bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of a penicillium GDP‐mannose: Glycopeptide mannosyltransferase solubilized with triton X‐100
Authors: Gander J. E. , Fang Faye
Published: May 2005

Membranes from Penicillium charlesii were separated into 6 fractions by sucrose density gradient ultracentrifugation. The least dense fraction (ρ = 1.1 g cm−3) contained GDP‐mannose: glycopeptide mannosyltransferases that transferred [14C] mannose onto mannopyranosyl‐(seryl/threoyl)‐polypeptide and phosphogalactomannan regions of peptidophosphogalactomannan. Approximately 90% of the [14C] mannose incorporated was isolated as mannobiose following treatment of peptidophosphogalactomannan with 0.5 N NaOH. The remainder was located in phosphogalactomannan. About 10% of the membrane‐bound mannosyltransferase activity was solubilized with 1% Triton X‐100. The soluble mannosyltransferase activity was purified by affinity chromatography on peptidophosphogalactomannan‐Sepharose 4B and ammonium sulfate fractionation. Mannose incorporation was shown to be a function of the concentration of added acceptor. No incorporation occurred in the absence of added acceptor or when MgCl2 was substituted for MnCl2. Peptidophosphogalactomannan, phosphogalactomannan, phosphomannan, and mannan, each obtained by appropriate treatment of peptidophosphogalactomannan from P. charlesii, served as mannosyl acceptors. In contrast, α‐mannosidase treated peptidophosphogalactomannan did not serve an acceptor of mannosyl residues. Up to 70% of the mannose from GDP‐mannose was transferred to added acceptor. Treatment of [14C] mannosyl‐labeled peptidophosphogalactomannan with 0.5 N NaOH released 90% of the [14C] mannose as phosphogalactomannan and the remainder was released as mannobiose. [14C] Mannose‐labeled phosphogalactomannan was subjected to acetolysis. Mannobiose was the major [14C]‐labeled product isolated. Significant quantities of [14C] mannose were isolated also. These results show that soluble mannosyltransferase catalyzes the formation of (1–6)‐linked mannosyl residues as well as the transfer of a mannosyl residue to a (1–6)‐linked mannosyl residue in the phosphogalactomannan. The specificity of the enzyme is shown by its inability to catalyze mannosyl transfer to α‐mannosidase treated peptidophosphogalactomannan, or to incorporate more than 2 mannosyl residues onto the phosphogalactomannan region. Presumably the second mannosyl residue is attached by a (1–2) linkage as the mannan contains only (1–6)‐ and (1–2)‐linked mannosyl residues (Gander et al: J Biol Chem 249:2063, 1974). No evidence was obtained for the participation of a lipid‐linked mannosyl‐containing intermediate in this system.
Cell antigens recognized by rabbit antibodies specific for oligomannosyl determinants
Authors: Sarkar Siddhartha , Menge Alan C.
Published: May 2005

Rabbit antibodies to cell wall mannans of various microbial strains and their mutants were found to be cross‐reactive to cell carbohydrates of mammalian sperm and 4–6‐day‐old blastocysts. Immunochemical studies indicate that oligomers of α1→2, α1→3, α1→6, and probably also α→4 linked mannose residues of sperm carbohydrates are available for antibody binding. At least 80% of binding activity of a yeast mannan antibody to sperm can be effectively inhibited by specific haptens or digestion with exo‐α‐D‐mannosidase, an enzyme activity highest in testicular tissue. In order to determine the role of this enzyme in the metabolism of the cross‐reactive mannan antigens of sperm, the relative amount of a specific α‐linked oligomannosyl determinant of bovine sperm from homozygous normals was compared to that of heterozygous carriers of α‐mannosidase deficiency.

Extensive cross‐reactivity between the microbial and mammalian oligomannosyl determinants suggest that these are conserved structures in cell carbohydrates, although the organization of these units in the microbial cell wall lipopolysaccharide has very little similarity to the carbohydrate moieties of mammalian glycoproteins.
Distribution of glycoconjugates in mouse fibroblasts with varying degrees of tumorigenicity
Authors: Winterbourne D. J. , Mora P. T.
Published: May 2005

Analysis of glucosamine labeled glycoconjugates in cultured cells has been made comparing 2 clones and the parent embryonic mouse cell line. Hyaluronic acid, heparan sulphate, and chondroitin sulphate as well as a complex mixture of glycopeptides were found in the medium, the trypsinate, and the trypsinized cells, although the distribution was not uniform. The 3 cell lines had very similar in vitro growth properties, including their plating efficiency in viscous medium. However, the tumorigenicity of the cells, determined in syngeneic mice, was found to differ. All 3 cell lines were found to have similar glycoconjugate distributions, although a slight relative increase in labeled hyaluronic acid was found in the more tumorigenic mass cell line than either of the clones. The possible significance of this increase is discussed.
Interaction of cartilage proteoglycans with hyaluronic acid
Authors: Hascall Vincent C.
Published: May 2005

