. The data are consistent with the view that sulfatides are formed in the Golgi apparatus of kidney and then transported to the plasma membrane.
Bacteriorhodopsin: Photosignal transduction and photoenergy transduction in different biological systems
Authors: Bogomolni Roberto A. , Stoeckenius Walther
Published: May 2005

Living organisms use light as a source of energy and as a source of information. They have developed highly specialized photoenergy and photosignal transducing devices which serve these functions. Membranes are essential parts of both photosignal and photoenergy transducing systems.

In photoenergy transduction a substantial part of the absorbed energy is conserved for times very long compared to the lifetime of excited states and converted finally to chemical free energy of ATP and other forms in which it can be stored for further use by the organism. In photosignal transduction light typically triggers an event which dissipates much more energy than is absorbed in the form of light. The additional energy had been stored previously by the organism through some energy transducing systems.
Radioiodination of the envelope proteins of newcastle disease virus
Authors: Li Joseph K.‐K. , Fox C. Fred
Published: May 2005

Lactoperoxidase‐catalyzed iodination selectively labels the two glycoproteins (VP1 and VP2) of Newcastle disease virus. The low‐molecular‐weight, nonglycosylated major viral protein, VP6, was not iodinated in the intact virus but was iodinated in disrupted virions, suggesting a localization on the inner, rather than the outer envelope surface. Studies on the distribution of virion proteins labeled with 125I and 3H‐isoleucine between detergent‐soluble and detergent‐insoluble fractions show that the virion proteins VP4, VP5, and VP6 are solubilized to a much lesser extent than are VP1 and VP2.
The bound nucleotide of actin
Authors: Cooke Roger
Published: May 2005

The extent of actin polymerization has been studied for samples in which the bound nucleotide of the actin was ATP, ADP, or an analog of ATP that was not split (AMPPNP). The equilibrium constants for the addition of a monomer to a polymer end were determined from the concentration of monomer coexisting with the polymer. An analysis of these results concludes that the bound ATP on G‐actin provides little energy to promote the polymerization of the actin. AMPPNP was incorporated into F‐actin and the interaction of F‐actin · AMPPNP with myosin was studied. F‐actin · AMPPNP activated the ATPase of myosin to the same extent as did F‐actin · ADP. However, the rate of superprecipitation was slower in the case of F‐actin · AMPPNP than in the control.
The binding of Mn2+ and ADP to myosin
Authors: Martonosi Anthony
Published: May 2005

The metal ion requirement of myosin‐ADP binding was investigated by use of Mn2+. Mn2+ binds to two sets of noninteracting sites on myosin which are characterized by affinity constants of 106 and 103, M−1 at 0.016 M KCl concentration. The maximum number of sites is 2 for the high affinity and 20–25 for the low affinity set. Binding of Mn2+ to the high affinity sites increases the affinity of ADP binding to myosin.

F‐actin inhibits ADP binding (Kiely, B., and Martonosi, A., Biochim. Biophys. Acta 172: 158–170 [1969]), but even at F‐actin concentrations much higher than that required to saturate the actin binding sites of myosin or its proteolytic fragments, significant ADP binding remained. The actin insensitive portion of ADP binding was inhibited by 10−4 M inorganic pyrophosphate or ATP. The results are discussed on the basis of a model in which actin and ADP bind to myosin at distinct but interacting sites.
Studies on the primary structure of 14 proteins from the large subunit of escherichia coli ribosomes with an improved protein sequenator and with mass spectrometry
Authors: Wittmann‐Liebold B. , Geissler A. W. , Marzinzig E.
Published: May 2005

Fourteen proteins from the large subunit of Escherichia coli ribosomes were analyzed in an improved sequenator. In addition to our previously described modifications of a Beckman sequenator, new valves which work free of a dead volume were constructed. By this and the previous improvements (e.g., a new vacuum system with a recorder, cool traps, automatic conversion) much better results were obtained than before. It was even possible to use (in addition to the standard methods, e.g., thin‐layer chromatography and amino acid analysis) mass spectrometry without preceding gas chromatography for identification of the released PTH amino acids. Our experience with the various methods, especially mass spectrometry, is described and the techniques are compared. The results obtained by the described methods on the amino acid sequences of the 14 ribosomal proteins are summarized.
Investigations of the possible role of proteases in altering surface proteins of virally transformed hamster fibroblasts
Authors: Hynes Richard O. , Pearlstein Edward S.
Published: May 2005

