bluuebunny/crossref_metadata_2025 huggingface dataset:
Circular dichroism of biological membranes–brain microsomes
Authors: Long Marianna M. , Masotti Lanfranco , Sachs George , Urry Dan W.
Published: May 2005

The circular dichroism and absorption spectra of particulate and solubilized brain microsomes are reported. The corrected molar ellipticity for the membrane suspension was −1.1 × 10+4, between the values of axonal and oxyntic cell membranes and those for sarcotubular vesicles. No conformational change was detected which correlated unambiguously with the active cation transport function of these microsomes. Divalent cations, however, elicited a significant change in the membranes' ultra structure. Correction of suspension data for the NaI microsomes with and without sonication gave virtually identical values providing optimism for the corrections used and validating the difference in corrected values for ths suspension with and without magnesium. This indicates that, in addition to causing a change in the state of aggregation, 2 mM magnesium ion may reasonably be considered to effect a change in protein conformation.
Structural requirements for mitochondrial mutagenesis
Authors: Mahler Henry R.
Published: May 2005

Ethidium bromide (3, 8‐diamino‐5‐ethyl‐6‐phenylphenanthridinium bromide) and euflavine (3, 6‐diamino‐10‐methylacridinium chloride) are superficially similar in structure and ability to intercalate into DNA. However, they exhibit qualitative differences in their ability to bring about a mitochondrial mutation (ρ+ → ρ−) in Saccharomyces cerevisiae. This investigation tried to establish and compare the essential structural prerequisites in three series of planar, heterocyclic dyes: the phenanthridines (P series), the acridines (A series), and molecules with different heteroatoms related to acridines (X series). Compounds capable of bringing about the mutation in the complete absence of growth and energy sources are restricted to di‐primary amines in the P series: quaternization of the ring nitrogen, and an aromatic side chain at C‐6 also appear essential. Compounds in the A series are mutagenic only with growing cells; quaternization (C1 through C4) is essential. The 10‐allyl derivative is unusual; it is highly effective even in buffer supplemented only with an energy source. The results are interpreted in terms of a model that requires interaction of the mutagen with the mitochondrial inner membrane as well as with its DNA.
The contractile proteins of dictyostelium discoideum
Authors: Spudich James A. , Clarke Margaret
Published: May 2005

We have purified actin and my osin‐like proteins from amoebae of Dictyostelium discoideum. These proteins are very similar in their physical and enzymatic properties to muscle actin and myosin. Most importantly, they form thin and thick filaments, respectively, and Dictyostelium actin activates Dictyostelium myosin ATPase activity. Actin from these amoebae appears to be identical in size to muscle actin. The Dictyostelium myosin consists of two heavy chains of about 210,000 daltons and two classes of light chains, about 18,000 and 16,000 daltons. The heavy chains are slightly larger than those of muscle myosin. Biochemical and structural studies of membrane association of the contractile complex suggests that some of the amoeba actin is membrane‐bound and acts as an attachment point for myosin and other actin filaments.
Assembly of lipid‐containing viruses
Authors: Compans Richard W. , Meier‐Ewert Herbert , Palese Peter
Published: May 2005

Enveloped viruses which form by budding at the cell surface possess a membrane consisting of a lipid bilayer and a small number of virus‐coded polypeptides. Since viral polypeptides become integral components of the plasma membrane during assembly, the process of synthesis and incorporation into membranes of these proteins may reflect the pathway of plasma membrane assembly. Electron microscopic studies have suggested that viral envelope proteins are incorporated into discrete, localized regions of the plasma membrane which serve as recognition sites for the viral nucleocapsid. In influenza virus‐infected cells, viral polypeptides are associated with cytoplasmic membranes as well as the plasma membrane. The major envelope glycoprotein appears to be synthesized in rough endoplasmic reticulum, and to migrate to smooth membranes after synthesis. Glycosylation is initiated in rough membranes and progresses further in smooth membranes. Unlike the glycoproteins, the major nonglycosylated polypeptide appears to be inserted directly into the plasma membrane. In the presence of 2‐deoxyglucose or high concentrations of glucosamine, aberrant viral glycoproteins are detected which appear to be unglycosylated or partially glycosylated; these are associated with membranes and incorporated into virus particles of reduced infectivity. Therefore normal glycosylation is not essential for incorporation of viral glycoproteins into cellular membranes or virus particles, but is required for normal biological activity. The role of the viral neuraminidase in assembly and release has been studied using mutants defective in neuraminidase at restrictive temperature. Under these conditions virus formation occurs, but the progeny form large aggregates at the cell surface. Colloidal iron hydroxide staining indicates that such virus particles contain neuraminic acid, and these residues appear to serve as receptors leading to the extensive aggregation.
Interactions between collagen chains and fiber formation
Authors: Fessler John H. , Tandberg William D.
Published: May 2005

The temperature‐dependent dissociation of neutral salt‐soluble collagen into its component chains was measured in 0.6–1.6 M urea solutions at pH 7.3. The temperature‐dependent association of the same radiocactively labeled collagen into fibers was measured in 0–0.4 M urea solutions, pH 7.3. The effect of urea on the temperature, Tm(G), for half dissociation into chains was small, and the value extrapolated to zero urea concentration was 39°C. In contrast, the effect of urea on the temperature, Tm(F), for half association into fibers was large, and the value at zero urea concentration was 30°C.

