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J W Gurd

Publications and source records attributed to J W Gurd.

At least 55 records · Page 3Linked to original sources
PubMed1987

Characterization of gp 50, a major glycoprotein present in rat brain synaptic membranes, with a monoclonal antibody.

Several cell lines secreting monoclonal antibodies (Mabs) against a major forebrain synaptic membrane (SM) glycoprotein, gp 50, have been raised. Western blots show that the Mabs react with a polypeptide doublet of Mrs 49 and 45 kDa. These polypeptides exist solely in a concanavalin A (Con A) binding form. Removal of the Con A receptors by digestion with endo-beta-N-acetylglucosaminidase H (endo H) lowers the Mrs of the glycoprotein doublet to 36.5 and 34 kDa. Western blots of 2D polyacrylamide gels indicate that gp 50 exists in several isoforms. Solid phase radioimmunoassay (RIA) and Western blots of brain subcellular fractions show the antigenic material to be concentrated in the SM fraction, but to be present in much lower amounts in synaptic junctions and postsynaptic densities. Gp 50 appears to be brain specific. Regional distribution studies show that it is present in all brain regions but is two-fold concentrated in cerebellum, brainstem and midbrain compared to forebrain. Immunocytochemical studies of several brain regions show that gp 50-like immunoreactivity is neuron specific and is concentrated in selected neuronal species, particularly granule cells. In both cerebellar and hippocampal granule cells gp 50-like immunoreactivity is localized in the perikarya and primary dendrites. Though immunocytochemistry did not show staining of synaptic regions this may be due to masking of the reactive epitope. The results are discussed in terms of the molecular properties of gp 50 and its subcellular localization in brain tissue.

Animals↗
PubMed1987

Characterization of fucosyl oligosaccharides associated with synaptic membrane and synaptic junctional glycoproteins.

Rats were administered [3H]fucose by intracranial injection and synaptic membranes (SMs) isolated 18 h later. Oligosaccharides associated with SM glycoproteins were prepared by hydrazinolysis and analyzed by a combination of affinity chromatography on concanavalin A (Con A)-agarose, ion-exchange chromatography on DEAE-cellulose, and gel permeation chromatography. Most (94%) of the [3H]fucose-labelled oligosaccharides were present in the fraction that did not bind to Con A. Of these 41% did not bind to DEAE-cellulose, indicating the absence of negatively charged groups and the remainder were resolved into four fractions of increasing acidity. Gel permeation chromatography of the fractions from the DEAE-cellulose column suggested that the major oligosaccharides corresponded to fucosylated triantennary structures containing varying amounts of sialic acid although more highly branched structures containing peripheral branches lacking one or more sugars may also have been present. Comparison of fucosyl oligosaccharides associated with SMs prepared from 10- and 28-day-old animals indicated that although the general oligosaccharide content was similar at both ages, membranes from younger animals were characterized by an increase in the proportion of highly acidic structures. Fucosylated glycans derived from synaptic junctional (SJ) glycoproteins were also characterized by a greater percentage of highly acidic structures than SMs. The results indicate that SMs and SJs are characterized by specific complements of fucosylated glycoprotein oligosaccharides.

Animals↗
PubMed1986

Decreased phosphorylation of synaptic glycoproteins following hippocampal kindling.

Rats with chronic electrode implants to region CA3 of the hippocampus were rapidly kindled by stimulation with a 10 s, 10 Hz train of biphasic square waves presented every 5 min, until generalized seizures developed (60-70 stimulations). The hippocampi were isolated from the brains of control animals (implanted but not stimulated), and experimental animals which had developed generalized seizures. Synaptic membranes (SM) were prepared. SM were incubated with [gamma-32P]ATP and the incorporation of 32P into proteins and glycoproteins isolated by affinity chromatography on concanavalin-A-agarose was investigated. There was no difference in the phosphorylation pattern of total SM proteins between groups. In contrast, the phosphorylation of a glycoprotein with an apparent molecular weight of 180,000 was decreased 20-40% in kindled animals. This result was replicated in three independent experiments. The results suggest that the phosphorylation of glycoprotein 180 may be related to neuroplastic events.

