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.     

Evidence that a Cerebellum-Enriched, Synaptic Junction Glycoprotein Is Related to Fodrin and Resists Extraction with Triton in a Calcium-Dependent Manner

Abstract: Subcellular fractions from rat cerebellum and other tissues were examined for the presence of a 240K glycoprotein, designated GP-A. Previous results have shown that GP-A is enriched in cerebellum synaptic junction (SJ) fractions when compared to parent synaptic plasma membrane (SPM) fractions and is not detected in forebrain SPM or SJ fractions. In the present studies, GP-A was not detected in myelin, mitochondria, purified nuclei, or cytosolic fractions from cerebellum, but was present in microsomal fractions. GP-A is partially soluble in the non-ionic detergent Triton X-100 and is completely soluble when cerebellum SPMs are treated with the ionic detergent N-lauryl sarcosinate. The solubilization of GP-A from cerebellum membranes was shown to be a function of bound calcium ions, e.g., pretreating SPMs with 100 μM-1mM Ca²⁺ decreased the solubility of GP-A in Triton by approximately threefold. GP-A is a major concanavalin A (Con A)-binding glycoprotein in cerebellum SJ fractions and migrates on sodium dodecyl sulfate (SDS) gels with a slower relative mobility than the 235K/ 230K fodrin doublet. Comparisons between purified fodrin and the 235K/230K doublet in cerebellum and fore-brain synaptic fractions by two-dimensional peptide mapping indicated that they were identical. The Con A-binding property of GP-A was exploited to purify it by affinity chromatography with agarose-Con A. Peptide mapping comparisons between affinity-purified GP-A and GP-A in SPM and SJ fractions indicated that GP-A in synaptic fractions is apparently homogeneous. Peptide map comparisons between GP-A and 235K fodrin polypeptide indicated that these two synaptic components are highly related (50% of their respective peptides are shared). The 235K fodrin polypeptide in SJs reacted with anti-fodrin antisera on Western blots; however, GP-A failed to cross-react. These observations, together with results from previous studies, indicate that GP-A is highly enriched in cerebellum compared to other neuronal and nonneural tissues. Moreover, GP-A is enriched in SJs relative to SPM fractions, is related to fodrin, and is most likely a cell-surface glycoprotein at asymmetric synapses in cerebellum. GP-A may be involved in neuronal recognition or synaptic transmission in the cerebellum. The important role of calcium in synaptic transmission, together with the decreased solubility of GP-A in Triton that results from micromolar concentrations of calcium, suggest that GP-A may play a role in stabilizing cerebellar synaptic junctions.     

Calmodulin-Dependent Protein Phosphorylation in Synaptic Junctions

Synaptic junctions (SJs) from rat forebrain were examined for Ca2+/calmodulin (CaM)-dependent kinase activity and compared to synaptic plasma membrane (SPM) and postsynaptic density (PSD) fractions. The kinase activity in synaptic fractions was examined for its capacity to phosphorylate endogenous proteins or exogenous synapsin I, in the presence or absence of Ca2+ plus CaM. When assayed for endogenous protein phosphorylation, SJs contained approximately 25-fold greater amounts of Ca2+/CAM-dependent kinase activity than SPMs, and fivefold more activity than PSDs. When kinase activities were measured by phosphorylation of exogenous synapsin I, SJs contained fourfold more activity than SPMs, and 10-fold more than PSDs. The phosphorylation of SJ proteins of 60- and 50-kilodalton (major PSD protein) polypeptides were greatly stimulated by Ca2+/CaM; levels of phosphorylation for these proteins were 23- and 17-fold greater than basal levels, respectively. Six additional proteins whose phosphorylation was stimulated 6-15-fold by Ca2+/CAM were identified in SJs. These proteins include synapsin I, and proteins of 240, 207, 170, 140, and 54 kilodaltons. The 54-kilodalton protein is a highly phosphorylated form of the major PSD protein and the 170-kilodalton component is a cell-surface glycoprotein of the postsynaptic membrane that binds concanavalin A. The CaM-dependent kinase in SJ fractions phosphorylated endogenous phosphoproteins at serine and/or threonine residues. Ca2+-dependent phosphorylation in SJ fractions was strictly dependent on exogenous CaM, even though SJs contained substantial amounts of endogenous CaM (15 micrograms CaM/mg SJ protein). Exogenous CaM, after being functionally incorporated into SJs, was rapidly removed by sequential washings. These observations suggest that the SJ-associated CaM involved in regulating Ca2+-dependent protein phosphorylation may be in dynamic equilibrium with the cytoplasm. These findings indicate that a brain CaM-dependent kinase(s) and substrate proteins are concentrated at SJs and that CaM-dependent protein phosphorylation may play an important role in mechanisms that underlie synaptic communication.     