We conclude that while body temperature provides excellent conditions for the matching of collagen chains to form molecules, the conditions are not optimal for the formation of highly ordered fibers. The large effects of 0.1 M urea suggest that other factors in vivo may help to destabilize mismatched molecular association during fiber growth. Alternately this might be facilitated by parts of the extension peptides of procollagen.
Assembly of the tail of bacteriophage T4
Authors: Kikuchi Yoshiko , King Jonathan
Published: May 2005

The protein products of at least 21 phage genes are needed for the formation of the tail of bacteriophage T4. Cells infected with amber mutants defective in these genes are blocked in the assembly process. By characterizing the intermediate structures and unassembled proteins accumulating in mutant‐infected cells, we have been able to delineate most of the gene‐controlled steps in tail assembly. Both the organized structures and unassembled proteins serve as precursors for in vitro tail assembly.

We review here studies on the initiation, polymerization, and termination of the tail tube and contractile sheath and the genetic control of these processes. These studies make clear the importance of the baseplate; if baseplate formation is blocked (by mutation) the tube and sheath subunits remain essentially unaggregated, in the form of soluble subunits.

Seventeen of the 21 tail genes specify proteins involved in baseplate assembly. The genes map contiguously in two separate clusters, one of nine genes and the other of eight genes. Recent studies show that the hexagonal baseplate is the end‐product of two independent subassembly pathways. The proteins of the first gene cluster interact to form a structure which probably represents one‐sixth of the outer radius. The products of the other gene cluster interact to form the central part of the baseplate.

Most of the phage tail precursor proteins appear to be synthesized in a non‐aggregating form; they are converted to a reactive form upon incorporation into preformed substrate complexes, without proteolytic cleavage. Thus reactive sites are limited to growing structures.
The formation of filamentous structures from iodinated neurotubules
Authors: Gaskin Felicia , Litman David J. , Cantor Charles R. , Shelanski Michael L.
Published: May 2005

The porcine neurotubule and its basic subunit were found to be modified in vitro by iodination of amino acids (principally tyrosine) using lactoperoxidase. Iodide ion, H2O2, or lactoperoxidase singly or in any pairwise combination had virtually no effect on neurotubules. However, when all three reagents were present, permitting covalent iodination, it was found that at 0.1 iodotyrosines per tubulin dimer the microtubules unravel to form structures which morphologically resemble strands of protofilaments twisted or wound around each other. These abnormal tubules are stable at room temperature and 4°C. Both monomers of tubulin are labeled to approximately the same extent. Iodinated tubulin (0.1 iodotyrosines/dimer) is unable to assemble in vitro under normal assembly conditions. Heavily iodinated microtubules (8 iodines per tubulin dimer) are similar in morphology to the slightly iodinated structures.
Interaction of the 5′‐ends of 28S RNA in dimerization of hamster ribosomes
Authors: Silverman David J. , Lubin Martin
Published: May 2005

Free ribosomes extracted from hamster cells and 28S RNA purified from these ribosomes are known to form dimers. We find that spleen phosphodiesterase inhibits ribosomal dimer formation, but only when a free 5′‐hydroxyl end group, produced by the action of alkaline phosphatase, is present. Hence, formation of dimer ribosomes probably involves interaction at or near the phosphorylated 5′‐ends of 28S RNA.

Dimer RNA molecules show a modal length, when measured on electron micrographs, of 2.1μm, which is about double the length of 28S RNA. Electron micrographs of 115S dimer ribosomes often show profiles consistent with our interpretation that in dimers the 28S RNA chains are loosely linked by their 5′‐ends.
Transient kinetic and isotopic tracer studies of the myosin adenosine triphosphatase reaction
Authors: Bagshaw C. R. , Trentham D. R.
Published: May 2005

From transient kinetic studies of the Mg2+‐dependent adenosine triphosphatase of myosin subfragment 1, prepared from rabbit skeletal muscle, a seven‐step mechanism has been proposed. Features of this mechanism include two‐step processes for ATP and ADP binding in which the binary complex isomerizes in addition to a rapid nucleotide association step. In the case of ATP a large negative standard free energy change is associated with the isomerization. An overall rate‐limiting isomerization of the myosin‐product complex prior to product release has been identified. Studies on the mechanism of cleavage of ATP bound to the active site indicate the process is readily reversible and can account for the observation that more than one oxygen of the product phosphate arises from water. This proposal has been substantiated by the finding that the oxygen atoms of the γ‐phosphoryl group of bound ATP also undergo extensive exchange with water.
Structure and function of cholera toxin and hormone receptors
Authors: Bennett V. , Craig S. , Hollenberg M. D. , O'Keefe E. , Sahyoun N. , Cuatrecasas P.
Published: May 2005