Animals↗
PubMed1986

Phosphorylation of synaptic membrane glycoproteins: the effects of Ca2+ and calmodulin.

Synaptic membranes were incubated with [gamma-32P]ATP, and glycoproteins were isolated by affinity chromatography on concanavalin A agarose. Glycoproteins accounted for 1.5-2.5% of the total 32P incorporated into synaptic membrane proteins. Ca2+ and calmodulin enhanced the phosphorylation of synaptic membrane glycoproteins approximately threefold. In the presence of Ca2+ and calmodulin, the rate of glycoprotein dephosphorylation was also increased three- to four-fold. Gel electrophoretic analysis identified several synaptic membrane glycoproteins that incorporated 32P, with the most highly labeled glycoprotein under basal phosphorylating conditions having an apparent Mr of 205,000 (gpiii). Ca2+ and calmodulin produced a marked increase in the phosphorylation of a glycoprotein with an apparent Mr of 180,000 (gpiv) and lesser increases in the labeling of three other glycoproteins. Membranes that had been labeled with [gamma-32P]ATP were extracted with Triton X-100 under conditions that yield a detergent-insoluble residue enriched in postsynaptic structures. The Triton X-100 insoluble residue accounted for 20-25% of the 32P associated with synaptic membrane glycoproteins. Gpiv and other glycoproteins, the phosphorylation of which was stimulated by calmodulin, were located exclusively in the Triton X-100 insoluble residue, whereas gpiii and other calmodulin-insensitive glycoproteins partitioned predominantly into the Triton X-100-soluble fraction. Phosphopeptide maps and phosphoamino acid analysis of gpiv isolated from synaptic membranes and a postsynaptic glycoprotein of apparent Mr of 180,000 (gp180) isolated from synaptic junctions indicated that the former protein was identical to the previously identified postsynaptic-specific gp180. In addition to phosphoserine and phosphothreonine, gpiv also contained phosphotyrosine, identifying it as a substrate for tyrosine-protein kinase as well as for Ca2+/calmodulin-dependent protein kinase.

Adenosine Triphosphate↗
PubMed1985

Phosphorylation of the postsynaptic density glycoprotein gp180 by endogenous tyrosine kinase.

Incubation of postsynaptic densities (PSDs) with [gamma-32P]adenosine triphosphate (ATP) results in the phosphorylation of a number of proteins. Of these, phosphoproteins with apparent molecular weights (Mr) of 51,000, 180,000, 300,000, 320,000 and 370,000 contain 32P which is resistant to digestion with hot KOH suggesting the presence of [32P]phosphotyrosine residues. Phosphoamino acid analysis of total 32P-labelled PSDs identified [32P]phosphotyrosine as well as phosphoserine and phosphothreonine as products of the phosphorylation reaction. The PSD-specific glycoprotein gp180 was isolated from 32P-labelled PSDs and shown to contain [32P]phosphotyrosine. The results identify tyrosine kinase as a component of purified PSDs and gp180 as an endogenous substrate for this enzyme.

Animals↗
PubMed1985

Phosphorylation of the postsynaptic density glycoprotein gp180 by Ca2+/calmodulin-dependent protein kinase.

Postsynaptic densities (PSDs) were prepared by the aqueous two-phase extraction of synaptic membranes in the presence of n-octyl glucoside. Incubation of postsynaptic densities with [gamma-32P]ATP resulted in the incorporation of 32P into a range of proteins. Isolation of glycoproteins from 32P-labelled PSDs by affinity chromatography on concanavalin A-agarose identified the postsynaptic glycoprotein of apparent Mr 180,000 (gp180) as a substrate for endogenous protein kinase(s). When the phosphorylation reaction was performed in the presence of Ca2+ and calmodulin, there was an overall 13-fold increase in the phosphorylation of PSD proteins. The largest effects of calmodulin were associated with two proteins of molecular weights 51,000 and 60,000, which showed average calmodulin-dependent increases in phosphorylation of 68-fold. The phosphorylation of gp180 was increased 7.5-fold in the presence of calmodulin. Fifty percent of maximum phosphorylation of proteins and glycoproteins occurred with a free Ca2+ concentration of 0.3 X 10(-6) M. The amounts 12.6 micrograms/ml and 9.1 micrograms/ml of calmodulin were required for 50% of maximum phosphorylation of proteins and glycoproteins, respectively. Peptide mapping experiments identified three major phosphorylation sites in gp180. The phosphorylation of all three sites was increased in the presence of calmodulin. Phosphoamino acid analysis of gp180 revealed that [32P]phosphoserine and [32P]phosphothreonine were both produced during the phosphorylation reaction, with phosphoserine being the predominant product. The phosphorylation of both amino acids was increased in the presence of calmodulin. [32P]phosphotyrosine was also identified as a product of the phosphorylation of gp180.

