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R B Marchase

Publications and source records attributed to R B Marchase.

16 recordsLinked to original sources
PubMed1992

Identification of a membrane protein from T84 cells using antibodies made against a DIDS-binding peptide.

The outwardly rectified chloride channel of secretory epithelial cells is inhibited by disulfonic stilbene (DS) compounds such as 4,4'-diisothiostilbene-2,2'-disulfonic acid (DIDS) [R. J. Bridges, R. T. Worrell, R. A. Frizzell, and D. J. Benos, Am. J. Physiol. 256 (Cell Physiol. 25): C902-C912, 1989]. A 13-amino acid peptide (P49) corresponding to the putative DS binding site region of the murine anion exchange protein was synthesized, and polyclonal antibodies were generated against it and then purified over a P49 affinity column. The resulting monospecific antibodies reacted on Western blots with a 95- to 100-kDa protein from human erythrocytes and a 55- to 60-kDa protein from the human colonic tumor cell line, T84. The reaction with T84 protein did not appear to represent recognition of an anion exchanger because anion efflux from T84 cells was independent of external Cl-. In addition, monoclonal antibodies raised against human band 3 recognized the band 3 protein in human red cell ghost preparations but recognized nothing in T84 cell membrane preparations. In T84 cells, DIDS protected the 60-kDa protein from antibody binding. The anti-P49 antibody blocked outwardly rectified Cl- channels incorporated into planar lipid bilayer membranes from rat colon. Immunocytochemical data reveal specific binding of the anti-P49 antibody to perinuclear cytoplasmic vesicles. Forskolin caused these antibody-labeled vesicles to migrate from the perinuclear region to the plasma membrane under conditions and with a time course identical to that seen for stimulation of Cl- transport in these cells. Our results suggest that the protein may be a part of a chloride channel complex of secretory epithelial cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Gangliosides as modulators of cell function.

Gangliosides, sialic acid-containing glycosphingolipids, are found in the outer leaflet of the plasma membrane of all vertebrate tissues and species. This report presents a brief introduction to the gangliosides and reviews the chemistry and topography of their biosynthesis. It also presents an overview of the present evidence supporting a physiological significance for the gangliosides in a variety of experimental systems. This includes consideration of their potential roles in development and cell adhesion. In addition, experimental examples in which gangliosides appear to influence signal transduction processes through their interactions with plasma membrane proteins are discussed.

Animals↗
PubMed1991

beta-Phosphorothioate analogs of nucleotide sugars are resistant to hydrolytic degradation and utilized efficiently by glycosyltransferases.

The alpha- and beta-phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a non-bridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyltransferase reactions. The alpha analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the beta S analogs were approximately 10 times more resistant. The beta S analog and native UDP-Glc were found to have comparable Km's when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent Vmax of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4 beta-galactosyltransferase exhibited a Vmax with (beta S)UDP-Gal that was only slightly lower than that with UDP-Gal and a Km that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4 beta-galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the beta S analog in each system.

Animals↗
PubMed1991

Receptor synthesis and routing to the plasma membrane.

Hormones and other extracellular proteins exert their effects on the cells that they influence by interacting with receptors in the plasma membranes of these target cells. For these interactions to occur, the receptor proteins must be efficiently synthesized, processed, and transported to the plasma membrane. This review summarizes the events and organelles involved in this process.

Animals↗
PubMed1990

Resolution of phosphoglucomatase and the 62-kDA acceptor for the glucosylphosphotransferase.

The radioactive, photoactivatable labeling probe [beta-32P]5-azidouridine 5'-diphosphoglucose has recently been shown to label a 62-kDa protein in crude homogenates and in partially purified enzyme preparations without photoactivation. Here, we report that a portion of this radioactivity is due to labeling of phosphoglucomutase by contaminating levels of [32P]alpha Glc-1-P initially present at less than 1% of the total 32P. This conclusion is based in part on the ability of excess unlabeled alpha Glc-1-P and Glc-6-P, but not UDP-Glc, to block the labeling. In addition, the labeled protein in liver homogenates had a tryptic peptide pattern similar to that of authentic phosphoglucomutase. These findings, however, raised a second question. Assays for the UDP-Glc: glycoprotein glucosyl phosphotransferase (Glc phosphotransferase) have utilized [beta-32P]UDP-Glc and have resulted in the labeling of a small number of acceptors, including one of approximately 62 kDa. Despite the fact that these assays had routinely been performed in the presence of 1 mM alpha Glc-1-P, the coincidence in molecular weights led to these further studies. We conclude that the acceptor of approximately 62 kDa is distinct from phosphoglucomutase. This conclusion is based on differences in the time courses of incorporation, the specificity of blocking agents, the presence of covalently linked glucose, the products of acid hydrolysis and of beta-elimination, and isoelectric points.