Most proteoglycans are present in hyaline cartilage matrices as aggregates with as many as 100 molecules, each with average molecular weight of about 2 × 106, bound through specific, noncovalent interactions to individual strands of hyaluronic acid (HA). The interactions with HA are mediated by the HA‐binding region of the core protein, which is located at one end of each of the interactive proteoglycans. A fragment of the core protein, average molecular weight of about 6 × 104, which contains the HA‐binding site, can be isolated in an active form from trypsin digests of proteoglycan aggregates. The “active” HA‐binding site in this preparation interacts strongly with HA‐10 but weakly with HA‐8, (oligomers of HA derived from partial digests of HA with testicular hyaluronidase); HA‐9 derived from β‐glucuronidase digestion of HA‐10 also interacts strongly. No polysaccharide other than HA has been found to interact. Christner, Brown, and Dziewiatkowski (personal communication) modified the carboxyls on glucuronic acid groups in mixture of HA‐10 to HA‐30, and they found that the interaction with proteoglycan no longer occurred if about 60% of the total carboxyls were (a) methyl esterified, (b) reduced to glucose, or (c) substituted with glycine in amide linkage. Saponification of the methyl esters restored activity. Dansylation of lysine residues in the HA‐binding region preparation abolished binding activity. However, when the dansylation reaction was done in the presence of HA, the HA‐binding activity was protected. Acetylation of the same residues did not abolish binding activity but did prevent subsequent inactivation by dansylation. Hardingham, Ewins, and Muir (Biochem J 157:127–143, 1976) studied the effect of various amino acid modifiers on the interaction of intact proteoglycans with HA and showed that reaction of arginine residues with low concentrations of 2,3‐butanedione was particularly effective in destroying binding. In sum, the data above suggests that the HA‐binding region (a) contains accessible arginine residues necessary for activity, (b) contains lysine residues near the binding site which, when substituted with bulky groups such as dansyl, but not acetyl, sterically block interaction, and (c) requires a length of HA with at least 4.5 repeat disaccharides containing 3, and possibly 4, unmodified glucuronic acid carboxyls for interaction. The possible relevance of proteoglycan‐hyaluronic acid interaction to the observations that hyaluronic acid specifically inhibits proteogly can synthesis by cultured chondrocytes is discussed.
Glycosylation of VSV glycoprotein is similar in cystic fibrosis, heterozygous carrier, and normal human fibroblasts
Authors: Hunt Lawrence A. , Summers Donald F.
Published: May 2005

The single envelope glycoprotein of vesicular stomatitis virus was used as a specific probe of glycosyltransferase activities in fibroblasts from two cystic fibrosis patients, an obligate heterozygous carrier and a normal individual. Gel filtration of pronasedigested glycopeptides from both purified virions and infected cell‐associated VSV glycoprotein which had been labeled with [3H] glucosamine did not reveal any significant differences in the glycosylation patterns between the different cell cultures. All 4 cell lines were apparently able to synthesize the mannose‐ and glucosamine‐containing core structure and branch chains terminating in sialic acid which are characteristic of asparagine‐linked carbohydrate side chains in cellular glycoproteins. Analysis of tryptic glycopeptides by anion‐exchange chromotography indicated that the same 2 major sites on the virus polypeptide were recognized and glycosylated in all 4 VSV‐infected cell cultures. These studies suggest that the basic biochemical defect(s) in cystic fibrosis is not an absence or deficiency in enzymes responsible for the biosynthesis of complex carbohydrate side chains.
Biochemical characteristics, metabolism, and antitumor activity of several acetylated hexosamines
Authors: Bernacki R. J. , Sharma M. , Porter N. K. , Rustum Y. , Paul B. , Korytnyk W.
Published: May 2005

We have synthesized several potential inhibitors and/or modifiers of the carbohydrate portion of plasma membrane glycoconjugates. These include fluorinated and actylated analogs of D‐glucosamine, D‐galactosamine, and D‐mannosamine. These compounds have been tested to determine their effects on both [14C] glucosamine and [3H] leucine incorporation into glycoconjugate and on cell growth and viability using P‐288 murine lymphoma cells maintained in tissue culture. The most cytotoxic agent tested was 2‐acetamido‐2‐deoxy‐1,3,4,6‐tetra‐O‐acetyl‐β‐D‐glucopyranose or simply β‐pentaacetylglucosamine which prevented cell growth at 10−4–10−3 M. β‐Pentaacetylglucosamine cytotoxicity was correlated with its high lipid solubility, having an octanol/water partition coefficient of 0.424 as compared with 0.278 for the β‐anomer and 0.017 for N‐acetylglucosamine. In vitro metabolism studies with [14C]‐and/or [3H]‐labeled pentaacetylglucosamine have indicated intracellular de‐O‐acetylation leading to the biosynthesis of UDP‐N‐acetylglucosamine, followed by the incorporation of this sugar into cellular glycoprotein. Concomitant with the formation of increased amounts of this nucleotide sugar, intracellular UTP and CTP pools fell to one third normal within 3 h after the administration of 1 mM pentaacetylglucosamine. At present it is unclear whether the cytotoxicity of β‐pentaacetylglucosamine or other similar agents is due to alterations in nucleotide and nucleotide‐sugar pools causing a decrease in energy charge and polynucleotide biosynthesis or is due to a direct effect on membrane glycoconjugate biosynthesis.
Cell surface changes accompanying myoblast differentiation
Authors: Furcht L. T. , Wendelschafer‐Crabb G. , Woodbridge P. A.
Published: May 2005