Virally transformed fibroblasts have on their surfaces zero or reduced amounts of a large external transformation‐sensitive (LETS) glycoprotein. This protein is extremely sensitive to proteolysis. When prelabeled normal fibroblasts are cocultivated with transformed cells, the LETS glycoprotein of the normal cells shows an increased rate of turnover. Experiments are described which investigate the possibility that this phenomenon and the absence of LETS glycoprotein are due to proteolysis by the transformed cells. In particular, the role of plasminogen activation is examined by the use of protease inhibitors and plasminogen‐depleted serum. It is concluded that activation of plasminogen is not required for the disappearance of the LETS glycoprotein although the involvement of other proteases cannot be ruled out. The role of proteases in affecting cell growth and behavior is discussed.
Binding to specific receptors on oocyte plasma membranes by serum phosvitin‐lipovitellin
Authors: Yusko S. C. , Roth Thomas F.
Published: May 2005

Specific binding sites for the serum complex of phosvitin and lipovitellin have been shown to exist on the outer surface of rapidly growing chicken oocytes. The existence and specificity of these sites were demonstrated by competition for binding to unfixed oocyte membrane fragments and by displacement of already bound and labeled phosvitin‐lipovitellin from formaldehyde‐fixed membranes. Only unlabeled phosvitin‐lipovitellin competed with the 125I‐labeled complex for binding to the fragments or displacement of bound label; IgG isolated from egg yolks and bovine serum albumin were ineffective.
The cytochemical localization of adenylate cyclase activity in mucous and serous cells of the salivary gland
Authors: Kim Sun‐Kee
Published: May 2005

The present study was undertaken to localize adenylate cyclase activity in salivary glands by cytochemical means. For the study, serous parotid glands and mixed sublingual glands of the rat were used. Pieces of the fixed glands were incubated with adenosine triphosphate (ATP) or adenylyl‐imidodi‐phosphate (AMP‐PNP) as substrate: inorganic pyrophosphate or PNP liberated upon the action of adenylate cyclase on the substrates is precipitated by lead ions at their sites of production.

In both glands, the reaction product was detected along the myoepithelial cell membranes in contact with secretory cells, indicating that a high level of adenylate cyclase activity occurs in association with these cell membranes. The association with a high level of the enzyme activity might be related to the contractile nature of myoepithelial cells which are supposed to aid secretory cells in discharging secretion products.

A high level of adenylate cyclase activity was also detected associated with serous secretory cells (acinar cells of the parotid gland and demilune cells of the sublingual gland), but not with mucous secretory cells. In serous cells, deposits of reaction product were localized along the extracellular space of the apical cell membrane bordering the lumen. This is the portion of the cell membrane which fuses with the granule membranes during secretion. Since the granule membranes are not associated with a detectable level of adenylate cyclase activity, it appears that the enzyme activity becomes activated or associated with the granule membranes as they become part of the cell membrane by fusion. The association with a high level of adenylate cyclase activity appears to be related to the ability of the membrane to fuse with other membranes. It is likely, since the luminal membrane of mucous cells which does not fuse with mucous granule membranes during secretion is not associated with a detectable enzyme activity.
The HLB dependency for detergent solubilization of hormonally sensitive adenylate cyclase
Authors: Storm D. R. , Field S. O. , Ryan J.
Published: May 2005

The HLB dependency for the solubilization of membrane proteins and adenylate cyclase activity from a plasma membrane‐enriched fraction from rat liver has been determined. The HLB (hydrophilic/lipophilic/balance) number of a detergent is an empirical measure of its relative hydrophobicity. Detergent HLB numbers vary systematically with the length of the ethylene oxide chain for a homologous series of detergents such as the Triton X series. These detergents have a constant hydrophobic moiety, octylphenyl, and a variable polar portion, polyethoxyethanol. Basal‐NaF‐epine‐phrine‐, and glucagon‐stimulated adenylate cyclase activities were solubilized in the HLB range of 16.8–17.4. Solubilization was most effective in 0.01 M Tris buffers at pH 7.5 containing 1–5 mM mercaptoethanol, 1 mM MgCl2, and 0.1% Triton X‐305. The detergent to membrane protein ratio used in these studies was 3:1.