Animals↗
PubMed1985

In vivo phosphorylation of the postsynaptic density glycoprotein gp180.

Rats received intraventricular injections of [32P]PO4 and were killed after 30 min for the preparation of postsynaptic densities (PSDs). Gel electrophoretic analysis identified a number of PSD proteins that incorporated 32P under these conditions. Major proteins that were labelled with 32P had Mr of 185,000, 165,000, 140,000, 92,000, and 51,000. Of these p185, p165, and p140 were also labelled when PSDs were incubated with [gamma-32P]ATP in vitro. In contrast p92 and p51 were relatively poorly labelled under in vitro conditions. Analysis of glycoproteins isolated by chromatography on concanavalin A (Con A)-agarose demonstrated that greater than 70-80% of the 32P present in the glycoproteins eluted from Con A-agarose with alpha-methyl-D-mannopyranoside (Con A+ glycoproteins) was associated with the PSD specific glycoprotein gp180 following both in vivo and in vitro labelling. Phosphopeptide maps and phosphoamino acid analysis of gp180 indicated that similar sites were labelled in vitro and in vivo. Analysis of the subcellular distribution of glycoproteins that incorporated 32P during in vivo labelling demonstrated that gp180 was highly concentrated in PSDs, in accord with the previously suggested exclusive association of this glycoprotein with postsynaptic structures.

Animals↗
PubMed1983

Identification and localization of concanavalin A binding sites on isolated postsynaptic densities.

Synaptic fractions of decreasing morphological complexity were prepared by phase partitioning of synaptic membranes in an aqueous two-phase polymer system containing increasing concentrations of the neutral detergent n-octylglucoside (OG). The morphology, distribution of concanavalin A binding sites (CABS) and protein and glycoprotein composition of the resultant fractions were examined. The lowest concentration of OG employed (0.5% w/w) gave fractions enriched in relatively intact junctions retaining both pre- and postsynaptic structures. Increasing the detergent concentration resulted in the stepwise solubilization of pre- and postsynaptic structures until purified postsynaptic densities (PSDs) were obtained with 1% (w/w) OG. CABS were generally distributed on all membrane structures present in the 0.5% OG fraction, were restricted to synaptic structures in the fraction obtained with 0.75% OG, and were localized to the convex (outer) surface of purified PSDs. Gel electrophoretic analysis showed that the restriction of CABS to the region of the synapse was associated with a marked increase in the concentration of glycoproteins with apparent molecular weights of 180,000 and 130,000. These glycoproteins were retained, and further concentrated in the purified PSD fraction.

Animals↗
PubMed1983

Synaptic junctional glycoproteins are phosphorylated by cyclic-AMP-dependent protein kinase.

Synaptic junctions (SJs) isolated from rat brain are associated with protein kinase activity and a unique complement of high molecular weight gglycoproteins. Incubation of SJs with [gamma-32P]A+ glycoproteins which were retained by concanavalin A agarose (con A+ glycoproteins). Three major (apparent mol. wt. 180 K, 130 K and 110 K) and 2 minor (apparent mol. wt. 230 K and 145 K) glycoproteins were identified in the con A+ fraction. Of these, GP180 incorporated the most 32P and GP145 was not labeled. Peptide mapping experiments showed that each molecular weight class of glycoprotein was associated with a unique set of phosphorylated peptides. Cyclic AMP stimulated the incorporation of 32P into total SJ proteins and con A+ lycoproteins by 38% and 58%, respectively. GP130 showed the greatest increase in labelling in the presence of cyclic AMP (198% of control levels) although incorporation into all 4 glycoproteins was increased. Cyclic AMP selectively stimulated the incorporation of 32P into only 2 of the 6 phosphorylated peptides derived from GP130. These studies demonstrate that endogenous glycoproteins serve as substrates for intrinsic SJ protein kinases and identify this reaction as a potential means of modifying postsynaptic membrane function.

