bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractTwo 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

AbstractPrevious 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

AbstractBHK 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

AbstractA 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

AbstractWhen 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

AbstractSolubilized 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

AbstractWe 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

AbstractVesicular 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

AbstractThe 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

AbstractAbout 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

AbstractCholeragen 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

AbstractThe 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

AbstractThe 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

AbstractPlasma 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.
Cation‐dependent phosphatase activites in a rat pancreatic islet plasma membrane fraction prepared by one‐step gradient centrifugation
Authors: Lernmark Årke , Nielsen David A. , Steiner Donald F. 
Published: May 2005

AbstractA plasma membrane‐enriched fraction was prepared from homogenized rat pancreatic islets by a one‐step sucrose gradient centrifugation. Using 125I‐wheat germ agglutinin as a plasma membrane probe, a fraction was obtained at a sucrose density of about 1.10 that was enriched in 5′‐nucleotidase, Mg2+‐ATPase and alkaline phosphatase. The fraction contained little, if any, monoamino oxidase activity, insulin or DNA. Hydrolysis of 3‐0‐methyl‐fluoresceinphosphate was stimulated by K+ (10mM) at a pH optimum of pH 8.2. Hydrolysis of ATP‐γ‐32P in the presence of MgCl2 was of high specific activity and was optimum at pH 7.0 and 8.2. K+ did not affect ATP‐hydrolysis. At pH 8.2, a small fraction of the total Mg2+‐ATPase activity was inhibited by ouabain in the presence of Na+ and K+. Since K+‐stimulated phosphatase activity does not correlate with Mg2+‐ATPase, the two assay systems define separate enzymatic processes.