Calmodulin Binding Proteins of the Cholinergic Electromotor Synapse: Synaptosomes, Synaptic Vesicles, Receptor-Enriched Membranes, and Cytoskeleton

Calmodulin binding proteins (CBPs) have been identified using a gel overlay technique for fractions isolated from Torpedo electromotor nerve endings. Different fractions possessed characteristic patterns of CBPs. Synaptosomes showed five major CBPs--Mr 220,000, 160,000, 125,000, 55,000, and 51,000. Polypeptides of Mr 55,000 and 51,000 were found in the cytoplasm and the others are membrane-associated. The Triton X-100-insoluble cytoskeleton of synaptosomes was isolated in the presence or absence of calcium. The major CBPs had Mr of 19,000, 18,000, and 16,000. In the presence of calcium, no other CBPs were seen. In the absence of calcium, an Mr 160,000 polypeptide was present in the Triton cytoskeleton. Synaptic vesicles showed CBPs of Mr 160,000, 25,000, and 20,000. Membrane fragments enriched in acetylcholine receptors contained two major CBPs, Mr 160,000 and 125,000, together with a less prominent protein at Mr 26,000. A protein of Mr similar to that of fodrin was present in synaptosomes and acetylcholine receptor membrane fragments, but only in small amounts relative to the other polypeptides observed. The heavy and light chains of clathrin-coated vesicles from pig brain did not bind calmodulin, although strong labelling of an Mr 47,000 polypeptide was found. Results showed that calelectrin does not bind calmodulin. The possible identity of the calmodulin binding proteins is discussed.     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).     

Identification and Characterization of the Major Proteins of Mammalian Brain Synaptic Vesicles

Highly purified rat and cow brain synaptic vesicles contain major proteins with molecular weights of approximately 74,000, 60,000, 57,000, 40,000, 38,000, and 34,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of the major proteins on synaptic vesicles was confirmed by immunoprecipitation of intact rat brain synaptic vesicles with a synaptic vesicle-specific monoclonal antibody. The 40,000-Mr protein appeared to be identical to the 38,000-Mr integral membrane glycoprotein, p38 or synaptophysin, previously identified as a major component of mammalian synaptic vesicles. The isoelectric point of the 75,000-Mr proteins from either rat or cow brain synaptic vesicles is 5.0, and the pI of the 57,000-Mr protein is approximately 5.1 in both species. The similarity in size and charge of several major proteins in rat and cow synaptic vesicles suggests a high degree of structure conservation of these proteins in diverse mammalian species and raises the possibility that a set of functions common to most or all mammalian synaptic vesicles is mediated by these 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.     

Kainic Acid Differentially Affects the Synaptosomal Release of Endogenous and Exogenous Amino Acidic Neurotransmitters

Presynaptic actions of kainic acid have been tested on uptake and release mechanisms in synaptosome-enriched preparations from rat hippocampus and goldfish brain. Kainic acid increased in a Ca2+-dependent way the basal release of endogenous glutamate and aspartate from both synaptosomal preparations, with the maximum effect (40-80%) being reached at the highest concentration tested (1 mM). In addition, kainic acid potentiated, in an additive or synergic way, the release of excitatory amino acids stimulated by high K+ concentrations. Kainic acid at 1 mM showed a completely opposite effect on the release of exogenously accumulated D-[3H]aspartate. The drug, in fact, caused a marked inhibition of both the basal and the high K+-stimulated release. Kainic acid at 0.1 mM had no clear-cut effect, whereas at 0.01 mM it caused a small stimulation of the basal release. The present results suggest that kainic acid differentially affects two neurotransmitter pools that are not readily miscible in the synaptic terminals. The release from an endogenous, possibly vesiculate, pool of excitatory amino acids is stimulated, whereas the release from an exogenously accumulated, possibly cytoplasmic and carrier-mediated, pool is inhibited or slightly stimulated, depending on the external concentration of kainic acid. Kainic acid, in addition, strongly inhibits the high-affinity uptake of L-glutamate and D-aspartate in synaptic terminals. All these effects appear specific for excitatory amino acids, making it likely that they are mediated through specific recognition sites present on the membranes of glutamatergic and aspartatergic terminals. The relevance of the present findings to the mechanism of excitotoxicity of kainic acid is discussed.     