Myoblasts are mononucleated cells and associated with differentation undergo cell fusion and become multinucleated. The current studies have examined cell surface dynamic changes of Concanavalin A lectin receptor mobility and the role of hormones in modulating myoblast differentiation. A uniform distribution of Con‐A receptors is observed in undifferentiated cells when reacted with Con‐A at 37°C. Cells from differentiating cultures or fully differentiated myotubes reacted similarly at 37°C show a significant redistribution of Con‐A into patches, “caps,” and endocytic vesicles containing Con‐A. If undifferentiated and differentiated cells are first prefixed with glutaraldehyde then reacted with Con‐A continuous distribution of Con‐A is seen across the cell surface. This suggests redistribution of Con‐A and its receptors occurs in differentiated cells reacted with lectin at 37°C. It is further shown that insulin (10 μg/ml) significantly enhances myoblast differentiation but that this occurs after an apparent stimulation of proliferation. In contrast to insulin, dexamethasone (10 μM and 100 μM) profoundly inhibits myoblast differentiation while having different effects on proliferation; 10 μM dex stimulates cell growth while 100 μM dex suppresses cell proliferation. Lastly, an extracellular filamentous matirix which binds Con‐A is observed at the ultrastructural level in high density cultures. No significant redistribution of Con‐A is observed on this matrix in distinction to the redistribution observed on the cell membrane in differentiated cells.
Purification and characteristics of hydrophobic membrane protein(s) required for DCCD sensitivity of ATPase in mycobacterium phlei
Authors: Cohen Natalie S. , Lee Soon‐Ho , Brodie Arnold F.
Published: May 2005

The energy‐transducing N,N′‐dicyclohexylcarbodiimide‐sensitive (DCCD‐sensitive) ATPase complex consists of two parts, a soluble catalytic protein (F1), and an intrinsic membrane protein (F0). The bacterial coupling factor complex, BCF0‐BCF1, has recently been purified from Mycobacterium phlei, and used to reconstitute oxidative phosphorylation in detergent‐extracted membranes. The BCF0 moiety has been purified by being recovered from the purified BCF0‐BCF1 complex by affinity chromatography. BCF0 is a lipoprotein or lipoprotein complex with an approximate molecular weight of 60,000. The preparation contained 0.15 mg of phospholipid per milligram protein. There appear to be three polypeptides, with approximate molecular weights of 24,000, 18,000, and 8,000 as determined by sodium dodecylsulfate a crylamide gel electrophoresis. Purified BCF0 conferred DCCD sensitivity on a purified BCF1 preparation. Reconstitution of oxidative phosphorylation was achieved after incubation of detergent‐extracted membranes with purified BCF0 and purified BCF1.
Quantitative analysis of the microtubule system in isolated fish melanophores
Authors: Schliwa Manfred , Euteneuer Ursula
Published: May 2005

Isolated melanophores of the angelfish, Pterophyllum scalare, have been used in a morphometric analysis and a quantitative study of their microtubule system. Using transverse sections spaced at regular intervals, the changes associated with the process of pigment aggregation have been determined. Upon the concentration of pigment granules in the central cell region, almost half of the cytoplasmic portion is also withdrawn from the peripheral cell regions. Counts of microtubules within a cell sector in cells with pigment aggregated and dispersed, respectively, reveal (a) a constancy of the number of microtubules in this sector regardless of the distance from the cell center, and (b) a reduction of microtubule number in cells with pigment aggregated by about 58%. On the basis of these counts, the total number of microtubules has been calculated. In the dispersed state, about 2,400 microtubules extend between the center and the periphery of the cell, while their number is about 1,000 in the aggregated state.

Using a 13‐protofilament model of a microtubule and relevant data on size and molecular weight of microtubule subunits, the amount of tubulin present as microtubules is calculated. In the average, the cells contain 1.95·108 monomers corresponding to 1.78·10−8 mg tubulin. A tentative estimation of the concentration of tubulin inside a melanophore yields values of 6.1 mg/ml for the whole cell and 16.5 mg/ml for the cytoplasm alone (excluding membrane‐bound organelles). Based on this estimation, a comparison, with microtubule assembly in vitro is made.
Topology of amino‐phospholipids in the red cell membrane
Authors: Marinetti Guido V. , Crain Richard C.
Published: May 2005

The red cell membrane has an asymmetric arrangement of phospholipids. The amino‐phospholipids are localized primarily on the inner surface of the membrane and the choline phospholipids are localized to a large extent on the outer surface of the membrane. Evidence is presented based on the use of covalent chemical probes in sequence that the red cell membrane contains heterogeneous domains of PE and PS and that the transport systems for Pi and K+ are asymmetrically arranged. Certain amino groups of PE, PS, and/or protein localized on the outer membrane surface are involved in Pi transport and certain amino groups of PE, PS, and/or protein localized on the inner surface of the membrane are involved in K+ transport.