Criteria for solubilization included lack of sedimentation at 100,000 × g, the absence of particulate material in the supernatant when examined by electron microscopy, and inclusion of hormonally sensitive adenylate cylcase activity in Sephadex G‐200 gels. The apparent molecular weight of the solubilized enzyme was approximately 200,000 in the presence of Triton X‐305. The solubilized enzyme was stimulated 5‐fold by NaF, 7‐fold by glucagon, and 20‐fold by epinephrine compared to the particulate enzyme used in this study which was stimulated 10‐fold, 3,4‐fold, and 4‐fold by NaF, epinephrine, and glucagon, respectively. The solubilized enzyme is stable for several weeks when stored at −60° C.
Insulin binding and glucose transport in the R3230AC mammary adenocarcinoma
Authors: Harmon Joan Thilly , Hilf Russell
Published: May 2005

Cells dissociated from the R3230AC mammary adenocarcinoma from intact and diabetic rats were examined for insulin binding and glucose transport. The Kd for insulin binding, ∼ 10−10 M, was similar in all tumors studied. However, the apparent number of receptor sites per cell increased in cells from diabetic rats. Kinetic analysis of 3‐0‐methyl glucose (3‐OMG) entry showed both diffusional and passive carrier characteristics. Insulin (4 × 10−9 M) in vitro did not affect diffusional entry, whereas the hormone altered the passive carrier system, as reflected by an increase in Km and Vmax. Insulin decreased initial velocity of glucose transport at 4–6 mM glucose levels but increased initial velocity of glucose transport at 20 mM glucose. An explanation of the role of insulin on tumor growth in vivo from effects on glucose transport in vitro is proposed.
Cyclic nucleotides and cell growth
Authors: Seifert W.
Published: May 2005

Growth induction in resting fibroblast cultures by serum or growth factors induces a fast, transient cGMP peak which may constitute the intracellular signal for growth. A similar cGMP peak occurs when 3T3 cells arrested at the restriction point or in G0 by starvation for certain amino acids are induced for growth by readdition of the lacking nutrients. Both 3T3 and SV3T3 cells which are arrested randomly all around the cell cycle do not exhibit major changes in cyclic nucleotides after growth induction.

Determination of intracellular cAMP and cGMP levels in normal and transformed fibroblasts under different growth conditions shows that the transition between growing and resting state (G0 arrest) is accompanied and probably induced by characteristic changes in cAMP to cGMP ratios. cGMP is decreased 2‐5‐fold in resting as compared to growing cultures, and increased 10‐20‐fold in activated cultures 20 min after serum induction. No major cGMP change was observed in growing, confluent, or serum‐activated cultures of transformed cells.

Measurement of guanylcyclase under unphysiological conditions (2 mM Mn++) in crude and purified membranes from 3T3 and SV3T3 cultures did not show increased enzyme activity in the transformed cells. Significant differences may only show up when synchronized cells pass through the restriction point in G1 phase. As a hypothesis it is proposed that transformed cells have an activated guanylcyclase system or a relaxed cGMP‐pleiotypic response mechanism at the restriction point of their cell cycle.
Vasopressin‐sensitive kidney adenylate cyclase: Modulation of the hormonal response
Authors: Roy Christian
Published: May 2005