Excitatory Transmitter Amino Acid Release from the Ischemic Rat Cerebral Cortex: Effects of Adenosine Receptor Agonists and Antagonists

The effects of selective adenosine receptor agonists [N6-cyclopentyladenosine (CPA) and N-ethylcarboxamidoadenosine (NECA)] and antagonists [8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4-triazolo[1,5-c]quinazoline-5-im ine (CGS-15943A)] on aspartate and glutamate release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (for 20 min) was elicited by four-vessel occlusion. Excitatory amino acid releases were compared from control ischemic rats and drug-treated rats. Basal levels of aspartate and glutamate release were not greatly affected by pretreatment with the adenosine receptor agonists or antagonists. However, CPA (10(-10) M) and NECA (10(-9) M) significantly inhibited the ischemia-evoked release of aspartate and glutamate into cortical superfusates. The ability to block ischemia-evoked release of excitatory amino acids was not evident at higher concentrations of CPA (10(-6) M) or NECA (10(-5) M). The selective A1 receptor antagonist DPCPX also had no effect on release when administered at a low dosage (0.01 mg/kg, i.p.) but blocked the ischemia-evoked release of aspartate and glutamate at a higher dosage (0.1 mg/kg). Evoked release was inhibited by the selective A2 receptor antagonist CGS-15943A (0.1 mg/kg, i.p.). Thus, adenosine and its analogs may suppress ischemia-evoked release of excitatory neurotransmitter amino acids via high-affinity A1 receptors, whereas coactivation of lower-affinity A2 receptors may block (or reverse) the A1-mediated response.     

Most Synaptic Vesicles Isolated from Rat Brain Carry Three Membrane Proteins, SV2, Synaptophysin, and p65

We have prepared highly purified synaptic vesicles from rat brain by subjecting vesicles purified by our previous method to a further fractionation step, i.e., equilibrium centrifugation on a Ficoll gradient. Monoclonal antibodies to three membrane proteins enriched in synaptic vesicles--SV2, synaptophysin, and p65--each were able to immunoprecipitate specifically approximately 90% of the total membrane protein from Ficoll-purified synaptic vesicle preparations. Anti-SV2 precipitated 96% of protein, anti-synaptophysin 92%, and anti-p65 83%. These results demonstrate two points: (1) Ficoll-purified synaptic vesicles appear to be greater than 90% pure, i.e., less than 10% of membranes in the preparation do not carry synaptic vesicle-associated proteins. These very pure synaptic vesicles may be useful for direct biochemical analyses of mammalian synaptic vesicle composition and function. (2) SV2, synaptophysin, and p65 coexist on most rat brain synaptic vesicles. This result suggests that the functions of these proteins are common to most brain synaptic vesicles. However, if SV2, synaptophysin, or p65 is involved in synaptic vesicle dynamics, e.g., in vesicle trafficking or exocytosis, separate cellular systems are very likely required to modulate the activity of such proteins in a temporally or spatially specific manner.     

Characterization of γ-Aminobutyric Acid Binding Sites on Crude Synaptic Membranes

[3H]GABA binding to crude synaptic membranes of rat brain was studied in an attempt to identify GABA binding to its synaptic receptor in the presence of Na+. Membrane vesicles prepared from crude synaptic membrane fractions were useful as a tool to differentiate synaptic GABA receptors from GABA uptake sites. The crude synaptic membranes treated with Triton X-100 [membranes (TX)] involved two classes of GABA binding sites (KD = 38.7 and 78.0 nM) in the absence of Na+, but the high-affinity sites disappeared in the presence of Na+ and a single class of GABA binding sites (KD = 75.0 nM) was detected. The failure to detect an active uptake of [3H]GABA into the vesicles prepared from membranes (TX) suggests that the [3H]GABA binding in the presence of Na+ was related to synaptic GABA receptors. It is probable that Na+ could mask the presence of the high-affinity class of GABA receptor.     