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.     

Cyclic AMP-stimulated protein kinases at brain synaptic junctions.

We have examined endogenous cyclic AMP-stimulated phosphorylation of subcellular fractions of rat brain enriched in synaptic plasma membranes (SPM), purified synaptic junctions (SJ), and postsynaptic densities (PSD). The analyses of these fractions are essential to provide direct evidence for cyclic AMP-dependent endogenous phosphorylation at discrete synaptic junctional loci. Protein kinase activity was measured in subcellular fractions using both endogenous and exogenous (histones) proteins as substrates. The SJ fraction possessed the highest kinase activity toward endogenous protein substrates, 5-fold greater than SPM and approximately 120-fold greater than PSD fractions. Although the kinase activity as measured with histones as substrates was only slightly higher in SJ than SPM fractions, there was a marked preference of kinase activity toward endogenous compared to exogenous substrates in SJ fractions but in SPM fractions. Although overall phosphorylation in SJ fractions was increased only 36% by 5 micron cyclic AMP, there were discrete proteins of Mr = 85,000, 82,000, 78,000, and 55,000 which incorporated 2- to 3-fold more radioactive phosphate in the presence of cyclic AMP. Most, if not all, of the cyclic AMP-independent kinase activity is probably catalyzed by catalytic subunit derived from cyclic AMP-dependent kinase, since the phosphorylation of both exogenous and endogenous proteins was greatly decreased in the presence of a heat-stable inhibitor protein prepared from the soluble fraction of rat brain. The specific retention of SJ protein kinase(s) activity during purification and their resistance to detergent solubilization was achieved by chemical treatments which produce interprotein cross-linking via disulfide bridges. Two SJ polypeptides of Mr = 55,000 and 49,000 were photoaffinity-labeled with [32P]8-N3-cyclic AMP and probably represent the regulatory subunits of the type I and II cyclic AMP-dependent protein kinases. The protein of Mr = 55,000 was phosphorylated in a cyclic AMP-stimulated manner suggesting autophosphorylation as previously observed in other systems.     

Isolation and major components of core structure of postsynaptic density

The core structure of postsynaptic density (PSD-core) was prepared from rat cerebral synaptosomes by application of the isolation procedure of synaptic junctions (SJ) after trypsinization, which dissociated pre- and post-synaptic structures. The PSD-core was considered to consist mainly of cytoplasmic part of postsynaptic structure, and lack the proteins localized on the external surface of the synaptic plasma membrane, such as receptors for neurotransmitters, Con A-binding proteins and connecting molecule(s) between pre- and post-synaptic structures. The PSD-core proteins which increased greatly in their contents compared with those of SJ prepared from synaptosomes (Syn-SJ) were 120 k Mr Con A-binding protein (Con A-BP) and 30 k Mr protein. Electron microscopic histochemistry suggested that 120 k Con A-BP localized widely in the main structure of the PSD-core. Protein of 30 k Mr was not extracted from PSD-core with 6 M urea, whereas actin, major PSD protein, and tubulin were easily extractable. The 30 k Mr protein was the most resistant one to trypsinization in the SJ fraction. The results suggest that the 30 k Mr protein plays an important role in stabilization and integrity of the postsynaptic density.     

Autophosphorylation of Calmodulin-Kinase II in Synaptic Junctions Modulates Endogenous Kinase Activity