Cross‐linking studies with DFDNB show that the cross‐linked PE‐PE molecules are rich in plasmalogens. This suggests that clusters of plasmalogen forms of PE occur in the membrane. Both PE and PS are cross‐linked to membrane protein. These PE and PS molecules contain 24–28% 16:0 and 18:0 fatty acids and 12% fatty aldehydes. PE and PS molecules are cross‐linked to a spectrin‐rich fraction. It is proposed that the binding of spectrin to membrane PE and PS may help anchor spectrin to the inner surface of the membrane and regulate shape changes in the cell.

K+‐valinomycin forms a complex with TNBS and converts it from a non‐penetrating proble to a penetrating probe. Valinomycin enhances K+ leak and Pi leak in the red cells. SITS inhibits completely the valinomycin‐induced Pi leak and inhibits partially the valinomycin induced K+ leak. Valinomycin and IAA have additive effects on Pi leak. Ouabin has no effect on basal or valino‐mycin‐induced Pi leak. These data suggest that Pi leak and K+ leak occur by separate transport systems.

In summary, the amino‐phospholipids in the red cell membrane are asymmetrically arranged; some occur in clusters and some are closely associated with membrane proteins. Amino‐phospholipids also are believed to bind spectrin to the inner surface of the membrane and also may play a role in cation and anion leak.
Mitogens for murine embryo cell lines
Authors: Herschman Harvey R. , Passovoy Dennis S. , Pruss Rebecca M. , Aharonov Aharon
Published: May 2005

The growth‐promoting activities of fetal bovine serum, cortisol, phorbol myristate acetate, prostaglandin F2α, insulin, epidermal growth factor, and fibroblast growth factor were evaluated on four murine embryo cell lines (Swiss 3T3, Balb 3T3, M2, and C3H10T1/2). Each cell had an unique response spectrum to this collection of reported mitogens. Phorbol myristate acetate and prostaglandin F2α were active only on selected cell lines; cortisol was inactive on all four lines. Serum, epidermal growth factor, and fibroblast growth factor were able to stimulate cell division in all four lines, albeit to varying degrees for the different target cells.
The band 3 protein of the human red cell membrane: A review
Authors: Steck Theodore L.
Published: May 2005

Band 3 is the predominant polypetide and the purported mediator of anion transport in the human erythrocyte membrane. Against a background of minor and apparently unrelated polypeptides of similar electrophoretic mobility, and despite apparent heterogeneity in its glycosylation, the bulk of band 3 exhibits uniform and characteristic behavior. This integral glycoprotein appears to exist as a noncovalent dimer of two ∼ 93,000‐dalton chains which span the membrane asymmetrically. The protein is hydrophobic in its composition and in its behaviour in aqueous solution and is best solubilized and purified in detergent. It can be cleaved while membrane‐bound into large, topographically defined segments. An integral, outer‐surface, 38,000‐dalton fragment bears most of the band 3 carbohydrate. A 17,000‐dalton, hydrophobic glycopeptide fragment spans the membrane. A ∼ 40,000‐dalton hydrophilic segment represents the cytoplasmic domain. In vitro, glyceraldehyde 3‐P dehydrogenase and aldolase bind reversibly, in a metabolite‐sensitive fashion, to this cytoplasmic segment. The cytoplasmic domain also bears the amino terminus of this polypetide, in contrast to other integral membrane proteins. Recent electron microscopic analysis suggests that the poles of the band 3 molecule can be seen by freezeetching at the two original membrane surfaces, while freeze‐fracture reveals the transmembrane disposition of band 3 dimer particles.

There is strong evidence that band 3 mediates 1:1 anion exchange across the membrane through a conformational cycle while remaining fixed and asymmetrical. Its cytoplasmic pole can be variously perturbed and even excised without a significant alteration of transport function. However, digestion of the outer‐surface region leads to inhibition of transport, so that both this segment and the membrane‐spanning piece (which is slectively labeled by covalent inhibitors of transport) may be presumed to be involved in transport. Genetic polymorphism has been observed in the structure and immunogenicity of the band 3 polypeptide but this feature has not been related to variation in anion transport or other band 3 activities.
Molecular characteristics of the transferrin‐receptor complex of the rabbit reticulocyte
Authors: Hu Hsiang‐Yun Yang , Aisen Philip
Published: May 2005

A macromolecular complex of transferrin and a membrane component was isolated by gel filtration chromatography from Triton X‐100‐solubilized ghosts of reticulocytes previously incubated with 125I‐labeled transferrin. This complex is believed to be transferrin specifically associated with its primary receptor. Following the procedures of Clark [14], the complex in Triton X‐100 was found to behave as an asymmetric molecule with a molecular weight of approximately 250,000 and an axial ratio of 9:1. On SDS‐polyacrylamide gel electrophoresis the complex displays, in addition to transferrin, components of molecular weights 176,000 and 95,000, respectively. The larger component may be a dimer of the smaller. Each appears to crosslink, with dimethyl suberimidate, to transferrin. These results are compatible with the hypothesis that the transferrin receptor itself has a molecular weight near 175,000 and may be a dimer of two smaller components each of molecular weight near 95,000.
The program of friend cell erythroid differentiation: Early changes in Na+/K+ ATPase function
Authors: Mager Dixie , Bernstein Alan
Published: May 2005