Binding Sites for l-[3H]Glutamate on Hippocampal Synaptic Membranes: Three Populations Differentially Affected by Chloride and Calcium Ions

The effects of Cl- and Ca2+ were studied on the specific binding of L-[3H]glutamate to multiple sites on rat hippocampal synaptic membranes. Quisqualate (5 microM) or DL-2-amino-4-phosphonobutyrate (2-APB) (300 microM) was used to discriminate two previously identified classes of binding sites. Saturation isotherms and displacement curves constructed under different ionic conditions suggested that the effects of Cl- and Ca2+ could best be explained by postulating the existence of three major binding site populations in this preparation rather than two. The binding of L-glutamate to Glu A sites exhibits an absolute dependence on Cl-, and Ca2+ markedly increases the maximum density of these sites. Glu A sites bind quisqualate and 2-APB with relatively high affinity. Cl- (47 mM) more than doubles the maximum density of Glu B sites, but Ca2+ appears to have no effect. Glu B sites can be discriminated from the other classes by their relatively low affinity for quisqualate and 2-APB. There is reason to think that the Glu B population is heterogeneous. The novel Glu C population can be virtually selectively labeled by exposing 2-APB-sensitive binding sites to radioligand in Tris-HOAc buffer with Ca2+. Binding of L-[3H]glutamate to these sites is enhanced by both Cl- and Ca2+, but requires neither ion. Ca2+ appears to increase both the affinity of Glu C sites for L-glutamate and their maximum binding site density. In the presence of Ca2+ and Cl-, Glu C sites bind the radioligand with micromolar affinity (KD approximately 2 microM) and high capacity (Bmax approximately 160 pmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Myelin Proteins, Glycoproteins, and Myelin-Related Enzymes in Experimental Demyelination of the Rabbit Optic Nerve: Sequence of Events

Wallerian degeneration of the rabbit optic nerve was investigated by the technique of retinal ablation which precludes edema, hemorrhage, or macrophage infiltration. After 8 days of degeneration, marked degradation of axons and some myelin abnormalities appeared in the optic nerve, optic chiasma, and optic tract. Myelin lesions were maximal 32 days after retinal destruction. The amount of material stained with a myelin dye decreased drastically between 32 and 90 days after the operation. Biochemical parameters gave the following sequence of events. The concentration of the major periodic acid--Schiff staining glycoproteins was decreased after 2 days, and 6 days later the presence of cholesterol esters was detected in the optic tissue. After 16 days of Wallerian degeneration, the specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase not associated with myelin decreased, indicating a possible de-differentiation of oligodendrocytes. Degradation of myelin basic protein became significant at 32 days and the amount of myelin isolated decreased later. The loss of myelin basic protein coincided with a reduction of myelin periodicity as measured in purified fractions by electron microscopy. These results show that secondary myelin destruction in the absence of edema, hemorrhage, or macrophages is a very slow process, and in this situation myelin undergoes a selective and sequential loss of its constituents.     

Regulation of AMPA Receptor Activity, Synaptic Targeting and Recycling: Role in Synaptic Plasticity

The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors for the neurotransmitter glutamate are oligomeric structures responsible for most fast excitatory responses in the central nervous system. The activity of AMPA receptors can be directly regulated by protein phosphorylation, which may also affect the interaction with intracellular proteins and, consequently, their recycling and localization to defined postsynaptic sites. This review focuses on recent advances in understanding the dynamic regulation of AMPA receptors, on a short- and long-term basis, and its implications in synaptic plasticity.     

Selective Loss of N-Methyl-d-Aspartate-Sensitive l-[3H]Glutamate Binding Sites in Rat Brain Following Portacaval Anastomosis

Excitatory amino acids have been implicated in the pathogenesis of hepatic encephalopathy. In the present study, kainate, quisqualate and N-methyl-D-aspartate (NMDA) subclasses of L-glutamate receptors were measured in adult rat brain by quantitative receptor autoradiography following surgical construction of an end-to-side portacaval anastomosis (PCA). PCA resulted in sustained hyperammonemia and decreased binding of L-glutamate to the NMDA receptor when compared to sham-operated controls. Decreases in binding ranged from 17 to 39% in several regions of cerebral cortex, hippocampus, striatum, and thalamus. Binding to quisqualate and kainate receptor subtypes was not altered. PCA leads to astrocytic changes in brain but does not result in any measurable loss of neuronal integrity. It is therefore proposed that decreased glutamate binding to the NMDA receptor following PCA results from increased extracellular glutamate caused by decreased reuptake into perineuronal astrocytes and a compensatory down-regulation of these receptors. Such changes could be of pathophysiological significance in hepatic encephalopathy.     

