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Biomedical subjects

J W Gurd

Publications and source records attributed to J W Gurd.

At least 73 records · Page 4Linked to original sources
PubMed1980

The localization of lectin binding sites during photoreceptor synaptogenesis in the chick retina.

Carbohydrate-containing macromolecules have been localized in the outer plexiform layer of the embryonic and hatchling chick retina by horseradish peroxidase-conjugated wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA) lectins. Both WGA and RCA binding sites are present along developing photoreceptor synaptic membranes in the embryonic retina and the plasma membranes of developing neurites and glia. After photoreceptor synapse formation, RCA staining is restricted to non-synaptic membranes, but WGA staining is present on the pre- and post-synaptic membranes of receptor ribbon synapses in addition to non-synaptic membranes. These differing results between the accessibility of RCA and WGA binding sites on mature synaptic membranes in the chick retina suggests that RCA receptors on synaptic membranes are somehow masked after synapse formation and maturation, but that WGA receptors remain accessible. The effects of enzymatic digestion on WGA and RCA binding has been studied after prior treatment with neuraminidase. RCA staining of developing synaptic and non-synaptic membranes in the embryo remains the same after treatment with the enzyme, but in the hatchling RCA staining of non-synaptic membranes is enhanced, which suggests that galactosyl residues are relatively exposed on immature membranes but inaccessible to the lectin on mature membranes until neuraminidase acts to expose them by removing the terminal sialic acid residues. WGA staining on developing synaptic and non-synaptic membranes in the embryo is greatly diminished after neuraminidase pretreatment which suggests that a considerable amount of staining at this time is due to sialic acid in addition to N-acetylglucosamine. In the hatchling, photoreceptor synaptic membranes are no longer labeled with WGA and non-synaptic membrane staining is reduced after neuraminidase digestion, which suggests that after synapse formation synaptic membrane WGA labeling is primarily to sialyl residues, whereas most of the non-synaptic labeling is to N-acetylglucosamine residues.

Age Factors↗
PubMed1980

Subcellular distribution and partial characterization of the three major classes of concanavalin A receptors associated with rat brain synaptic junctions.

Synaptic junctional complexes from rat brain contain three major classes of glycoproteins which react with concanavalin A. They have apparent molecular weights of 110,000 (GP 110) 130,000 (GP 130), and 180,000 (GP 180). They are present in postsynaptic densities but are not found in microsomes, axolemma, synaptic vesicles, or myelin and are present in low concentrations in the Triton X-100 extract obtained during the preparation of synaptic junctions suggesting that they are uniquely localized to the postsynaptic apparatus. Reaction of the individual glycoproteins, partially purified by affinity chromatography on concanavalin A-agarose followed by polyacrylamide gel electrophoresis, showed that GP 130 contained the most receptor sites for concanavalin A per unit of protein followed by GP 180 and GP 110. Of the receptor sites for concanavalin A, 60-70% were subject to hydrolysis by endoglycosidase H indicating that the lectin reacts primarily with polymannose asparagine linked oligosaccharides. Each of the glycoproteins also reacted to varying degrees with the lectins from Lotus tetragonolobus (specific for alpha-L-fucose), wheat germ (N'-acetyl-D-glucosamine and (or) sialic acid), and lentils (mannose, N'-acetyl-D-glucosamine). Chromatography of 125I-labelled concanavalin A positive glycoproteins on wheat germ Sepharose resolved GP 110 and GP 180 into wheat germ positive and negative components indicating the presence of some structural heterogeneity within these molecular weight classes.

Animals↗
PubMed1980

Characterization of mononucleosomes and associated glycoproteins from Ehrlich ascites tumour cells.

Mononucleosomes generated by the digestion of ascites nuclei with micrococcal nuclease were resolved into three types by polyacrylamide gel electrophoresis. All three types were present in early and late digest and a precursor-product relationship was not apparent. Each mononucleosome type was associated with a unique pattern of subnucleosome DNA fragments and differences were apparent in histone and nonhistone proteins. On the basis of reactivity ot 125I-labelled concanavalin A and labelling with [3H]glucosamine, glycoproteins were identified as components of two of the mononucleosome types. The principal glycoprotein species associated with mononucleosomes had an apparent molecular weight of 130 000.

Animals↗
PubMed1979

Molecular and biosynthetic heterogeneity of fucosyl glycoproteins associated with rat brain synaptic functions.

The composition and biosynthesis of fucosyl glycoproteins present in rat brain synaptic membranes and synaptic junctions were investigated. Reaction with 125I-labelled fucose-binding protein (Lotus tetragonolobus) following sodium dodecyl sulphate gel electrophoresis identified 6--8 fucosyl glycoproteins in synaptic membranes but only three major high molecular classes (Mr = 180 000, 130 000 and 110 000) in synaptic junctions. Affinity chromatography on concanavalin A-Sepharose resolved each of the synaptic junctional fucosyl glycoproteins into concanavalin A-positive and negative components indicating the presence of at least six high molecular weight fucosyl glycoproteins in synaptic junctions. Following the administration of [3H]fucose synaptic membranes, synaptic junctions and post-synaptic densities incorporated isotope, the order of relative specific activities being synaptic membranes greater than synaptic junctions greater than post-synaptic densities. Fractionation of [3H]fucose-labelled synaptic junctions on concanavalin A-Sepharose revealed a time-dependent increase in the percentage of isotope associated with the concanavalin A-positive glycoproteins. The results demonstrate both molecular and biosynthetic heterogeneity of fucosyl glycoproteins associated with synaptic junctions.