Treatment of Friend erythroleukemia cells with several different chemical agents causes an early decrease in the 86Rb+ influx mediated by Na+/K+ adenosine triphosphatase (ATPase). These agents, which induced Friend cells to differentiate, include dimethylsulfoxide (DMSO), ouabain, hypoxanthine, and actinomycin D. The magnitude of the early decrease in 86Rb+ influx correlates with the proportion of cells in cultures of inducible Friend cell clones which later go on to synthesize hemoglobin. Compounds which do not incude differentiation in these cells, such as xanthine, exogenous hematin, and erythropoietin, do not cause a change in 86Rb+ influx. A change in the intracellular K+ ion concentration does not occur during induction by DMSO because, although there is a decrease in K+ content per cell soon after induction, there is a parallel decrease in cell volume. These results and previous observations from this laboratory are discussed in terms of the posible involvement of the Na+/K+ ATPase in Friend cell differentiation.
Analysis of the major polypeptides of spectrin by tryptic digestion
Authors: Maddy Alun H. , Dunn Michael J.
Published: May 2005

The two major polypeptides of erythrocyte membrane spectrin have been isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The tryptic peptide maps of the two polypeptides have been prepared by thin‐layer chromatography and electrophoresis. Radioactive peptides have been prepared by 14C‐carboxymethylation and chloramine T‐catalysed 125I iodination. Maps of both sets of peptides demonstrate a marked similarity between the two parent polypeptides.
Metabolic stability of the nucleoside transport system of novikoff rat hepatoma cells
Authors: Marz Richard , Wohlhueter Robert M. , Plagemann Peter G. W.
Published: May 2005

Rates of transport of uridine and thymidine, estimated with a rapid sampling technique, did not change with culture age. Inhibition of cellular RNA and protein synthesis for periods up to 6 h, did not lead to a loss of nucleoside transport activity. Mild treatment of cell suspensions with trypsin or neuraminidase had no effect on the kinetics of thymidine transport. Thus we conclude, contrary to previous reports, that nucleoside transporters are metabolically stable and that the decreases in nucleoside uptake rates observed with decreased protein synthesis reflect loss of nucleoside kinase activities. These kinases (which have narrow substrate specificity) rather than the membrane‐associated, transport apparatus (which has broad substrate specificity) are the most likely sites for regulation of nucleoside uptake.
Properties and action mechanism of the toxic lectin modeccin: Interaction with cell lines resistant to modeccin, abrin, and ricin
Authors: Olsnes Sjur , Sandvig Kirsten , Eiklid Kristin , Pihl Alexander
Published: May 2005

The toxic lectin modeccin, which inhibits protein synthesis in eukaryotic cells, is cleaved upon treatment with 2‐mercaptoethanol into two peptide chains which move in polyacrylamide gels at rates corresponding to molecular weights 28,000 and 38,000. After reduction, the toxin loses its effect on cells, while its ability to inhibit cell‐free protein synthesis increases. Like abrin and ricin it inhibits protein synthesis by inactivating the 60S ribosomal subunits.

Modeccin binds to surface receptors containing terminal galactose residues. Competition experiments with various glycoproteins indicate that the modeccin receptors are different from the abrin receptors. In addition, they were present on HeLa cells in much smaller numbers. Moreover, mutant lines resistant to abrin and ricin were not resistant to modeccin and vice‐versa.

The toxin resistance of various mutant cell lines could not be accounted for by a reduced number of binding sites on cells. The data are consistent with the view that the cells possesss different populations of binding sites with differences in ability to facilitate the uptake of the toxins and that in the resistant lines the most active receptors have been reduced or eliminated.
Use of photoaffinity nucleotide analogs to determine the mechanism of ATP regulation of a membrane‐bound, cAMP‐activated protein kinase
Authors: Owens James R. , Haley Boyd E.
Published: May 2005

Using a radioactively tagged, photoaffinity analog of cAMP, 8‐azidoadenosine‐3′,5′‐cyclic monophosphate (8‐N3 cAMP), and [γ32P] ATP, the membranebinding properties of both the regulatory and catalytic subunits of the cAMP‐activated protein kinase of human erythrocyte membranes were investigated. [32P] 8‐N3 cAMP was used to locate and quantify regulatory subunits. Increased phosphorylation of specific membrane proteins by [γ32P] ATP was used to determine the presence of the catalytic subunit. The data support a mechanism which operates through a tight membrane‐bound regulatory subunit and a catalytic subunit that is released from the membrane when cAMP is present and the Mg · ATP concentration is below approximately 10 μM. The catalytic subunit is not required for the Mg · ATP inhibition of 8‐N3 cAMP binding. Experiments with a photoaffinity analog of ATP, 8‐azidoadenosine triphosphate (8‐N3ATP), support the hypothesis that ATP hydrolysis and phosphorylation are not involved in the regulation. The data indicate that the regulatory subunit contains an ATP regulatory site which inhibits 8‐N3 cAMP binding and the release of the catalytic subunit. These results indicate that the membrane‐bound type I enzyme (type IM) differs significantly from the soluble (type IS) enzyme studied in other tissues. These enzymes are compartmentalized by being in different cellular locations and are regulated differently by Mg · ATP.
Glycophorins A, B, and C: A family of sialoglycoproteins. Isolation and preliminary characterization of trypsin derived peptides
Authors: Furthmayr Heinz
Published: May 2005