Identification of Lectin-Purified Neural Glycoproteins, GPs 180, 116, and 110, with NMDA and AMPA Receptor Subunits: Conservation of Glycosylation at the Synapse

Abstract: The postsynaptic apparatus is associated with a number of glycoproteins with apparent molecular masses of 180, 116, and 110 kDa, which are highly concentrated in and may be uniquely associated with this structure. These glycoproteins, purified by concanavalin A lectin-affinity chromatography, showed immunoreactivity in the present study with subunit-specific antibodies to glutamate receptors as follows: GP 180, NMDA receptor subunits NR2A/NR2B; GP 116, NMDA receptor NR1 (1a); and GP 110, pan-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (pan-AMPA) receptors. Sensitivities to the glycosidases peptide N -glycosidase F and endo -β- N -acetylglucosaminidase H on both western blots and silver-stained gels suggested that the glutamate receptors were at least major constituents of the glycoprotein bands. Similar detailed glycosylation was observed for all three glycoproteins, with neutral oligosaccharides being dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for ∼50% of the neutral sugars, with Man 5 (at almost 20% of the neutral sugars) always the major glycan. Other abundant neutral oligosaccharides were of the complex type. Similar sensitivities to peptide N -glycosidase F and endo -β- N -acetylglucosaminidase H were observed for cell line-expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly processed oligosaccharides will be expressed. This may be indicative of glycosylation sites in these receptors that are inaccessible to the later processing enzymes and favours the oligomannosidic class of glycans in functional roles.     

Amino Acids in the Substantia Nigra of Rats with Striatal Lesions Produced by Kainic Acid

In an attempt to estimate the pool size of glutamate and other amino acids in γ-aminobutyric acid (GABA)-containing neurons, we determined the content of 12 amino acids in the bilateral substantia nigra of rats, in which unilateral striatal lesions had been made with kainic acid two weeks earlier. The assay of the amino acids (including glutamate, aspartate, glutamine, asparagine, glycine, and GABA) and ethanolamine was based on HPLC and fluorimetric detection after precolumn derivatization with o-phthaldialdehyde. The levels of all measured amino acids (except those of tyrosine, threonine, and ethanolamine) were decreased in the affected striatum, but only the levels of aspartate, taurine, and GABA were lowered in the ipsilateral substantia nigra. These results indicate that the pool size of the various amino acids in the striatonigral GABAergic pathway is small compared to their nigral content, and that in addition to GABA a significant fraction of aspartate and taurine may be confined to nerve terminals in the substantia nigra.     

Aspartate Aminotransferase for Synthesis of Transmitter Glutamate in the Medulla Oblongata: Effect of Aminooxyacetic Acid and 2-Oxoglutarate

The effects of aminooxyacetic acid (AOAA), a transaminase inhibitor, and 2-oxoglutarate, a precursor to glutamate by the activity of aspartate aminotransferase (AAT), on slices of rat medulla oblongata, cerebellum, cerebral cortex, and hippocampus were studied. The slices were superfused and electrically stimulated. There was a Ca2+-dependent stimulus-evoked release of endogenous glutamate, gamma-aminobutyric acid (GABA), and beta-alanine in all regions examined. AOAA (10(-4) and 10(-3) M) decreased the release of glutamate in the medulla oblongata and cerebellum but not in the hippocampus. L-Canaline, a specific inhibitor of ornithine aminotransferase, did not affect the glutamate release in the medulla. 2-Oxoglutarate (10(-3) M) increased the release of glutamate in the medulla oblongata and cerebellum but not in the cerebral cortex and hippocampus. Treatment with AOAA (10(-4) M) almost abolished the activities of AAT in all regions studied. AOAA (10(-4) and 10(-3) M) increased the stimulus-evoked release of GABA in the cerebellum, cerebral cortex, and hippocampus, whereas the stimulus-evoked release of beta-alanine was decreased by this agent in all regions studied. These results suggest the participation of AAT in the synthesis of the transmitter glutamate in the medulla oblongata and cerebellum of the rat.     