The enterotoxin from Vibrio cholerae is a protein of 100,000 mol wt which stimulates adenylate cyclase activity ubiquitously. The binding of biologically active 125I‐labeled choleragen to cell membranes is of extraordinary affinity and specificity. The binding may be restricted to membrane‐bound ganglioside GMI. This ganglioside can be inserted into membranes from exogenous sources, and the increased toxin binding in such cells can be reflected by an increased sensitivity to the biological effects of the toxin. Features of the toxin‐activated adenylate cyclase, including conversion of the enzyme to a GTP‐sensitive state, and the increased sensitivity of activation by hormones, suggest analogies between the basic mechanism of action of choleragen and the events following binding of hormones to their receptors. The action of the toxin is probably not mediated through intermediary cytoplasmic events, suggesting that its effects are entirely due to processes involving the plasma membrane. The kinetics of activation of adenylate cyclase in erythrocytes from various species as well as in rat adipocytes suggest a direct interaction between toxin and the cyclase enzyme which is difficult to reconcile with catalytic mechanisms of adenylate cyclase activation. Direct evidence for this can be obtained from the comigration of toxin radioactivity with adenylate cyclase activity when toxin‐activated membranes are dissolved in detergents and chromatographed on gel filtration columns. Agarose derivatives containing the “active” subunit of the toxin can specifically adsorb adenylate cyclase activity, and specific antibodies against the choleragen can be used for selective immunoprecipitation of adenylate cyclase activity from detergentsolubilized preparations of activated membranes. It is proposed that toxin action involves the initial formation of an inactive toxin‐ganglioside complex which subsequently migrates and is somehow transformed into an active species which involves relocation within the two‐dimensional structure of the membrane with direct pertubation of adenylate cyclase molecules (virtually irreversibly). These studies suggest new insights into the normal mechanisms by which hormone receptors modify membrane functions.
The exchange of erythrocyte membrane phospholipids with rat liver extracts in vitro
Authors: Steck Theodore L. , Wackman Nancy , Tarlov Alvin R.
Published: May 2005

Intact rat or human erythrocytes and their isolated (ghost) membranes were incubated with the high speed supernatant fraction of homogenates derived from 32P‐labeled rat livers. Phospholipid molecules were transferred between the red cell membranes and the liver extracts, as reflected by the convergence of their specific radioactivities with time. Whereas ghosts usually approached isotopic equilibrium with the liver supernatant fraction during a few hours of incubation at 37° C, the exchange of phospholipids by intact cells was no more than one‐half, even after 18 hr. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin were all exchanged in both intact cells and ghosts, albeit to different extents. (A control experiment, incubating 32P‐labeled rat erythrocytes or ghosts with unlabeled rat liver extracts, also demonstrated the exchange of all four major phospholipids.) These data may signify that the phospholipids on the cytoplasmic side of the membrane of intact erythrocytes do not exchange with the phospholipids in exogenous liver extracts. If so, all four major phospholipid classes would appear to be present to some extent at both membrane surfaces. The first inference is in agreement with several other studies on this membrane, while the second inference is not.
Lesion‐Induced synaptogenesis in brain: A study of dynamic changes in neuronal membrane specializations
Authors: Cotman Carl W.
Published: May 2005

When incoming fibers to a given brain region are damaged and degenerate, the remaining undamaged fibers can, in some cases, form new synapses, and restore physiologically functional circuitry. Synaptic membrane events underlie this reconstruction: the connection between membranes is broken and reformed. In order to understand these membrane events, it is necessary to know the molecular composition of the synapse and the nature of the interaction between pre‐ and postsynaptic membranes. The synaptic membranes are probably joined by proteins extending from their surfaces. The postsynaptic membrane has on its outer surface an array of lectin receptors, probably glycoproteins. On its inner surface, juxtaposed to the bilayer, the membrane has an electron‐dense structure called the postsynaptic density which, from studies on the isolated structure, is composed of a few polypeptides. On the basis of the molecular composition and structure of CNS synapses and ultrastructural studies of the lesion‐induced synaptogenesis, some of the underlying dynamic events at synaptic membranes are inferred. New synapses are formed either by reutilization of the old contact sites or by generation of new ones. The protein and carbohydrates in the cleft are enzymatically degraded and a new synapse is generated in response to ingrowing fibers by the addition or reutilization of the specialized proteins of postsynaptic membrane, which differentiate a small segment of the postsynaptic membrane.
Substrate heterogeneity of component a of the human erythrocyte membrane
Authors: Roses Allen D.
Published: May 2005

Component a of the erythrocyte membrane is a specific substrate for endogenous protein kinase activity and its phosphorylation is significantly decreased under assay conditions in myotonic muscular dystrophy (Roses, A. D., and Appel, S. H., J. Membr. Biol. 20:51–58 (1975)). We have demonstrated substrate heterogeneity of two fractions of component a separated by concanavalin A (Con‐A) sepharose chromatography. The fraction of component a that is retarded by Con A and eluted with α‐methyl‐D‐glucoside does not accept the transfer of phosphate from [γ‐32P] ATP as a substrate for endogenous protein kinase activity. The nonretarded fraction contains > 90% of the radioactive label. These experiments also confirm the carbohydrate heterogeneity of component a (Findley, J. B. C., J. Biol. Chem. 249:4398 (1974)).
Avian tumor virus interactions with chicken fibroblast plasma membranes
Authors: Moldow Charles F. , McGrath Michael , Santen Lenore Van
Published: May 2005

A method is described which will rapidly measure the binding of avian tumor viruses (ATV) to plasma membrane receptors. With this procedure it may be shown that Rous sarcoma virus pseudotypes bind to protease‐labile, heat‐stable structures on the surface of chicken embryo fibroblast (CEF) plasma membranes. The binding sites for ATV subgroups A and B appear distinct, and membranes from genetically resistant CEF bind as well those of sensitive CEF.
Estimation of cell surface associated protease activity and its application to lymphocytes
Authors: Tökés Zoltán A. , Kiefer Hansruedi
Published: May 2005

A new method has been developed to estimate proteolytic activity available at the cell surface. Radioiodinated protein substrates are covalently linked to modified polystyrene‐divinylbenzene beads with various diameters. These beads are presented to viable cells. Secreted enzyme activity is estimated when no contact occurs between beads and cells. Surface associated proteolytic activity is estimated by the increased rate of iodinated peptide release due to a contact between beads and cells.