Two new sialoglycoproteins, glycophorin B and glycophorin C, were isolated from erythrocyte membranes by extraction with lithium diiodosalicylate, partition in aqueous phenol, gel filtration in detergent, and preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The two proteins were characterized by amino acid and carbohydrate analysis, separation of tryptic peptides, and isolation and purification of the amino terminal glycopeptide from each polypeptide chain. Glycophorin B is found in two forms in electrophoretograms of normal erythrocyte membranes corresponding to monomer and dimer, as has been similarly described for glycophorin A. By using antibodies to a carboxy terminal determinant of glycophorin A, and direct staining of gels with antibodies and 125I‐protein A from Staph. aureus, as well as by two‐dimensional immunoelectrophoreis, only the two known forms of glycophorin A are detectable. The data confirm and extend the notion that the sialoglycoproteins in human red cells are dimeric molecules which are either preformed in the membrane or which can readily be generated in vitro. Only glycophorin A and glycophorin C are sensitive to trypsin while in situ in the intact red blood cells.
Phosphorylation and dephosphorylation of spectrin
Authors: Fairbanks Grant , Avruch Joseph , Dino Judith E. , Patel Vikram P.
Published: May 2005

The phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP‐independent, salt‐stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co‐fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane‐bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of 32Pi from [32P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [32P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions – notably, ATP and 2,3‐DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated (1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and (2) by membrane deformation that alters enzyme–spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to the resolution of major questions raised by previous work: the relationships between membrane‐bound and cytoplasmic forms of the enzymes, the nature of their physical interactions with the membrane, and the regulation of their activities in defined cell‐free systems.
Actin—membrane interactions: Association of G‐actin with the red cell membrane
Authors: Cohen Carl M. , Jackson Pamela L. , Branton Daniel
Published: May 2005

Chemically tritiated actin from rabbit skeletal muscle was used to investigate the association of G‐actin with the red cell membrane. The tritiated actin was shown to be identical to unmodified actin in its ability to polymerize and to activate heavy meromyosin ATPase. Using sealed and unsealed red cell ghosts we have shown that G‐actin binds to the cytoplasmic but not the extracellular membrane surface of ghosts. Inside‐out vesicles which have been stripped of endogenous actin and spectrin by low‐ionic‐strength incubation bind little G‐actin. However, when a crude spectrin extract containing primarily spectrin, actin, and band 4.1 is added back to stripped vesicles, subsequent binding of G‐actin can be increased up to 40‐fold. Further, this crude spectrin extract can compete for and abolish G‐actin binding to unsealed ghosts. Actin binding to ghosts increases linearly with added G‐actin and requires the presence of magnesium. In addition, actin binding is inhibited by cytochalasin B and DNAase I. Negative staining reveals an abundance of actin filaments formed when G‐actin is added to reconstituted inside‐out vesicles but none when it is added to unreconstituted vesicles. These observations indicate that added G‐actin binds to the red cell membrane via filament formation nucleated by some membrane component at the cytoplasmic surface.
An avidin‐biotinyl‐propranolol complex for β‐adrenergic receptor characterization
Authors: Meier Kathryn E. , Ruoho Arnold E.
Published: May 2005

The synthesis of biotinyl‐hexaglycyl‐NEDA (abbreviation:BGN), a biotinyl derivative of propranolol, is described. This bifunctional molecule binds with high affinity to the biotin‐binding protein, avidin. The duck erythrocyte was used as a model β‐receptor system. Formation of an avidin‐BGN‐β‐receptor complex was demonstrated in intact erythrocytes, in erythrocyte ghosts, and in the digitonin‐solubilized β‐receptor. The avidin‐BGN complex will be used for localization and purification of the β‐receptor.
A serological assay for the detection of cell surface receptors of nerve growth factor
Authors: Zimmermann Astrid , Sutter Arne , Samuelson John , Shooter Eric M.
Published: May 2005

When single‐cell suspensions prepared from embroyonic day 8 (E8) chick sensory ganglia are incubated with nerve growth factor (NGF), anti‐NGF antiserum, and complement, an NGF‐dependent cytotoxic kill of 20 (±3)% of the ganglia cells is observed. This percentage is increased by a factor of two when only the neuronal cells are tested. No kill is observed on the nonneuronal cell population representing 50% of the ganglia dissociate. When E8 sensory ganglia cells are cultured in the presence of NGF following cytotoxic kill, the large, phase‐bright NGF‐reponsive neurons are missing from the culture. These results indicate that the cells recognized in the cytotoxicity assay have to carry NGF‐binding sites of type I, which is the one with the higher affinity of the two types of NGF‐binding sites (I and II) present on sensory ganglia cells. This conclusion is further supported by the following data: (a) half maximal cytotoxicity is reached already at a concentration of NGF which is below the KD of binding site I; (b) a washing step which removes all NGF bound to type II receptors while leaving a high percentage of type I receptors occupied has no effect on the percentage of ganglia cells killed.