Animals↗
PubMed1983

Developmental changes in the oligosaccharide composition of synaptic junctional glycoproteins.

Synaptic junctions (SJs) were isolated from the forebrains of rats ranging in postnatal age from 10 days to greater than 1 year. SJ glycoproteins that react with Concanavalin A (Con A) were isolated by chromatography on Con A-agarose and separated by gel electrophoresis. The concentrations of the major SJ Con A binding (Con A+) glycoproteins (apparent Mr 180,000, 130,000, and 110,000) increased between 10 and 28 days, with GP180 and GP110 showing greater relative increases than GP130. Con A binding oligosaccharides associated with 10-day SJs were sensitive to digestion with endoglycosidase C11 and alpha-mannosidase, indicating that they were of the high-mannose type, as previously shown for 28-day SJs. Con A+ oligosaccharides from rats of increasing postnatal age were analyzed by chromatography on Biogel P-4. Two major oligosaccharides, containing five and eight mannose residues, were present in SJs of all ages examined. During development the ratio of man5 to man8 oligosaccharides increased, so that man5 constituted the predominant species in 28-day and adult SJs. Peptide mapping experiments showed that GP180, GP130, and GP110 were each associated with a unique polypeptide composition. Little or no change in peptide composition of the major SJ glycoproteins occurred during development.

Age Factors↗
PubMed1983

Identification of intrinsic sialidase and sialoglycoprotein substrates in rat brain synaptic junctions.

Sialidase activity associated with rat brain synaptic junctions (SJ) and synaptic membranes (SM) was determined. Both fractions released sialic acid from exogenous glycopeptides and gangliosides. SJ accounted for 5-10% of the total sialidase activity recovered from SM following extraction with Triton X-100, and the specific activity of SJ sialidase was 60% of that of the parent SM fraction. Intrinsic SJ sialidase hydrolysed 12-15% of the sialic acid associated with endogenous SJ glycoproteins. Sialic acid residues associated with SJ glycoproteins were labelled with sodium borotritide and SJ proteins fractionated by affinity chromatography on concanavalin A-agarose. SJ glycoproteins that reacted with concanavalin A (con A+ glycoproteins) accounted for 25% of the total SJ [3H]sialic acid. Intrinsic SJ sialidase hydrolysed 20% of the [3H]sialic acid associated with these glycoproteins. Each molecular weight class of con A+ glycoprotein previously shown to be a specific component of the postsynaptic apparatus contained sialic acid and was acted on by intrinsic SJ sialidase.

Animals↗
PubMed1982

Concanavalin A receptors associated with rat brain synaptic junctions are high mannose-type oligosaccharides.

Glycoproteins were isolated from a rat brain synaptic junction fraction by affinity chromatography on Concanavalin A-agarose. The isolated glycoproteins were digested with pronase and radiolabeled with 125I-Bolton Hunter reagent, and 125I-Concanavalin A-binding glycopeptides were isolated by chromatography on Concanavalin A-agarose. Treatment of the 125I-Concanavalin A-binding glycopeptides with either alpha-mannosidase or endo-beta-N-acetylglucosaminidase-C11 abolished their interaction with Concanavalin A. The pronase digest was reacted with endo-beta-N-acetylglucosaminidase-C11 and released oligosaccharides were reduced with NaB3H4. Following affinity chromatography on Concanavalin A-agarose, Concanavalin A-binding [3H]oligosaccharides were chromatographed on Biogel P4. Two major oligosaccharides corresponding to standard carbohydrates containing eight and five mannose residues were identified. Treatment of these oligosaccharides with alpha-mannosidase converted them to smaller saccharides having a mobility on Biogel P4 columns equal to the standard disaccharide mannose-beta-1-4-N'-acetylglucosamine. These results demonstrate that the Concanavalin A receptor activity associated with CNS synaptic junctions resides in asparagine-linked oligosaccharides of the high-mannose type.