Previous studies have purified from brain a Ca2+/calmodulin-dependent protein kinase II (designated CaM-kinase II) that phosphorylates synapsin I, a synaptic vesicle-associated phosphoprotein. CaM-kinase II is composed of a major Mr 50K polypeptide and a minor Mr 60K polypeptide; both bind calmodulin and are phosphorylated in a Ca2+/calmodulin-dependent manner. Recent studies have demonstrated that the 50K component of CaM-kinase II and the major postsynaptic density protein (mPSDp) in brain synaptic junctions (SJs) are virtually identical and that the CaM-kinase II and SJ 60K polypeptides are highly related. In the present study the photoaffinity analog [alpha-32P]8-azido-ATP was used to demonstrate that the 60K and 50K polypeptides of SJ-associated CaM-kinase II each bind ATP in the presence of Ca2+ plus calmodulin. This result is consistent with the observation that these proteins are phosphorylated in a Ca2+/calmodulin-dependent manner. Experiments using 32P-labeled peptides obtained by limited proteolysis of 60K and 50K polypeptides from SJs demonstrated that within each kinase polypeptide the same peptide regions contain both autophosphorylation and 125I-calmodulin binding sites. These results suggested that the autophosphorylation of CaM-kinase II could regulate its capacity to bind calmodulin and, thus, its capacity to phosphorylate substrate proteins. By using 125I-calmodulin overlay techniques and sodium dodecyl sulfate-polyacrylamide gel electrophoresis we found that phosphorylated 50K and 60K CaM-kinase II polypeptides bound more calmodulin (50-70%) than did unphosphorylated kinase polypeptides. Levels of in vitro CaM-kinase II activity in SJs were measured by phosphorylation of exogenous synapsin I. SJs containing highly phosphorylated CaM-kinase II displayed greater activity in phosphorylating synapsin I (300% at 15 nM calmodulin) relative to control SJs that contained unphosphorylated CaM-kinase II. The CaM-kinase II activity in phosphorylated SJs was indistinguishable from control SJs at saturating calmodulin concentrations (300-1,000 nM). These findings show that the degree of autophosphorylation of CaM-kinase II in brain SJs modulates its in vitro activity at low and possibly physiological calmodulin concentrations; such a process may represent a mechanism of regulating this kinase's activity at CNS synapses in situ.     

Solubilisation of a Glutamate Binding Protein from Rat Brain

Rat brain synaptic plasma membranes were solubilised in either 1% Triton X-100 or potassium cholate and subjected to batch affinity adsorption on L-glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L-glutamate in 0.1% detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L-[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L-glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD = 1.0 and 1.8 microM for Triton X-100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X-100-solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L-glutamate, L-aspartate, and sulphur-containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists--quisqualate, N-methyl-D-aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since solubilisation with Triton X-100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.     

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.     

Development of Synaptic Glycoproteins: Effect of Postnatal Age on the Synthesis and Concentration of Synaptic Membrane and Synaptic Junctional Fucosyl and Sialyl Glycoproteins

Abstract: 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 NaIO₄NaB[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 ag-glutinin 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.     

Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide

The major proteins in isolated synaptic junctions (SJs) and postsynaptic densities (PSDs) have been compared to actin, tubulin, and the major neurofilament (NF) protein by two-dimensional gel electrophoresis and tryptic peptide map analysis. These studies show: (a) tubulin is present in SJ and PSD fractions and is identical to cytoplasmic tubulin, (b) actin in these fractions is very similar to the gamma- and beta-actin found predominantly in nonmuscle cells, and (c) the major PSD protein is distinct from all other known fibrous proteins.     

L-[3H]Glutamate Binding to Hippocampal Synaptic Membranes: Two Binding Sites Discriminated by Their Differing Affinities for Quisqualate

The excitatory glutamate analogs quisqualate and ibotenate were employed to distinguish multiple binding sites for L-[3H]glutamate on freshly prepared hippocampal synaptic membranes. The fraction of bound radioligand that was displaceable by 5 microM quisqualate was termed GLU A binding. That which persisted in the presence of 5 microM quisqualate, but was displaceable by 100 microM ibotenate, was termed GLU B binding. GLU A binding equilibrated within 5 min and remained unchanged for up to 80 min. GLU B binding appeared to equilibrate at least as rapidly, but incubation with ligand unmasked latent binding sites. Saturation binding curves were best fitted by single exponentials, which yielded KD values of about 200 nM (GLU A) and 1 microM (GLU B). On the average, GLU B binding sites were about twice as abundant in these membranes as were GLU A sites. Rapid freezing of the membranes, followed by storage at -26 degrees C and rapid thawing markedly diminished GLU A binding, but nearly tripled GLU B binding. Both site bound L-glutamate with 10-30 times the affinity of D-glutamate. The GLU A site also bound L-glutamate with about 10 times the affinity of L-aspartate and discriminated poorly between L- and D-aspartate. In contrast, the GLU B site bound L-aspartate with an affinity similar to that for L-glutamate, and with an order-of-magnitude greater affinity than D-aspartate. The structural specificities of the GLU A and GLU B binding sites suggest that these sites may correspond to receptors on hippocampal pyramidal cell dendrites that are activated by iontophoretically applied L-glutamate.     