Affinity Labels↗
PubMed1990

Parafusin, an exocytic-sensitive phosphoprotein, is the primary acceptor for the glucosylphosphotransferase in Paramecium tetraurelia and rat liver.

Parafusin, the major protein in Paramecium tetraurelia to undergo dephosphorylation in response to secretory stimuli, appears to be the primary acceptor for the glucosylphosphotransferase in this species based on five independent criteria: identical molecular size of 63 kD; identical isoelectric points in the phosphorylated state of pH 5.8 and 6.2; identical behavior in reverse-phase chromatography; immunological cross-reactivity with an affinity-purified anti-parafusin antibody; the presence of a phosphorylated sugar after acid hydrolysis. It appears likely that the dephosphorylation observed with secretion reflects the removal of alpha Glc-1-P from parafusin's oligosaccharides and is consistent, therefore, with a regulatory role for this cytoplasmic glycosylation event. The glucosylphosphotransferase acceptor in rat liver is also immunoprecipitated by the anti-parafusin antibody and is very similar in physical characteristics to the paramecium protein. This conservation suggests a role for parafusin in mammalian exocytosis as well, at a step common to both the regulated and constitutive secretory pathways.

Animals↗
PubMed1990

Relative accessibility of N-acetylglucosamine in trimers of the adenovirus types 2 and 5 fiber proteins.

Fiber is an adenovirus capsid protein responsible for virus attachment to the cell surface and contains O-linked N-acetylglucosamine (GlcNAc). Results of both amino acid analysis and Dionex chromatography indicated that 3 to 4 and 1.7 to 2.5 mol of GlcNAc are attached per mol of affinity-purified adenovirus type 2 (Ad2) and Ad5 fibers, respectively. Fiber shares an epitope with nuclear pore proteins containing O-linked GlcNAc, as shown by reactivity to monoclonal antibody RL2 directed against these pore proteins. GlcNAc on fiber was found to serve as an acceptor for the transfer of galactose from UDP-galactose by 4 beta-galactosyl-transferase in Ad2 and Ad5 but not in Ad7; quantitation by labeling with UDP-[U-14C]galactose in this reaction gave a 100-fold-lower estimate of the GlcNAc content of fiber, suggesting that these monosaccharides are buried within fiber trimers and are not accessible to the transferase. Affinity chromatography on lectin-bound Sepharose beads showed that Ad2 and Ad5 fibers bound weakly to wheat germ agglutinin and did not bind to ricin or concanavalin A; weak binding to wheat germ agglutinin suggests either that GlcNAc is not easily accessible or that there are not sufficient GlcNAcs for efficient binding. These data suggest that O-linked GlcNAc might be important for Ad2 and Ad5 fiber assembly or stabilization.

Acetylglucosamine↗
PubMed1979

Ligatin from embryonic chick neural retina.

Ligatin, a filamentous protein previously found in suckling rat ileum, has been purified from plasma membranes of embryonic chick neural retina. The isolated plasma membranes are covered in part by 4.5-nm filaments that can be released from the membranes by treatment with Ca++. Subsequent dialysis against EGTA followed by sieve chromatography results in purification of the 10,000-dalton ligatin monomer. When labeled either with radioisotopes or with fluorescamine, the monomer is shown to electrophorese as a single discrete band in polyacrylamide gels. However, during standard fixing and staining procedures it diffuses from the gels and thus is not visualized. Ligatin's amino acid composition is distinguished by its high content of polar residues, especially Glx and Asx, and by the presence of phosphorylated serine. Upon re-addition of Ca++, purified ligatin monomers polymerize to form filaments 3 nm in Diam, identical to those formed by purified ileal ligatin. However, in both retina and ileum, the filaments observed on plasma membranes are greater than 3 nm in Diam. In ileum, this enlargement results from ligatin's function as a baseplate for the attachment of another protein, a beta-N-acetylhexosaminidase, to the cell surface. In retina, a corresponding difference in diameter between filaments seen in vivo and those formed from repolymerized ligatin alone and the co-solubilization of other proteins with ligatin suggest that ligatin may also function there as a baseplate for other cell surface proteins. The proteins associated with ligatin in retina differ morphologically from beta-N-acetylhexosaminidase and do not possess this enzymatic activity.