This method was applied to various lymphocyte preparations. In the absence of serum, mouse spleen lymphocytes produce three‐ to fourfold higher proteolytic activity than lymph node cells. This activity is completely inhibited by serum diluted 1:10. Since the proteolysis is so marked in the case of spleen cells, one must conclude that lymphocytes removed from the serum and treated in buffered mediums at 37° C have enzymatically altered surface properties.

Cell surface associated enzyme activity was measured using rat lymph node lymphocytes with less than 0.1% contamination by granulocytes. This predominantly thymus derived, T cell population had 30% increase in proteolysis due to contact between cells and solid‐phase localized substrate of casein. The released enzymatic activity was inhibited by diisopropylfluorophosphate, but its effect on the surface associated enzyme activity remains questionable since it perturbs several membrane functions.
Cell shape changes and transmembrane receptor uncoupling induced by tertiary amine local anesthetics
Authors: Nicolson Garth L. , Poste George
Published: May 2005

Tertiary amine local anesthetics (dibucaine, Tetracaine, procaine, etc.) modify cell morphology, concanavalin A (Con A)‐mediated agglutinability and redistribution of Con A receptors. Con A agglutination of untransformed mouse 3T3 cells was enhanced at low concentrations of local anesthetics, and the dynamics of fluorescent‐Con A indicated that ligand‐induced clustering was increased in the presence of the drugs. In contast, these drugs inhibited Con A‐induced receptor capping on mouse spleen cells. These effects can be duplicated by combinations of vinblastine (or colchicine) and cytochalasin B suggesting that local anesthetics act on microtubule cell surface receptor mobility and distribution. It is proposed that tertiary amine local anesthetics displace plasma membrane‐bond Ca2+, resulting in disengagement of microfilament systems from the plasma membrane and increased cellular Ca2+ concentration to levels which disrupt microtubular organization. The possible involvement of cellular Ca2+ in cytoskeletal destruction by local anesthetics was investigated utilizing Ca2+‐specific ionophores A23187 and X537A. In media containing Ca2+ and cytochalasin B these ionophores caused effects similar to tertiary amine local anesthetics.
Does a bacterial elongation factor share a common evolutionary ancestor with actin?
Authors: Rosenbusch Jurg P. , Jacobson Gary R. , Jaton Jean‐Claude
Published: May 2005

Protein synthesis elongation factor Tu from E. coli shares several physical, chemical, and functional properties with actin‐like proteins. Limited tryptic degradation indicartes that the two polypeptides have a similar molecular architecture. These observations suggest that they could have evolved from a common ancestor, although more information will be necessary to prove or disprove this hypothesis. A partial sequence, comprising 22 aminoacid residues from the aminoterminal end of the large tryptic fragment of elongation factor Tu is presented.
Characterization of a periplasmic protein related to sn‐glycerol‐3‐phosphate transport in escherichia coli
Authors: Argast Manfred , Schumacher Güunter , Boos Winfried
Published: May 2005

The cold osmotic shock procedure releases a protein (GLPT) from the cell envelope of Escherichia coli that is related to the transport of sn‐glycerol‐3‐phosphate in this organism. The evidence for this correlation is as follows: (1) GLPT is under the regulatory control of the glpR gene. (2) Some glpT mutants that were isolated as phosphonomycin resistant clones do not synthesize GLPT. Revertants of these mutants (growth on sn‐glycerol 3‐phosphate) again synthesize GLPT. (3) Some amber mutations in glpT reduce the amount of GLPT while suppressed strains produce normal amounts. (4) Transfer of a plasmid carrying the glpT genes into a strain lacking GLPT and sn‐glycerol‐3‐phosphate transport restores both functions in the recipient. Transport and GLPT synthesis in the plasmid carrying strain are increased 2‐ to 3‐fold over a fully induced wild‐type strain, but appear to be constitutive. GLPT is a soluble protein of molecular weight 160,000 composed of 4 identical subunits. The 160,000 molecular weight complex is stable in 1% sodium dodecylsulfate at room temperature. Upon boiling in 1% sodium dodecylsulfate GLPT dissociates into its subunits. Likewise, 8 M urea at room temperature dissociates GLPT into its subunits. Dialysis of dissociated GLPT against phosphate or Tris‐HCl buffer, pH 7.0, allows renaturation to the tetrameric form. The protein is acidic in nature (isoelectric point 4.4).

In contrast to the typical transport‐related periplasmic‐binding proteins, no conditions could be found where pure GLPT exhibited binding activity toward its supposed substrate, sn‐glycerol‐3‐phosphate.

In vivo new appearance of transport activity for sn‐glycerol‐3‐phosphate transport occurs only shortly before cell division. However, GLPT synthesis does not fluctuate during the cell cycle. The available evidence indicates a cell‐division‐dependent processing of GLPT in the cell envelope as a reason for the alteration in transport activity.