Immune labeling and purification of a 71-kDa glutamate-binding protein from brain synaptic membranes. Possible relationship of this protein to physiologic glutamate receptors

Immunoblot studies of synaptic membranes isolated from rat brain using antibodies raised against a previously purified glutamate-binding protein (GBP) indicated labeling of an approximately 70-kDa protein band. Since the antibodies used were raised against a 14-kDa GBP, the present studies were undertaken to explore the possibility that the 14-kDa protein may have been a proteolytic fragment of a larger Mr protein in synaptic membranes. Protease activity during protein purification was prevented by introducing five protease inhibitors, and a three-step purification procedure was developed that yielded a high degree of purification of glutamate-binding proteins. The major protein enriched in the most highly purified fractions was a 71-kDa glycoprotein, but a 63-kDa protein was co-purified during most steps of the isolation procedure. The glutamate-binding characteristics of these isolated protein fractions were very similar to those previously described for the 14-kDa GBP, including estimated dissociation constants for L-glutamate binding of 0.25 and 1 microM, inhibition of glutamate binding by azide and cyanide, and a selectivity of the ligand binding site for L-glutamate and L-aspartate. The neuroexcitatory analogs of L-glutamate and L-aspartate, ibotenate, quisqualate, and D-glutamate, inhibited L-[3H]glutamate binding to the isolated proteins, as did the antagonist of L-glutamate-induced neuronal excitation, L-glutamate diethylester. On the basis of the lack of any detectable glutamate-related enzyme activity associated with the isolated proteins and the presence of distinguishing sensitivities to analogs that inhibit glutamate transport carriers in synaptic membranes, it is proposed that the 71-kDa protein may be a component of a physiologic glutamate receptor complex in neuronal membranes.     

Developmental Changes in Calmodulin-Kinase II Activity at Brain Synaptic Junctions: Alterations in Holoenzyme Composition

Synaptic junctions (SJs) from rat forebrain were isolated at increasing postnatal ages and examined for endogenous protein kinase activities. Our studies focused on the postnatal maturation of the multifunctional protein kinase designated Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II). This kinase is comprised of a major 50-kilodalton (kDa) and a minor 60-kDa subunit. Experiments examined the developmental properties of CaM-kinase II associated with synaptic plasma membranes (SPMs) and synaptic junctions (SJs), as well as the holoenzyme purified from cytosolic extracts. Large developmental increases in CaM-kinase II activity of SJ fractions were observed between postnatal days 6 and 20; developmental changes were examined for a number of properties including (a) autophosphorylation, (b) endogenous substrate phosphorylation, (c) exogenous substrate phosphorylation, and (d) immunoreactivity. Results demonstrated that forebrain CaM-kinase II undergoes a striking age-dependent change in subunit composition. In early postnatal forebrain the 60-kDa subunit constitutes the major catalytic and immunoreactive subunit of the holoenzyme. The major peak of CaM-kinase II activity in SJ fractions occurred at approximately postnatal day 20, a time near the end of the most active period of in vivo synapse formation. Following this developmental age, CaM-kinase II continued to accumulate at SJs; however, its activity was not as highly activated by Ca2+ plus calmodulin.     

Isolation of Postsynaptic Densities from Day-Old Chicken Brain

Synaptic plasma membranes from chicken brain were used to isolate a postsynaptic density (PSD) fraction using an aqueous two-phase polymer system and the detergent n-octyl glucoside. The protein and glycoprotein composition and the morphology of the day-old chicken brain PSD fraction were compared with a PSD fraction isolated from 12-week-old chicken brain. The PSD fraction from day-old chicken brain contained predominantly PSDs although, like the fraction from 12-week-old chicken, there was some membrane contamination. The major polypeptides in the day-old chicken fraction resolved by polyacrylamide gel electrophoresis comigrated with alpha- and beta-tubulin (Mr 57,000 and 55,000) and actin (Mr 45,000). The major PSD polypeptide (mPSDp) of 12-week-old chicken forebrain, which has a molecular weight of 52,000 was not a major component in day-old chicken. A polypeptide of molecular weight 63,000 was also far more prominent in the 12-week-old chicken PSD fraction whereas the reverse was true for a polypeptide of 31,000. Day-old chicken brain PSDs contained at least 14 concanavalin A-binding glycoproteins of high (greater than 85,000) molecular weight, the two most prominent having molecular weights of 170,000 and 180,000. In contrast to the polypeptide composition, the glycoprotein pattern of day-old chicken PSDs was very similar to that of the 12-week-old bird. Intraperitoneally injected [3H]fucose was incorporated into the glycoproteins of synaptic plasma membranes and PSDs from day-old chickens.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Purification and Molecular Characterization of the Brain Synaptic Membrane Glutamate-Binding Protein