Amino Acids↗
PubMed1978

Properties of a double gradient model for retinotectal specificity.

The properties of a double gradient model for retinotectal specificity are discussed. The model utilizes only two complementary molecules, each located on both retina and tectum, to determine position along the dorsoventral axis. Two possible modes of interaction between these molecules are assumed. One of these allows all possible bonds between a retinal cell and tectal loci to be formed. This results in a rigid retinotectal projection in which the adhesion of each retinal cell to its normal tectal locus is maximal. The other assumes stochastic interactions between the molecules, and results in retinotectal specificity only if additional constraints are imposed on the system. Either of these modes of interaction predicts adhesive preferences for retinal cells to tectal halves similar to those observed experimentally.

Animals↗
PubMed1977

Biochemical investigations of retinotectal adhesive specificity.

The preferential adhesion of chick neural retina cells to surfaces of intact optic tecta has been investigated biochemically. The study uses a collection assay in which single cells from either dorsal or ventral halves of neural retain adhere preferentially to ventral or dorsal halves of optic tecta respectively. The data presented support the following conclusions: (a) The adhesion of ventral retina to dorsal tecta seems to depend on proteins located on ventral retina and on terminal beta-N-acetylgalactosamine residues on dorsal tecta. (b) The adhesion of dorsal retina to ventral tecta seems to depend on proteins located on ventral tecta and on terminal beta- N-acetylgalactosamine residues on dorsal retina. (c) A double gradient model for retinotectal adhesion along the dorsoventral axis is consistent with the data presented. The model utilizes only two complementary molecules. The molecule suggested to be concentrated dorsally in both retina and tectum seems to require terminal beta-N-acetylgalactosamine residues for adhesion. Its activity is not affected by protease. A molecule fitting these qualifications, the ganglioside GM(2), could not be detected in a gradient, but lecithin vesicles containing GM(2) adhered preferentially to ventral tectal surfaces. The second molecule, concentrated ventrally in both retina and tectum, is a protein and seems capable of binding terminal beta-N- acetylgalactosamine residues. One enzyme, UDP-galactose:GM(2) galactosyltransferase, has been found to be more concentrated in ventral retina than dorsal, but only by 30 percent.

Acetylgalactosamine↗
PubMed1977

Biochemical investigations of retinotectal specificity.

An in vitro assay for retinotectal specificity has been described. The results show that, in the chick embryo, cells dissociated from the dorsal retina preferentially adhered to ventral tectal surfaces while cells from the ventral retina preferentially adhered to dorsal tectal surfaces. These adhesive preferences thus mimic the retinotectal specificity observed in vivo. The assay has been extended for use with plasma membrane preparations from retinal cells. Experiments in which retinal cells or tectal surfaces were treated with purified proteases and glycosidases have partially characterized the moieties responsible for the observed specificities. These results are consistent with a double gradient in the dorsal-ventral axis of complementary proteins and carbohydrates. The carbohydrate moiety would be expected to terminate in an acetylated hexosamine and to be more concentrated dorsally in both retina and tectum. A protein that is complementary to the hexosamine terminus would be localized in the ventral part of retina and tectum.

Animals↗
PubMed1975

A molecular approach to retinotectal specificity.

An assay is described to examine the hypothesis that retinal neurons adhere preferentially to that part of the optic tectum near theri normal synaptic termini. The method measures the adherence of isotopically labelled cell bodies from either the dorsal or ventral half of the neural retina of chick embryos to dorsal and ventral tectum halves. When a labelled cell suspension is prepared from a dorsal half-retina, more cells adhere to the ventral half of the tectum. When the cells are from the ventral part of the retina, more bind to the dorsal half of the tectum. This preferential adhesion mimics the retinotectal projection found in vivo and supports an interpretation of neuronal specificity dependent on cell surface adhesive properties. Molecular mokels are presented that utilize glycosyltransferases and their substrates as the basis for adhesive recognition. Two of these models suggest that quantitative changes in the distribution of transferases and their substrates determine retinotectal specificity. The third proposes qualitative variations in these molecules across the retina and tectum.

Animals↗