Animals↗
PubMed1979

Developmental alteration of rat brain synaptic membranes. Reaction of glycoproteins with plant lectins.

Synaptic membranes were isolated from the forebrains of rats of increasing postnatal ages. Developmentally related changes in the structure and concnetration of synaptic membrane glycoproteins were indicated by: (1) a 2--3 fold increase in glycoprotein sialic acid between 5 and 60 days; (2) a similar increase in the number of membrane receptors for the lectins concanavalin A and wheat germ agglutinin; (3) transient increases between 10 and 17 days in the receptors for lentil and castor bean lectins and (4) an age dependent stimulation of castor bean lectin binding by neuraminidase. Labelling of SDS polysacrylamide gels of synaptic membranes with [125I]concanavalin A or wheat germ agglutinin revealed specific, age dependent changes in the lectin binding properties of individual molecular weight classes of glycoproteins. Differences in the glycoproteins composition of synaptic junctional complexes isolated from 10 and 28-day-old brains were also revealed by lectin binding studies. The results indicate that the number and structure of oligosaccharides associated with synaptic membrane glycoproteins are under developmental regulation.

Acetylcholinesterase↗
PubMed1977

Synaptic plasma membrane glycoproteins: molecular identification of lectin receptors.

Synaptic plasma membranes isolated from rat cerebral cortex reacted with concanavalin (con A), wheat germ agglutinin (WGA), Lens culinaris phytohaemagglutinin (LcH), and Ricinus commounus agglutinin (RCA). Competition studies indicated that specific topographical relationships exist between receptors for con A and LcH and for WGA AND LcH. Reaction of [125I]con A with synaptic membrane proteins following polyacrylamide gel electrophoresis identified eight distinct molecular weight classes of glycoproteins possessing receptor activity for con A, ranging in apparent molecular weight from 27 000 to 165 000. Each of these also reacted to varying degrees with LcH, WGA, and RCA, indicating that a diverse population of carbohydrate moieties is associated with each molecular-weight class of glycoprotein. Following gel electrophoresis, competition between lectins did not occur, suggesting that each lectin reacts with a distinct group of carbohydrates and that specific relationships between these groups are destroyed by the solubilization and electrophoretic procedure. Synaptic junctional complexes isolated by Triton-X-100 extraction of synaptic membranes exhibited a simplified glycoprotein composition with only three major molecular weight classes of glycoproteins possessing receptor activity for con A being present.

Animals↗
PubMed1976

Identification of liver plasma membrane glycoproteins which bind to 125I-labelled concanavalin A following electrophoresis in sodium dodecyl sulfate.

Following electrophoresis of ovalbumin in sodium dodecyl sulfate (SDS) this glycoprotein bound 125I-labelled concanavalin A (Con A). The reaction was specific and proportional to the amount of glycoprotein present on the gel. This technique was used to study the Con-A-binding glycoproteins of liver cell surfaces. Mouse liver plasma membranes were purified and subfractionated to yield two fractions corresponding to the bile canalicular surface and the surface between adjacent hepatocytes (Evans, W.H. (1970) Biochem. J. 116, 833-842). Both fractions bound 125I-labelled Con A, the former binding two to three times more lectin than the latter. Following SDS gel electrophoresis individual membrane glycoproteins reacted with 125I-labelled Con A. Both membrane subfractions yielded qualitatively similar Con A binding profiles, seven binding proteins being present in each. The results are consistent with a generally uniform distribution of glycoproteins over the hepatocyte surface. The reaction of lectins with glycoproteins following SDS gel electrophoresis should find general application in the study of membrane composition.

Animals↗
PubMed1973

Properties of a 5'-nucleotidase purified from mouse liver plasma membranes.

1. Extraction of a mouse liver plasma-membrane fraction with a detergent buffer, N-dodecylsarcosinate-Tris buffer (sarcosyl-Tris buffer), solubilized 90% of the protein and 70% of the 5'-nucleotidase activity. 2. The proteins of the sarcosyl-Tris buffer extract were fractionated by a rate-zonal centrifugation in a sucrose-detergent gradient. The major protein peak sedimented ahead of phospholipids, which mainly remained in the overlay. Glycoproteins were separated ahead of the protein peak. 3. The 5'-nucleotidase activity peak was associated with 5% of the protein applied to the gradient, and contained relatively few protein bands. 4. The 5'-nucleotidase was purified further by gel filtration on Sepharose and Sephadex columns equilibrated with sarcosyl-Tris buffer, to give a single glycoprotein band on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. The purified enzyme was lipid-free. 5. Electrophoresis in polyacrylamide gels in sarcosyl-Tris buffers showed that the enzymic activity was coincident with the protein band. 6. The molecular weight suggested for the enzyme activity by gel filtration or centrifugation in sucrose gradients was 140000-150000. Sometimes, a minor enzyme peak of lower molecular weight was obtained. 7. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate indicated that as the polyacrylamide concentration was increased from 5 to 15%, the apparent molecular weight of the enzyme decreased from 130000 to 90000. 8. The evidence that 5'-nucleotidase is composed of two active and similar, if not identical, glycoprotein subunits and the role of detergent in effecting the separation of membrane proteins and glycoproteins are discussed. 9. Substrate requirements, pH optima and the nature of inhibition by an analogue of adenosine diphosphate are reported.

Animals↗