Using the cytotoxicity assay the presence of high‐affinity binding sites of type I can be demonstrated on sensory ganglia cells from E8 chick embryos but not from E4 embryos and not on liver and heart cells from E8 embryos. Further, type I receptor‐bearing cells were detectable in the brain using this assay. At E8, NGF receptors could be detected on cells of the forebrain and the tectum but not on brain stem cells. Cytotoxic kill of forebrain cells was found to be especially high at E8 and E9, and decreased by E10.
The spontaneous release of a high‐molecular‐weight aggregate containing immunoglobulin G from the surface of Ehrlich ascites tumor cells
Authors: Rittenhouse Harry G. , Ar Diane , Lynn Matthew D. , Denholm David K.
Published: May 2005

The spontaneous release of tumor cell antigens from the cell surface into the circulation has been proposed as a mechanism whereby tumors may escape the immune response of the host. In this study we have found that Ehrlich ascites tumor cells after removal from the host (mouse) spontaneously release significant amounts of cell surface components during incubation for 1 h in cold isotonic buffer. Immunodiffusion studies revealed that immunoglobulin G (IgG) and a complement component (C3) are included in this spontaneously released material. These surface‐bound humoral immune components are apparently released in the form of a high‐molecular‐weight aggregate (cell coat particle) as shown by ultracentrifugation and ultrafiltration experiments. Precipitation of IgG from the cell coat particle preparation with antibodies directed against mouse IgG followed by detergent gel electrophoresis of the immune precipitate revealed five major bands in addition to the heavy and light chains of IgG. These results suggest that host IgG is tightly bound to several other components at the cell surface, perhaps in the form of immune complexes.

IgG is localized on the tumor cell surface in a highly heterogeneous pattern with the appearance of patches and caps in some cells as shown by immuno‐fluorescence analysis. The possibility that humoral immune components bind to the tumor cell surface and result in the shedding of high‐molecular‐weight aggregates of cell surface antigens into extracellular fluids is discussed.
Murine leukemia cell hybrids: The quantity of TL antigens expressed by parental and hybrid cells fails to correlate with their sensitivity to TL antibody and complement
Authors: Cohen Edward P. , Liang Weitze
Published: May 2005

The quantity of thymus‐leukemia (TL) antigens expressed by murine leukemia cells is significantly greater than that expressed by somatic hybrids of such cells. Based upon the results of 125I‐lactoperoxidase labeling and antibody absorption procedures, and corrected for size differences between the two cell types, the quantity of TL antigens expressed by RADA‐1 cells, a radiation‐induced murine leukemia cell line of strain A/J mice, is approximately 5.0 times greater than that of somatic hybrids of RADA‐1 and LM(TK)− cells. LM(TK)− cells are a thymidine kinase‐deficient TL(‐) mouse fibroblast cell line. The quantity of TL antigens expressed is related only in part to their susceptibility to lysis by TL antibodies and guinea pig complement (GPC). RADA‐1 cells resist lysis. The quantity of TL antigens expressed by RADA‐1 cells is analogous to that formed by nonneoplastic thymocytes obtained from F1 hybrids of two strains of TL(+) and TL(‐) mice; cells from both strains are sensitive to TL antiserum and GPC. ASL‐1 cells, a spontaneously occurring leukemia cell line of A/J mice, express TL antigens in significantly higher quantities than any of the cell types examined. Exposed to TL antisera, the quantity of TL antigens of ASL‐1 cells, but not that of hybrid cells, gradually diminishes. ASL‐1 cells convert over a 6‐h period of exposure to antibody and guinea pig complement (GPC) resistance; hybrid cells remain sensitive. However, ASL‐1 cells converted to TL antibody and GPC resistance continue for a time to express TL antigens in quantities similar to that of sensitive F1 thymocytes and resistant RADA‐1 cells. RADA‐1 X LM(TK)− hybrid cells, which are sensitive to TL antibodies and GPC, express the lowest quantities of TL antigens of any of the cell types examined. It is likely that differences in the quantities of TL antigens expressed by different cell lines reflect genetic mechanisms controlling TL antigen expression. The failure of TL antisera to affect the quantities of TL antigens expressed by hybrid cells is taken as an indication that genetic controls governing antigen expression may be distinguished from those involved in regulating responsiveness to specific antiserum.
Role of physiologic autoantibody in the removal of senescent human red cells
Authors: Kay Marguerite M. B.
Published: May 2005

The mechanism by which mononuclear phagocytes distinguish mature “self” from senescent “self” was investigated. Evidence is presented indicating that human mononuclear phagocytes distinguish senescent RBC from mature RBC on the basis of selective Ig attachment to the membranes of senescent cells. This Ig, eluted from senescent human RBC, was shown to be IgG and free of other Igs by immunodiffusion, immunoelectrophoresis, and polyacrylamide gel electrophoresis. The IgG was polyclonal with respect to light chains. The eluted IgG reattaches to homologous stored RBC, but not to mature autologous or allogeneic RBC, via the Fab region. It then initiates phagocytosis of these stored RBC by mononuclear phagocytes. Evidence suggests that the IgG is directed against altered membrane receptors. Thus, this IgG may be a “physiologic” autoantibody and contribute to the maintenance of homeostasis by performing regulatory functions.
A possible modulation of acetylcholine receptors of embryonic chick muscle cells by α‐bungarotoxin
Authors: Elson Hannah Friedman
Published: May 2005

Acetylcholine receptors were assayed with α‐bugarotoxin on embryonic chick skeletal muscle growing in primary cell culture. Toxin was bound specifically to muscle cells and could be competed with D‐tubocurarine. Two dissociation constants were obtained by equilibrium binding: 7.2 × 10−9M and 2.7 × 10−7M at 25°C. Two sets of rate constants were also obtained from dissociation kinetics. There are five times more low affinity sites on cells than high affinity sites. The average density of high‐affinity receptors is about 200/μm2.