Transport in whole cells is sensitive to the cold osmotic shock procedure, demonstrating the participation of an essential periplasmic component. However, isolated membrane vesicles that are devoid of periplasmic components, including GLPT, are fully active in sn‐glycerol‐3‐phosphate transport. Therefore, we conclude that GLPT is essential in overcoming a diffusion barrier for sn‐glycerol‐3‐phosphate established by the outer membrane. Attempts to isolate mutants that are transport negative in whole cells due to a defect in GLPT but are active in isolated membrane vesicles have failed so far. All GLPT mutants tested, whether or not they synthesize GLPT, are not active in isolated membrane vesicles.

Iodination of whole cells with [125I] followed by osmotic shock reveals that several shock‐releasable proteins including GLPT become radioactively labeled. This indicates that some portions of GLPT are accessible to the external medium.
The folate and thiamine transport proteins of lactobacillus casei
Authors: Henderson Gary B. , Zevely Edward M. , Kadner Robert J. , Huennekens F. M.
Published: May 2005

Two separate binding proteins, one specific for folate and the other for thiamine, have been isolated from membrane fragments of Lactobacillus casei. Purification to homogeneity was achieved by fractionation of the Triton‐solubilized proteins with microgranular silica (Quso G‐32) and Sephadex G‐150. Amino acid analyses revealed that the folate (Mr = 25,000) and thiamine (Mr = 29,000) binders have unusually low polarity constants, 0.32 and 0.26, respectively. Evidence obtained with intact cells has established a direct role for these binding proteins in transport of the corresponding vitamins: (A) In each case, the processes of binding and transport showed similarities in substrate affinities and repression by excess vitamin in the growth medium. (B) Competition studies employing amethopterin, 5‐formyl tetrahydrofolate, and 5‐methyl tetrahydrofolate (for folate) and thiamine monophosphate and thiamine pyrophosphate (for thiamine) have shown that the ability of these compounds to inhibit the transport of the corresponding vitamins is paralleled by their ability to inhibit binding. (C) Amethopterin‐resistant mutants which are defective in folate transport have a comparable defect in ability to bind folate. (D) Amethopterin‐resistant cells which (compared with the parent cell line) contain folate transport systems with altered affinities for amethopterin also contain binding proteins whose affinities for amethopterin have changed by equivalent amounts. (E) Both the transport and binding of folate by one of the mutants were stimulated (approximately 3‐fold) in parallel by the addition of mercaptoethanol.
The mechanism of sugar‐dependent repression of synthesis of catabolic enzymes in escherichia coli
Authors: Gonzalez Jose E. , Peterkofsky Alan
Published: May 2005

Previous studies have indicated that the Escherichia coli adenylate cyclase (AC) activity is controlled by an interaction with the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS). A model for the regulation of AC involving the phosphorylation state of the PTS is described. Kinetic studies support the concept that the velocity of AC is determined by the opposing contributions of PEP‐dependent phosphorylation (V1) and sugar‐dependent dephosphorylation (V2) of the PTS proteins according to the expression % VAC = 100/[1 + (Max V2/Max V1)]. Physiological parameters influencing the rate of the PTS are discussed in the framework of their effects on cAMP metabolism. Factors that increase cellular concentration of PEP (and stimulate V1) appear to enhance AC activity while increases in extracellular sugar concentration (which stimulate V2) or internal levels of pyruvate (which inhibit V1) inhibit the activity of this enzyme.
Sialic acid uptake by BHK cells and subsequent incorporation into glycoproteins and glycolipids
Authors: Hirschberg Carlos B. , Yeh Mary
Published: May 2005

BHK cells can be grown in the presence of growth medium to which radiolabeled sialic acid has been added. After 24 h, 85% of the radioactivity in the cells is covalently bound to glycoproteins and glycolipids. No metabolism of the radiolabeled sialic acid could be detected.
Characterization of the Fc receptors of the murine leukemia L1210
Authors: Cooper Sheldon M. , Sambray Yugalkishore
Published: May 2005

A glycoprotein extract prepared from the plasma membranes of L1210 cells was passed over columns of Sepharose 4B to which either heat‐aggregated human IgG or F(ab′)2 fragments had been coupled. The intact IgG column bound 35.7% of the applied counts, whereas the F(ab′)2 columns bound 2.8%. The bound glycoproteins were eluted with citrate buffer (pH 3.2) and analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Three peaks with apparent molecular weights of 65,000, 45,000, and 28,000 daltons were identified and purified by electroelution from polyacrylamide gels. The isolated proteins were able to bind to the same subclasses of mouse IgG myeloma proteins as the intact L1210 cells, indicating that these molecules are related to L1210 surface Fc receptors. Amino acid analyses of the 3 proteins were markedly similar suggesting that the observed molecular heterogeneity might be due to carbohydrate differences. Neuraminidase digestion of the isolated proteins resulted in mobility shifts on polyacrylamide gel electrophoresis which were consistent with the interpretation that either the isolated proteins have considerably different sialic acid contents, or that removal of the sialic acid results in disaggregation of an Fc receptor molecule.
Role of mannosyl phosphoryl polyisoprenol in biosynthesis of mammary glycoproteins
Authors: Vijay Inder K. , Fram Steven R.
Published: May 2005

When a membrane preparation from the lactating bovine mammary gland is incubated with GDP‐[14C] mannose, mannose is incorporated into a [14C] mannolipid, a [Man‐14C] oligosaccharide‐lipid, and metabolically stable endogenous acceptor(s). The rate of mannosyl incorporation is the fastest into [14C] mannolipid, intermediate in [Man‐14C] oligosaccharide‐lipid, and least into [Man‐14C] endogenous acceptor(s).