Vasopressin‐sensitive pig kidney adenylate cyclase is sensitive to several effectors, such as Mg2+, other divalent cations, and guanyl nucleotides. The purpose of the present study was to compare the main characteristics of adenylate cyclase activation by vasopressin, Mg2+, and GMPPNP, respectively. Mg2+·ions were shown to exert at least three different effects on adenylate cyclase. The substrate of the adenylate cyclase reaction is the Mg‐ATP complex. Mg2+ interacts with an enzyme regulatory site. Finally, Mg2+ can modulate the hormonal response, with Mg2+ions affecting the coupling function–that is, the quantitative relationship between receptor occupancy and adenylate cyclase activation. At all the magnesium concentrations tested, from 0.25 mM to 16 mM, adenylate cyclase activation was not a direct function of receptor occupancy. At low Mg2+ concentrations, adenylate cyclase activation dose‐response curve to the hormone tended to be superimposable to the hormone dose‐binding curve. These results suggest a role of magnesium at the coupling step between the hormone‐receptor complex and adenylate cyclase response. Cobalt, but not calcium, ions could exert the same effects as Mg2+ ions on this coupling step.

GMPPNP induced considerable adenylate cyclase activation (15 to 35 times the basal value). Activation by GMPPNP was highly time and temperature dependent. At 30° C, a 20 to 60 min preincubation period in the presence of GMPPNP was needed to obtain maximal activation. The higher the dose of GMPPNP in the medium, the longer it took to reach equilibrium. At 15° C, activation was still increasing with time after 3 hr preincubation in the presence of the nucleotide. GMPPNP was active in a 10−8 M to 10−5 M concentration range. Unlike the results obtained with lysine vasopressin, the kinetic characteristics of dose‐dependent adenylate cyclase activation curves by GMPPNP were unaffected by varying Mg2+ concentrations except for the increase in velocity when raising Mg2+ concentration. It was not clear whether or not the activation processes by the hormone and by GMPPNP had common mechanisms.
Effect of cholinergic ligands on the lipids of acetylcholine receptor‐rich membrane preparations from torpedo californica
Authors: Martinez‐Carrion M. , Raftery M. A. , Thomas J. K. , Šator V.
Published: May 2005

Ion permeation, triggered by ligand‐receptor interaction, is associated with the primary events of membrane depolarization at the neuromuscular junction and synaptic connections. To explore the possible sites of ion permeation, the long‐lived fluorescent probe pyrene (fluorescence lifetime ∼400 nsec) has been inserted into the lipid phase of acetylcholine receptor‐rich membrane (AcChR‐M) preparations from Torpedo californica. The pyrene probe is susceptible to both fluidity and permeability changes in the lipid bilayer. These changes are detected by variations in the rate of decay of the excited singlet state of pyrene after pulsation with a 10‐nsec ruby laser flash. Variations of these lifetimes in the membrane preparations alone or in the presence of quenchers show that binding of cholinergic agonists and antagonists, neurotoxins, and local anesthetics to AcChR‐M produces varying effects on the properties of the pyrene probe in the lipid phase.

It is concluded that binding of cholinergic ligands to the receptor does not significantly alter the fluidity or permeability of the lipids in the bilayer in contact with pyrene. On the other hand, local anesthetics do affect these properties.
Protein transport: A selective membrane mechanism
Authors: Roth Thomas F. , Cutting John A. , Atlas Susan B.
Published: May 2005

Proteins are selectively sequestered by a number of cell types. However, only in oocytes is the process sufficiently aggravated and specific to be readily studied. In these cells certain serum proteins are taken up in proportions different from those found in the serum.

In vitro incubations of hormonally stimulated and synchronous mosquito oocytes show that the only protein capable of initiating the transport process is the female specific yolk protein. Heterologous proteins such as IgG, bovine serum albumin, cytochrome C, and ferritin are inactive. The female specific protein is a phosphoglycolipoprotein. It is synthesized in the fat body, a liver analog in the insect, and passed into the serum before being transported into the oocytes. Preliminary kinetic analysis shows the uptake process to be specific with an apparent Km of about 10−7 M. Glycolytic inhibitors stop protein uptake.