Animals↗
PubMed1982

Biochemical and morphological comparison of postsynaptic densities prepared from rat, hamster, and monkey brains by phase partitioning.

A new procedure for the preparation of postsynaptic densities (PSDs) is described. A synaptic membrane fraction was homogenized in an aqueous two-phase polymer system containing Poly(ethylene glycol) 6000 (5% wt/wt) and Dextran T500 (6% wt/wt) containing 1% 1-o-n-octyl-beta-D-glucoside. Following a brief centrifugation to separate the phases, highly purified PSDs banded at the interface of the two phases. Using this procedure PSDs have been isolated from rat and hamster cerebral cortex and from the frontal cortex, cerebral cortex, hippocampus, and pooled caudate/putamen regions of Macaca mulatta Rhesus monkeys. The isolated PSDs appeared as curved bars when sectioned or as discs when viewed en face in the electron microscope. The hamster PSDs were associated with large numbers of small rod-like structures 4.5 nm thick and 28 nm long. Similar structures were present, although in fewer numbers, in the rat and monkey preparations. Polyacrylamide gel electrophoresis showed that the PSDs contained a complex population of proteins with major components having molecular weights of 180,000, 130,000, 110,000, 94,000, 65,000, 60,000, and 51,000. Reaction of polyacrylamide gels with 125I-concanavalin A (Con A) identified two major (apparent Mr 180,000 and 130,000) and three minor (apparent Mr 230,000, 145,000, and 110,000) Con A-binding glycoproteins in the PSD fractions. Although some quantitative variation between species and brain regions was apparent, the overall protein and glycoprotein composition was similar for all fractions studied.

Animals↗
PubMed1981

Selective effects of LSD and hyperthermia on the synthesis of synaptic proteins and glycoproteins.

Protein synthesis in rabbit brain was inhibited following the intravenous injection of LSD. The incorporation of [35S]methionine into brain microsomal and synaptic fractions was decreased by 35-45% relative to control values. A selective increase was observed, however, in the relative labeling of a protein of molecular weight 75,000. Our previous studies have shown that LSD induces an increase in body temperature (i.e. hyperthermia) in rabbits. When LSD-induced hyperthermia was blocked the general reduction in labeling of microsomal and synaptic proteins was still apparent but the selective increase in relative labeling of the 75,000 dalton protein was not. Induction of hyperthermia by means other than LSD (i.e. elevation of ambient temperature) produced selective increases in the relative labeling of microsomal and synaptic proteins of molecular weight 75,000 and 95,000. These proteins are similar in molecular weight of two of the major 'heat shock' proteins whose synthesis is induced in several cultured cell lines following elevation of ambient temperature. Fractionation of [35S]methionine-labeled synaptic membranes by lectin affinity chromatography and analysis of [3H]fucose labeling patterns indicated that, in contrast to the general reduction in labeling of brain proteins, the synthesis of synaptic glycoproteins was not altered by LSD. The synthesis of glycosylated proteins present in other subcellular fractions was, however, reduced. These results suggest that LSD induced selective changes in the synthesis of brain proteins and that the synthesis of synaptic glycoproteins may be relatively resistant to drug administration.

Animals↗
PubMed1981

Lectin cytochemistry of carbohydrates on cell membranes of rat cerebellum.