The [14C] mannolipid has been partially purified and characterized. Mild acid hydrolysis of this compound gives [14C] mannose, whereas alkaline hydrolysis yielded [14C] mannose phosphate as the labeled product. The t½ of hydrolysis of the mannolipid under the acidic and basic conditions are comparable to values obtained for mannosyl phosphoryl dolichol in other systems. The mannolipid is chromatographically indistinguishable from calf brain mannosyl phosphoryl polyisoprenol and chemically synthesized β‐mannosyl phosphoryl dolichol. Exogenous dolichol phosphate stimulates the synthesis of mannolipid in mammary particulate preparations 8.5‐fold. Synthesis of mannolipid is freely reversible; in the presence of GDP, the transfer of mannosyl moiety from endogenously labeled mannolipid to GDP‐mannose is obtained. All of these results indicate that the structure of mannolipid is mannosyl phosphoryl polyisoprenol. Even though the precise chain length of the polyisoprenol portion has not been established, it is tentatively suggested to be dolichol.

Partially purified [14C] mannolipid can directly serve as a mannosyl donor in the synthesis of [Man‐14C] oligosaccharide‐lipid and [Man‐14C] endogenous acceptor(s). Pulse and chase kinetics utilizing GDP‐mannose to chase the mannosyl transfer from GDP‐[14C] mannose in the mammary membrane incubations caused an immediate and rapid turnover of [14C] mannose from [14C] mannolipid while the incorporation of label in [Man‐14C] oligosaccharide‐lipid and radioactive endogenous acceptor(s) continued for a short period before coming to a halt.

Both gel filtration and electrophoresis indicate that the endogenous acceptor(s) are a mixture of 2 or more glycoproteins since incubation with proteases releases all of the radioactivity into water soluble low‐molecular‐weight components, perhaps glycopeptides.

All of the above evidence is consistent with the following precursor‐product relationship:

GDP‐mannose ⇆ mannosyl phosphoryl polyisoprenol → mannosyl‐oligosaccharide‐lipid → mannosyl‐proteins.

The exact structure of the oligosaccharide‐lipid and the endogenous glycoproteins is unknown.
Reconstitution of neutral amino acid transport from partially purified membrane components from ehrlich ascites tumor cells
Authors: Cecchini Gary , Payne Gregory S. , Oxender Dale L.
Published: May 2005

Solubilized protein fractions have been obtained from plasma membranes of Ehrlich ascites cells either by extraction with 0.5% Triton X‐100 or by extraction with 2% cholate. Partial purification of the solubilized protein fraction has been obtained by utilizing a combination of ammonium sulfate precipitation and column chromatography. Leucine‐binding activity has been detected in the Triton X‐100 solubilized membrane fraction. The leucine‐binding activity was measured by equilibrium dialysis and was saturable with high levels of leucine or phenylalanine and is not strongly effected by alanine. These properties are similar to those previously identified as System L. In addition, the cholate extracted protein fraction was partially purified and reconstituted into liposomes. Sodium dependent uptake of alanine and leucine could be demonstrated in the reconstituted vesicles. Concentrative uptake was dependent upon a sodium gradient. A membrane potential produced by valinomycin mediated potassium diffusion in the presence of sodium also stimulated amino acid transport in reconstituted liposomes.
Glycoprotein synthesis as a function of epithelial cell arrangement: Biosynthesis and release of glycoproteins by human breast and prostate cells in organ culture
Authors: Tökés Zoltán A. , Dermer Gerald B.
Published: May 2005

We demonstrate that a technique is available to investigate glycoprotein synthesis in organ cultures of human breast and prostate surgical specimens where the 3‐dimensional epithelial cell arrangement remains intact. Malignant breast and prostate epithelium maintained their capacity to synthesize glycoproteins for at least 3 days as followed by the incorporation of [3H] glucosamine into macromolecules. Over 70% of incorporation was by malignant cells as judged by autoradiography. Labeled glycoproteins were released into glandular lumina and consequently into the culture fluid. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed predominantly one group of macromolecules released with an apparent molecular weight of 48,000 ± 6,000 daltons. This glycoprotein was found in all of the breast specimens studied, which included 1 medullary, 1 infiltrating lobular, and 8 infiltrating duct carcinomas. The pattern was independent of the availability of estrogen receptors. A similar glycoprotein was also observed in the culture media from a Grade I and a Grade II well‐differentiated infiltrating prostate carcinoma. Incorporation was below the level of detection in 4 of 6 cases of benign prostatic hyperplasia. A more complex pattern of labeled glycoproteins was found in the media of a Grade II and a Grade III poorly‐differentiated prostate carcinoma. The established human mammary carcinoma cell line MCF‐7 synthesized and released a similar 48,000 molecular weight glycoprotein but additional components with larger molecular weights were also released. An intriguing interpretation that 3‐dimensional tissue integrity restricts some glycorprotein synthesis is discussed. Cells grown in 2‐dimensional monolayers could escape from such a topographic restriction and express additional families of glycoproteins.
Glycoprotein and protein precursors to plasma membranes in vesicular stomatitis virus infected HeLa cells
Authors: Atkinson Paul H.
Published: May 2005