The receptor‐mediated binding steps in the transport process are most easily studied in the chicken because of the enormous amount of oocyte membrane available from a given oocyte and because up to 1 gm of protein is normally transported per day per oocyte. IgG and the hen specific phosvitin lipovitellin are two of the physiologically important proteins that are transported intact into the chicken oocytes. The uptake appears selective as shown by studies with iodinated proteins. Ferritin conjugated to IgG is shown by electron microscopy to bind to isolated plasma membranes only where coated pits have formed, whereas ferritin alone is not seen localized on any membrane surface. These very specialized regions of the membrane are similar to micropinocytotic pits but, in addition, possess on their cytoplasmic side dense ridges that form the coat. Transport involves binding to the coated pits, the pinching off of the pits, and the subsequent movement of the coated vesicles in the cytoplasm.
Purification of plasma membranes from rat mammary gland by a density perturbation procedure
Authors: Huggins John W. , Carraway Kermit L.
Published: May 2005

A highly purified plasma membrane fraction was obtained from a microsomal subfraction of rat mammary gland after treatment with digitonin to increase its density. The purified membranes were enriched 70‐fold overall in 5′‐nucleotidase with essentially no contamination from glactosyltransferase, succinate‐INT reductase, or NADPH‐cytochrome c reductase. The mermbranes were also highly enriched in sialoglycoproteins.
A study of adenosine 3′–5′ cyclic monophosphate binding sites of human erythrocyte membranes using 8‐azidoadenosine 3′–5′ cyclic monophosphate, a photoaffinity probe
Authors: Owens James R. , Haley Boyd E.
Published: May 2005

An earlier report (1a) has shown the utility of 8‐N3cAMP (8‐azidoadenosine‐3′, 5′‐cyclic monophosphate) as a photoaffinity probe for cAMP binding sites in human erythrocyte membranes. The increased resolution obtained using a linear‐gradient SDS polyacrylamide gel system now shows that: (1) both cAMP and 8‐N3cAMP stimulate the phosphorylation by [γ‐32P]‐ATP of the same red cell membrane proteins; (2) the protein of approximately 48,000 molecular weight whose phosphorylation by [γ‐32P]‐ATP is stimulated by cAMP and 8‐N3cAMP migrates at a solwer rate than the protein in the same molecular weight range which is heavily photolabeled with [32P]‐8‐N3cAMP; (3) other cyclic nucleotide binding sites exist besides those initailly reported; (4) the variation in the ratio of incorporation of 32P‐8‐N3cAMP into the two highest affinity binding sites appears to be the result of a specific proteolysis of the larger protein.
Topography of outer membrane assembly in salmonella
Authors: Mühlradt P. F.
Published: May 2005

The topography of lipopolysaccharide insertion into the outer membrane of Salmonella is discussed in context with a review of recent findings pertaining to general properties of the outer membrane, such as asymmetry and lateral mobility of surface components.
Morphological and biochemical abnormalities in hearts of cardiac mutant salamanders (Ambystoma mexicanum)
Authors: Lemanski Larry F.
Published: May 2005

The effect of homozygosity for recessive gene c in Ambystoma mexicanum is the absence of a heartbeat even though initially heart development appears normal. Mutant embryos (c/c) are first distinguishable from their normal siblings (+/+; +/c) at stage 34 (7 days after fertilization) when the normals develop contracting hearts. The mutant hearts at this stage, upon gross examination, appear structurally normal but fail to beat. Nevertheless, the mutants survive through stage 41, which is about 20 days beyond the heartbeat stage, and they exhibit normal swimming movements, indicating that gene c does not affect skeletal muscle. Electron microscopic studies of normal hearts show some myofibrils to be present at stage 34; by stage 41, the normal myocardial cells have become highly differentiated muscle cells. Although some mutant heart cells contain a few thin 60 A and thick 150 A filaments, organized myofibrils are absent. Instead, amorphous proteinaceous collections are prominent. Heavy meromyosin (HMM) binding experiments were performed on mutant hearts to determine whether the myocardial cells contain actin. Mutant myocardial cells that are glycerinated but not treated with HMM contain intact amorphous bodies. After incubation in HMM, the amorphous collections are no longer present and large numbers of decorated actin filaments appear. The results suggest that the amorphous proteinaceous collections contain actin in a nonfilamentous form, and the addition of HMM induces this actin to polymerize into filaments. SDS‐polyacrylamide gel electrophoresis of mutant heart tissue supports this conclusion by showing a prominent 43,000 dalton band suggestive of actin. The electrophoresis experiments also demonstrate a significant reduction of myosin heavy chain (200,000 daltons) in mutant hearts when compared to normal, and this latter observation is confirmed by radioimmunoassay experiments. Muscle tropomyosin (34,000 daltons), prominent in normal hearts, is virtually nonexistent in mutants. Thus, it appears that this single gene mutation affects the accumulation and organization of several different muscle proteins, including actin, myosin, and tropomyosin.
Suppression of stimulating cell activity by microtubule‐disrupting alkaloids
Authors: Ranney D. F. , Pincus J. H.
Published: May 2005