A glutamate-binding protein from rat brain synaptic plasma membranes has been purified to apparent homogeneity. This protein has a Mr of 14,300 based on amino acid and carbohydrate analyses. The protein is enriched with tryptophan residues, which contribute substantially to its hydrophobic nature. It also has a relatively high content of acidic amino acids, which determine is low isoelectric point (4.82). The protein exhibits either a single, high-affinity class of sites for L-[3H]glutamate binding (KD = 0.13 microM) when binding is measured at low protein concentrations, or two classes of sites with high (KD = 0.17 microM) and low affinities (KD = 0.8 microM) when binding is measured at high protein concentrations. These observations suggest preferential binding of L-glutamate to a self-associating form of the protein. The displacement of protein-bound L-[3H]glutamic acid by other neuroactive amino acids has characteristics similar to those observed for displacement of L-glutamate from membrane binding sites. Chemical modification of the cysteine and arginine residues results in an inhibition of glutamate binding activity. The possible function of this protein in the physiologic glutamate receptor complex of neuronal membranes is discussed.     

Solubilization, Characterization, and Partial Purification of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid-, Quisqualate-, Kainate-Sensitive l-Glutamate Binding Sites from Porcine Brain Synaptic Junctions

L-[3H]Glutamate binding sites were solubilized from porcine brain synaptic junctions by Triton X-114 in the presence of KCl. The solubilized binding sites bound L-[3H]glutamate reversibly with KD and Bmax values of 1.48 +/- 0.18 microM and 178.2 +/- 15.9 pmol/mg of protein, respectively. These binding sites appeared to be integral membrane glycoproteins, with sugar moieties recognized by wheat germ agglutinin. A 49.3-fold purification of these binding sites was achieved by Triton X-114 solubilization, anion-exchange chromatography, and affinity chromatography using wheat germ agglutinin-Sepharose. The apparent molecular mass of the partially purified binding sites was 620 +/- 50 kDa. L-[3H]Glutamate bound to the solubilized preparation could be effectively displaced by agonists of non-N-methyl-D-aspartate (NMDA) L-glutamate receptors but not by NMDA or alpha-amino-4-phosphonobutyrate. The rank order for the competitive ligands in displacing L-[3H]glutamate was: quisqualate greater than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid greater than L-glutamate greater than kainate.     

Distribution of [3H]Dihydrotetrabenazine Binding in Bovine Striatal Subsynaptic Fractions: Enrichment of Higher Affinity Binding in a Synaptic Vesicle Fraction

[3H]Dihydrotetrabenazine bound to a single class of binding sites in bovine striatal synaptic vesicles with an apparent dissociation constant of 3-9 nM. This is comparable to the inhibitory potency of dihydrotetrabenazine in catecholamine transport assays. In contrast to these results, [3H]dihydrotetrabenazine bound to at least two classes of sites in all other subsynaptic fractions investigated. The higher affinity class of sites was comparable in affinity to that of synaptic vesicles, whereas the lower affinity sites exhibited an apparent dissociation constant of 95-400 nM. Higher affinity sites were most abundant in the synaptic vesicle fraction, and little higher affinity binding was observed in mitochondrial and myelin fractions, or in highly purified synaptic plasma membranes. Lower affinity binding was not enriched in any subsynaptic fraction and was the only class of binding sites detected in homogenates of liver and diaphragm. The distribution of the presynaptic vesicle marker synaptophysin corresponded with that of higher affinity but not lower affinity binding. These results are consistent with the expectation that the higher affinity sites are associated primarily with synaptic vesicles and other neuronal entities that are in communication with these organelles.     

Microsomal Opiate Receptors: Characterization of Smooth Microsomal and Synaptic Membrane Opiate Receptors

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.