Vesicular stomatitis virus is known to mature at HeLa cell plasma membranes. To study the process, cells, infected with vesicular stomatitis virus, were fractionated after short term labeling studies (1 min pulse, 1 min chase) to determine the assembly kinetics of G protein and M protein into plasma membranes. Newly synthesized M protein was found released in the supernatant from which free polysomes were sedimented during sucrose gradient analysis of these polysomes. If this M protein is particle bound, it must have a density of less than 1.08 g/ml. About 40% of this M protein so labeled was not sedimentable at 165,000 X g for 16 h. This newly synthesized M protein had not yet assembled into plasma membrane and thus must represent an internal pool. This and previous studies show that it has a subsequent transit time to the plasma membrane of about 2 min. Once associated with plasma membranes, M protein decayed in an approximately logarithmic fashion indicating that newly synthesized M randomly mixes (and turns over) with preexisting M protein. G protein was particle bound in a 1 min pulse, 1 min chase, and was never found released in a soluble form. At the later time when fucose is added to G protein, the oligosaccharide moiety is near to complete, and on completion is about 2,000 in molecular weight. Evidence is presented showing that fucose is probably attached to the N‐acetylglucosamine of the protein carbohydrate linkage. G protein to which fucose had just been added was located internally on a membranous fraction of density 1.14 g/ml in sucrose; its subsequent transit time from this pool (which in uninfected cells is between 1–2% of the total cell fucosyl glycoprotein) was about 15 min. Because their densities were different and their transit times were different, internal newly synthesized M and fucosyl G protein which assemble into plasma membranes were not on the same internal membranous component. Association of M protein with the plasma membranes may thus occur from a nonsedimentable soluble cytoplasmic pool by a process of direct adsorption.
Mechanism of resistance to ricin toxin in selected mouse lymphoma cell lines
Authors: Nicolson Garth L. , Poste George
Published: May 2005

The role of impaired toxin uptake in conferring cellular resistance to the plant toxin RCAII (ricin) has been examined using a murine BW5 147 lymphoma line and a toxin‐resistant variant (BW5 147 RicR.3) selected by repeated exposure to RCAII. The toxin‐resistant variant is 250 times more resistant to RCAII in long‐term growth experiments and 1,000 times more resistant in short‐term protein synthesis assays. Experiments with ferritin‐conjugated 125I‐labeled RCAII (ferritin‐125I‐RCAII) indicated that toxin binding to sensitive and resistant cells is similar at low toxin concentrations where maximum differential cytotoxicity occurs but that major difference exist with respect to toxin uptake. In sensitive cells toxin is internalized via endocytosis, and as seen previously in other systems subsequent rupture of some of the toxin‐containing endocytotic vesicles releases toxin into the cytoplasm, where it inhibits protein synthesis. The process of toxin transfer to the cytoplasm is presumed to account for the one‐hour lag before toxin‐induced inhibition of protein synthesis can be detected. Endocytotic uptake of toxin is impaired in resistant BW5147RicR.3 cells, and they are unaffected by toxin concentrations that inhibit protein synthesis and kill sensitive parental cells. Killing of resistant cells at low toxin concentrations was accomplished by encapsulating RCAII into lipid vesicles capable of fusing with the plasma membrane. Direct introduction of toxin into resistant cells using lipid vesicles as carriers produced rapid inhibition (< 15 min) of protein synthesis and eliminated the lag in toxin action seen in sensitive cells exposed to free toxin. These findings are discussed in relation to the mechanism of toxin action and proposals that toxin activity requires structural modification of the toxin molecule at the cell surface before transport into the cell.
Triton shells of intact erythrocytes
Authors: Sheetz Michael P. , Sawyer David
Published: May 2005

About 40% of human erythrocyte membrane protein is resistant to solubilization in 0.5% Triton X‐114. These components comprise a structure called a Triton shell roughly similar in size and shape to the original erythrocyte and thus constitute a cytoskeleton. With increasing concentrations of Triton the lipid content of the Triton shell decreases dramatically, whereas the majority of the protein components remain constant. Exceptions to this rule include proteins contained in band 3, the presumed anion channel, and in band 4 which decrease with increasing Triton concentration. The Triton‐insoluble complex includes spectrin (bands 1 and 2), actin (band 5), and bands 3′ and 7. Component 3′ has an apparent molecular weight of 88,000 daltons as does 3; but unlike 3, it is insensitive to protease treatment of the intact cell, has a low extinction coefficient at 280 nm, and is solubilized from the shells in alkaline water solutions. Component 7 also has a low extinction coefficient at 280 nm. Spectrin alone is solubilized from the Triton shells in isotonic media. The solubilized spectrin contains no bound Triton and coelectrophoreses with spectrin eluted in hypotonic solutions from ghosts. Electron micrographs of fixed Triton shells stained with uranyl acetate show the presence of numerous filaments which appear beaded and are 80–120 Å in diameter. The filaments cannot be composed mainly of actin, but enough spectrin is present to form the filaments. Triton shells may provide an excellent source of material useful in the investigation of the erythrocyte cytoskeleton.
Mechanism of action of choleragen
Authors: Vauhan Martha , Moss Joel
Published: May 2005