Multiple Amphiphysin II Splice Variants Display Differential Clathrin Binding: Identification of Two Distinct Clathrin-Binding Sites

Abstract: Amphiphysin I and II are nerve terminal-enriched proteins that display src homology 3 domain-mediated interactions with dynamin and synaptojanin. It has been demonstrated that the amphiphysins also bind to clathrin, and we have proposed that this interaction may help to target synaptojanin and dynamin to sites of synaptic vesicle endocytosis. To understand better this potential functional role, we have begun to characterize clathrin-amphiphysin interactions. Using PCR from adult human cortex cDNA, we have cloned a number of amphiphysin II splice variants. In in vitro binding assays, the amphiphysin II splice variants display differential clathrin binding and define a 44-amino acid region mediating the interaction. Amphiphysin II truncation and deletion mutants identify two distinct clathrin-binding domains within this region: one with the sequence LLDLDFDP, the second with the sequence PWDLW. Both domains are conserved in amphiphysin I, and saturation binding analysis demonstrates that both sites bind clathrin with approximately equal affinity. The elucidation of clathrin as a splice-specific binding partner for amphiphysin II begins to address the potential functional role(s) for the multiple amphiphysin II splice variants and further supports an important function for clathrin-amphiphysin interactions in protein targeting during endocytosis.     

Comparison of the Surface Proteins and Glycoproteins On Erythrocytes of Calves Before and During Infection With Babesia Bovis

The surface proteins and glycoproteins on red cells from normal and Babesia bovis-infected calf blood have been compared. Several radiolabeling probes were used to label specifically external membrane molecules which were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by autoradiography or fluorography. No differences were observed among the Coomassie Blue-stained membrane proteins of erythrocytes from individual uninfected calves. Comparison of red cells from these animals also indicated no qualitative differences in the surface proteins with accessible tyrosyl residues labeled by lactoperoxidase-catalyzed radioiodnation, although some quantitative variation in the uptake of radioactivity into particular proteins was observed. the major radioiodinated bands on normal bovine erythrocytes had Mr of 165, 130, 90, and 45 kiloDaltons. However, labeling of surface glycoproteins by the periodate/[³H]NaBH₄ and galactose oxidase (± neuraminidase)/[³H]NaBH₄ methods showed significant differences in the surface proteins of red cells from individual uninfected calves. of 14 animals tested, 5 had major labeled glycoproteins of unique Mr. No changes were observed in radioiodinated surface proteins of total red cell samples from infected calves with 0.5-6% parasitemia. Radioiodination of concentrated infected red cells from the same samples (concentrated by selective hypotonic lysis of uninfected erythrocytes in KC1) resulted in the labeling of 3 new surface proteins, with Mr of 118, 115, and 60 kiloDaltons. the same new ¹²⁵I-labeled bands were identified on infected cells from 3 avirulent strains of B. bovis used in vaccine production. Furthermore, in concentrated infected cells there was very poor radiolabeling of major bands strongly labeled on uninfected cells (Mr 165, 130, and 90 kiloDaltons), suggesting parasite-induced loss of these proteins. Although there were some differences in ³H-labeled surface glycoproteins of red cells from normal and. B. bovis -infected blood, they were restricted to minor labeled bands and were not seen consistently. the labeled surface glycoproteins of concentrated infected cells were very similar to those of the uninfected red blood cells from infected blood.     

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.     

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.     

膜上tRNA结合蛋白的分离与初步鉴定

用ＴｒｉｔｏｎＸ－１１４分相法分离啤酒酵母的膜总蛋白,经过酵母ｔＲＮＡ分子交联的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,用０－０．８ｍｏｌ／Ｌ（ＮＨ４０２ＳＯ４梯度缓冲液洗脱ｔＲＮＡ结合的蛋白质。凝胶阻滞电泳实验室鉴定出两种主要的与ｔＲＮＡ分子特异性结合的蛋白质。