Microtubule‐disrupting alkaloids and protein fixatives were used to investigate the nature of an active process that must occur within stimulator cells in order for them to intiate a unidirectional mixed lymphocyte response (MLR). Brief treatment of the stimulator cells (SC) with glutaraldehyde (0.15%), formalin (0.6%), or lanthanum chloride (10−3 M) abolished their capacity to activate responder cells (RC). Pre‐treatment of SC with the microtubule‐disrupting alkaloids, colchicine (c) (10−4 to 10−6) or clochicine + vincristine (c+v) (10−4 to 10−6 M) also abrogated their stimulating capacity. This capacity was not restored by the addition of supernates from untreated cultures, thereby excluding the possibility that the alkaloids acted by decreasing the release of soluble stimulatory factors from SC. The introduction of alkaloid‐inactivated, mitomycin‐treated RC as drug carriers did not affect the mitogenic response of untreated RC to concanavalin A. This excluded a significant leakage of alkaloids from the treated SC and uptake by RC during culture. Lumicolchicine produced no decrease in the stimulating capacity of SC. This suggested that the suppression induced by low concentrations of colchicine resulted from its specific disruption of microtubules. None of the above treatements quantitatively reduced the antigenicity of SC, as evaluated by humoral and cell‐mediated lysis of the treated cells. Also, these treatements produced no significant changes in the specific binding of concanavalin A by SC. These indicate there is a functional interaction of microtubular structures with cell surface antigens that appears to regulate either the capacity of SC to associate with RC, or the ability of SC to form and stabilize stimulatory antigenic configurations on the cell surface.
Nature and origin of patterns of changes in cell shape in embryos
Authors: Jacobson Antone G. , Gordon Richard
Published: May 2005

Spatial patterns of the future elongation of cells exist in the early embroyo. In the newt, such a pattern of changers of cell shape contributes to the formation of the neural plate. Regardless of where neural plate. Regardless of where neural plte cells are transplanted, they change shape as prescribed by tge pattern. Embryonic induction has a role in establishing this pattern.
Interaction of charged lipid vesicles with planar bilayer lipid membranes: Detection by antibiotic membrane probes
Authors: Cohen Joel A. , Moronne Mario M.
Published: May 2005

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface‐charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylocholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmertically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle‐BLM interactions are discussed.
Measurement of the lateral mobility of cell surface components in single living cells by fluorescence recovery after photobleaching
Authors: Jacobson K. , Derzko Z. , Wu E‐S. , Hou Y. , Poste G.
Published: May 2005

The use of fluorescence recovery after photobleaching (FRAP) techniques to monitor the lateral mobility of plant lectin‐receptor complexes on the surface of single, living mammalian cells is described in detail. FRAP measurements indicate that over 75% of the wheat germ agglutinin receptor (WGA‐receptor) complexes on the surface of human embryo fibroblasts are mobile. These WGA‐receptor complexes diffuse laterally (as opposed to flow) on the cell surface with a diffusion coefficient in the range of 2 × 10−11 to 2 × 10−10 cm2/sec. Both the percentage of mobile WGA‐receptor complexes and the mean diffusion coefficient of these complexes are higher than that obtained from earlier FRAP measurements of the mobility of concanavalin A‐receptor (Con A‐receptor) complexes in a variety of cell types. The possible reasons for the differing mobilities of WGA and Con A receptors are discussed.