Wheat germ agglutinin (WGA), Ricinis communis agglutinin (RCA) and Lens culinaris (LC) lectins have been used to characterize carbohydrates of neuronal membrane systems in rat and chick cerebellum. WGA and RCA both label Golgi membrane cisternae on the side of the membrane facing the cisternal space but they do not label other elements of the granular and agranular endoplasmic reticulum (ER). WGA labels the plasma membrane generally and WGA binding sites are concentrated within the synaptic cleft and at specialized sites along the axolemma at the node of Ranvier. RCA labelling of plasma membranes is sparse. Neuraminidase treatment of tissue slices prior to lectin cytochemistry does not alter the Golgi membrane distribution of WGA and RCA binding sites and other ER membranes remain unlabelled. The plasma membrane staining with WGA is decreased and that with RCA is increased after neuraminidase pretreatment. The pattern of lectin labelling with LC resembles that seen with concanavalin A (con A) in that all elements of the ER within cell bodies label on the side of the membrane facing the cisternal space. A major difference when compared to con A labelling is that the hypolemmal cisternae lying adjacent to the plasma membrane of Purkinje cell axons and dendrites do not label with LC. Collectively, these results suggest that elements of the ER of Purkinje cells are heterogeneous with respect to their lectin binding properties in a manner which is consistent with the formation of more complex oligosaccharides on membranes of the cell periphery.

Animals↗
PubMed1981

The effects of development on activity, specificity and endogenous substrates of synaptic membrane sialidase.

Synaptic plasma membranes were prepared from cortices of rats varying in post-natal age between 4 and 30 days. Sialic acid associated with synaptic plasma membrane glycoproteins and gangliosides increased 75% and 50%, respectively, between 4 and 30 days. The amount of sialic acid released from these membrane constituents by intrinsic synaptic sialidase increased 2-4-fold over the same period. Incubation of synaptic plasma membranes with exogenous gangliosides or glycopeptides demonstrated a 2-3-fold increase in sialidase activity during development. The major gangliosides present in synaptic plasma membranes at all ages were GT1, GD1a, GD1b and GM1. Intrinsic sialidase hydrolyzed 50-70% of endogenous GT1 and GD1a gangliosides at all ages. Endogenous GD1b ganglioside was poorly hydrolyzed in young rats and its susceptibility to enzymic hydrolysis increased during development. When exogenous GD1a and GD1b were used as substrates a preferential increase in activity against GD1b occurred during development, the ratio of activity (GD1a/GD1b) decreasing from 3.6 to 1.6 between 7 and 30 days. 10- and 30-day-old synaptic plasma membranes contained complex mixtures of sialoglycoproteins, an increase in the relative concentrations of lower molecular weight sialoglycoproteins occurring during development. Intrinsic sialidase present in 10- and 30-day-old synaptic plasma membranes acted upon all molecular weight classes of sialoglycoproteins.

Animals↗
PubMed1981

Development of synaptic glycoproteins: effect of postnatal age on the synthesis and concentration of synaptic membrane and synaptic junctional fucosyl and sialyl glycoproteins.

Synaptic plasma membranes (SPM) and synaptic junctions (SJ) were isolated from the cortices of rats varying in age between 5 and 28 days. Gel electrophoresis of SPM and SJ indicated a marked increase in the concentration of the "PSD protein" (M. W. 52,000) with development. The biosynthesis of glycoproteins was measured following the intracranial injection of [3H]fucose or [3H]N'-acetylmannosamine. The incorporation of [3H]fucose into synaptic fractions decreased two- to threefold between 10 and 28 days whereas little change in the incorporation of [3H]N'-acetylmannosamine occurred over the same period. Gel electrophoretic analyses of labeled synaptic membranes indicated major increases in the relative incorporation of radiolabeled precursors into glycoproteins with apparent molecular weights of 74,000, 65,000, 50,000, and 40,000 with increasing age. Identification of fucosyl and sialyl glycoproteins following reaction with 125I-fucose-binding protein or labeling of sialic acid with NaIO4/NaB[3H4] demonstrated similar increases in the concentrations of these glycoproteins. Synaptic junctions contained three major glycoproteins with apparent molecular weights of 180,000, 130,000 and 110,000. The reaction of these glycoproteins with 125I-fucose-binding protein increased one- to twofold between 10 and 28 days but little variation in their relative distribution or synthesis occurred over this period. The reaction of synaptic junctional glycoproteins GP 180 and GP 110 with 125I-wheat germ agglutinin increased between 10 and 28 days. The results indicate that the molecular composition of the synapse continues to evolve after the initial synaptic contact has been formed.

Aging↗