A time course of toxin binding to receptors at 37°C vs 25°C in growth medium revealed that under conditions permitting growth and metabolism, toxin bound to cells was lost. The possibility that the growth medium was in‐activating toxin molecules was ruled out by showing that unbound toxin molecules in the medium were fully capable of binding to fresh cultures.
Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: The endocytic activation hypothesis
Authors: Fox C. Fred , Das Manjusri
Published: May 2005

The addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity 125I‐EGF, and the fate of the affinity labeled EGF‐receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF‐induced receptor internalization and EGF‐induced stimulation of 3H‐thymidine uptake into cellular DNA show that there is a direct correlation between EGF‐induced receptor internalization and EGF‐induced stimulation of DNA synthesis, but not between EGF binding and EGF‐induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF‐induced processes requires constant EGF‐induced internalization of receptor for a requisite 6–8 h period as an obligatory step in production of “second messenger” in the action of this hormone.
Detection of erythrocyte membrane structural abnormalities in lecithin: Cholesterol acyltransferase deficiency using a spin label approach
Authors: Maraviglia B. , Herring F. G. , Weeks G. , Godin D. V.
Published: May 2005

The membrane fluidity of erythrocytes from patients with Lecithin: cholesterol acyltransferase (LCT) deficiency was studied by means of electron spin resonance. The temperature dependence of the separation of the outer extrema of the spectra of 2‐(3‐carboxy‐propyl)‐4,4‐dimethyl, 2‐tridecyl‐3‐oxazolidinyloxyl spin probe was monitored for normal, presumed carrier and clinically affected subjects. The temperature profile of controls was significantly different form that of the presumed carriers and the clinically affected individuals. The results show that the compositional abnormalities previously noted in erythrocyte membranes from patients with LCAT deficiency are associated with alterations in the physicochemical state of the membrane. An investigation of the spectral lineshapes below 10°C allowed a distinction to be made at the membarne level between clinically affected subjects and clinically normal heterozygous carriers. Alterations in the temperature dependence of electron spin resonance parameters may provide a sensitive index of red cell membrane alterations in pathological states of generalized membrane involvement.
A high‐affinity ATP‐binding site on 30S dynein
Authors: Blum J. J. , Hayes A.
Published: May 2005

The enhancing effect of low concentrations (eg, 8 μM) of bis(4‐fluoro‐3‐nitrophenyl)sulfone (FNS) on 30S dynein ATPase activity is increased when 1 mM dithiothreitol (DTT) is present. The effect of FNS + DTT is optimal at pH 7.5. Activation of the latent ATPase activity of 30S dynein by FNS + DTT is partially prevented by 1–3 μM ATP. Adenylylimidodiphosphate (AMP‐PNP) is less effective than ATP, while β,γ‐methylene‐adenosine triphosphate (AMP‐PCP), though a much stronger inhibitor of ATPase activity than AMP‐PNP, does not protect against enhancement. These results demonstrate the presence of a high‐affinity ATP‐binding site on 30S dynein.
Threshold effects on the lectin‐mediated aggregation of synthetic glycolipid‐containing liposomes
Authors: Rando Robert R. , Bangerter Faan Wen
Published: May 2005

Cholesterol analogs containing sugar residues linked by spacer groups to the cholesterol O can be incorporated into egg yolk lecithin small unilamellar liposomes. The synthetic glycolipid analogs distribute evenly on both sides of the bilayer. These liposomes are aggregated by the appropriate lectin. For example, when the sugar residue is a β‐galactoside the liposomes are aggregated by ricin and when it is an α‐mannoside they are aggregated by Con A. The lectin‐mediated aggregation of these liposomes is reversed by the addition of the appropriate sugar. The rates but not the extents of aggregation of these liposomes are highly sensitive to the amount of glycolipid incorporated. Below approximately 5% glycolipid incorporation the rate of the lectin‐mediated aggregation of these liposomes is exceedingly slow, whereas above this level rapid aggregation proceeds. At all concentrations studied the synthetic glycolipids are incorporated in a unimodal fashion so that the observed threshold effects cannot be based on possible differences in the manner in which the glycolipids are incorporated at different concentrations. This conclusion is based on (1) studies with galactose oxidase that show that the percentage of galactose oxidation in a liposome prepared from a galactosyl‐containing glycolipid is independent of glycolipid concentration, and (2) studies on the aggregation of liposomes containing mixed glycolipids in which the glycolipids are shown to behave independently. The importance of a critical density of membrane‐bound receptors in order for aggregation to occur is discussed.