Choleragen exerts its effect on cells through activation of adenylate cyclase. Choleragen initially interacts with cells through binding of the B subunit of the toxin to the ganglioside GM1 on the cell surface. Subsequent events are less clear. Patching or capping of toxin on the cell surface may be an obligatory step in choleragen action. Studies in cell‐free systems have demonstrated that activation of adenylate cyclase by choleragen requires NAD. In addition to NAD, requirements have been observed for ATP, GTP, and calcium‐dependent regulatory protein. GTP also is required for the expression of choleragen‐activated adenylate cyclase. In preparations from turkey erythrocytes, choleragen appears to inhibit an isoproterenol‐stimulated GTPase. It has been postulated that by decreasing the activity of a specific GTPase, choleragen would stabilize a GTP‐adenylate cyclase complex and maintain the cyclase in an activated state. Although the holotoxin is most effective in intact cells, with the A subunit having 1/20th of its activity and the B subunit (choleragenoid) being inactive, in cell‐free systems the A subunit, specifically the A1 fragment, is required for adenylate cyclase activation. The B protomer is inactive. Choleragen, the A subunit, or A1 fragment under suitable conditions hydrolyzes NAD to ADP‐ribose and nicotinamide (NAD glycohydrolase activity) and catalyzes the transfer of the ADP‐ribose moiety of NAD to the guandino group of arginine (ADP‐ribosyltransferase activity). The NAD glycohydrolase activity is similar to that exhibited by other NAD‐dependent bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A), which act by catalyzing the ADP‐ribosylation of a specific acceptor protein. If the ADP‐ribosylation of arginine is a model for the reaction catalyzed by choleragen in vivo, then arginine is presumably an analog of the amino acid which is ADP‐ribosylated in the acceptor protein. It is postulated that choleragen exerts its effects on cells through the NAD‐dependent ADP‐ribosylation of an arginine or similar amino acid in either the cyclase itself or a regulatory protein of the cyclase system.
Catecholamine binding to CNS adrenergic receptors
Authors: U'Prichard David C. , Snyder Solomon H.
Published: May 2005

The properties of 3H‐catecholamine binding to α‐ and β‐adrenergic receptors in CNS are reviewed. 3H‐epinephrine and 3H‐norepinephrine label one class of α‐receptors throughout the brain, with high affinities for agonists and some antagonists. Agonist affinities at this site are increased in low temperature conditions but are reduced by guanine nucleotides and monovalent cations. Divalent cations reverse both effects. This α‐receptor may be coupled to adenylate cyclase by GTP and/or sodium, and uncoupled by divalent cations. 3H‐epinephrine labels β2, but not β1, receptors in CNS, especially in bovine cerebellum. The same β‐receptor does not show agonist‐specific GTP‐sensitivity, but does exhibit Na+‐sensitivity. This receptor appears to be linked to adenylate cyclase, and sodium rather than GTP may be the coupling agent.
Membrane effects of imidoesters in hereditary stomatocytosis
Authors: Mentzer W. C. , Lam G. K. H. , Lubin B. H. , Greenquist A. , Schrier S. L. , Lande W.
Published: May 2005

The marked increase in cation (Na+, K+) permeability that results in swollen, cup‐shaped red cells in the hereditary stomatocytosis syndrome can be corrected in vitro with a bifunctional crosslinking reagent, dimethyl adipimidate (DMA). 45Ca influx in intact RBC, 45Ca efflux in red ghosts, and 45Ca retention in red ghosts are normal and not influenced by DMA. Endocytosis in resealed red ghosts is strikingly impaired but becomes normal if cells are first treated with 2 mM DMA. Protein kinase mediated phosphorylation of membrane proteins by AT32P–only 20–40% of normal control values in both short‐term (5 min) and more extended (60 min) incubations–is not improved by DMA. After reaction of 14C‐DMA with stomatocytes, radiolabel is found associated with phosphatidyl serine and phosphatidyl ethanolamine and is also widely distributed among membrane proteins. Cation permeability of stomatocytes is corrected at DMA concentrations (1 mM) that result in barely detectable crosslinking of aminophospholipids or proteins, suggesting that either crosslinking of a minor component present in only small quantities or intramolecular (rather than intermolecular) crosslinking is responsible for the permeability effects. DMA, whose maximal crosslinking dimension is 7.3–9 Å, is the most effective bifunctional imidoester of those tested. Shorter (dimethyl malonimidate) or longer (dimethylsuberimidate) reagents are either less effective than DMA or totally without effect.
Plasma membrane proteins and glycoproteins from chinese hamster cells sensitive and resistant to actinomycin D
Authors: Peterson Robert H. F. , Biedler June L.
Published: May 2005

Plasma membrane proteins and glycoproteins have been isolated from Chinese hamster cells of the spontaneously transformed DC‐3F parental cell line and the DC‐3F/AD X line with a high level of acquired resistance to actinomycin D. Plasma membrane preparations from both cell lines band at 1.16 g/ml after isopycnic centrifugation. We present evidence to indicate differences in the leucylpeptide backbones of the antibiotic‐sensitive cells and the drug‐resistant DC‐3F/AD X cells. In addition, there are differences in the plasma membrane glycopeptides of the two cell lines as revealed by sodium dodecyl gel electrophoresis. Drug‐resistant cells synthesize a surface glycopeptide which is much larger than the major one present on the drug‐sensitive cells. Both of these cell lines are devoid of 5′‐nucleotidase and alkaline phosphatase activities. The role of plasma membrane protein differences in drug‐resistant cells is discussed.