Synaptic Membrane Fractions Research Articles

Phosphatidylethanol formation via transphosphatidylation by rat brain synaptosomal phospholipase D.

Phosphatidylethanol (Peth) formation catalyzed by the transphosphatidylation activity of phospholipase D was demonstrated to occur in a rat brain synaptosomal enriched preparation. The optimal pH was determined to be 6.5, and the optimal ethanol concentration was determined to be 0.3-0.4 M with an apparent Km of 0.2 M. Peth formation was barely detectable in the absence of an appropriate activator and several unsaturated fatty acids were found to be effective activators. The concentrations of oleic acid required for maximum activation varied with the concentration of exogenous phosphatidylcholine present in the incubation mixtures. All detergents tested were significantly less active than the unsaturated fatty acids and divalent ions were not required for Peth formation. Phosphatidylcholine was the most effective phosphatidyl donor of the phospholipids tested. Peth forming activity was greatest in the synaptic membrane fraction of the various brain subfractions examined. The 12,000 g-100,000 g particulate fraction of lung, heart, and adipose tissue had activities similar to that of brain.

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 M rs 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-β-N-acetylglucosaminidase H (endo H) lowers the M rs 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 thet 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.

The Mr 93,000 polypeptide of the postsynaptic glycine receptor complex is a peripheral membrane protein.

The glycine receptor of mammalian spinal cord is an oligomeric membrane protein that, after affinity purification on aminostrychnine-agarose or immobilized antibody, contains three polypeptides of Mr 48,000, 58,000, and 93,000. Here, the association and the properties of the polypeptides of the rat glycine receptor were investigated. Upon phase partitioning in the nonionic detergent Triton X-114, the three receptor polypeptides behaved as a hydrophilic protein complex exhibiting phospholipid binding. Sucrose gradient centrifugation or gel filtration in the presence of dithiothreitol and Triton X-100 separated the Mr 93,000 polypeptide from the Mr 48,000 and 58,000 polypeptides, which harbor the antagonist binding site of the glycine receptor. Alkaline or dimethylmaleic acid anhydride treatment of crude synaptic membrane fractions resulted in extraction of the Mr 93,000 polypeptide. Lectin binding was observed for the Mr 48,000 and 58,000 glycine receptor subunits but not the Mr 93,000 polypeptide. These results indicate that the Mr 93,000 polypeptide is a peripheral membrane protein that is located at the cytoplasmic face of the postsynaptic glycine receptor complex.

Purification of γ-aminobutyric acid receptor, benzodiazepine receptor and C1 channel from bovine cerebral cortex by benzodiazepine affinity gel column chromatography

Purification of solubilized ?-aminobutyric acid (GABA) and benzodiazepine receptors by Nonidet P-40 from the bovine cerebral cortex was employed by the affinity column chromatography using a benzodiazepine, 1012-S, as immobilized ligand. Although this benzodiazepine affinity gel retained all of the solubilized benzodiazepine receptors from synaptic membranes applied to the column, 25.6% of the GABA receptor binding activity was recovered in the run-through fraction. After the washing with NaCl, the biospecific elution with 1 mM 1012-S resulted the elution of 31.3% of GABA receptor binding activity applied to the column. The highest purification fold thus obtained was 3789 (specific activity: 0.39 nmol/mg protein). Successive application of 1-2 mM NaSCN also resulted further elution of GABA receptor. Scatchard analysis of [(3)H]muscimol binding to these fractions indicated that the purified GABA receptor had high and low affinity binding sites as detected in solubilized and synaptic membrane fractions. In addition, it was found that the B(max) values for both binding sites in the 1012-S-eluted fraction increased significantly without changing the K(D) values. SDS-polyacrylamide gel electrophoretic profiles of the 1012-S-eluted fraction showed the existence of two major bands having the molecular weights of approx. 53,000 and 57,000, both of which were irreversibly photoaffinity-labeled with [(3)H]flunitrazepam. This result made a sharp contrast with the result obtained in the rat brain, in which the subunits of the purified receptor consisted of molecular weights of 48,500 and 54,500 and the former band was selectively photoaffinity-labeled with [(3)H]flunitrazepam. In contrast, the gel filtration using Sephadex G-200 of the purified GABA/benzodiazepine receptor complex from both animal species revealed the molecular weight of these receptor complexes was approx. 340,000. These results strongly suggest that the subunit structures of GABA/benzodiazepine receptors from the bovine and rat brains may be essentially different. On the other hand, the purification of GABA/benzodiazepine receptor complex and C1 channel solubilized by CHAPS, a non denaturating zwitterionic detergent, using the same benzodiazepine affinity column followed by DEAE-Sephacel ion exchange column chromatography indicated that the purified GABA receptor was functionally coupled with both benzodiazepine receptor and C1 channel. These results also indicate that GABA(A) receptor which is structurally coupled with benzodiazepine receptor and C1 channel is readily purified by the use of this benzodiazepine affinity gel, whereas GABA(B) receptor which is not associated with both components is recovered in its run-through fraction, respectively.

Immunological factors in neuronal degeneration with particular reference to motor neurone disease.

Cellular and humoral immunoreactivity to neuronal antigens was investigated in patients with motor neurone disease (MND). Lymphocytes from patients with MND and normal healthy controls were cultured with a membrane fraction prepared from cultured spinal cord neurones. 4 out of 14 patients with MND and 0 out of 9 normal controls showed a significantly increased stimulation index. An enzyme-linked immunoabsorbent assay (ELISA) was established to detect antibodies to synaptic membrane fraction prepared from human motor cortex. Sera from MND patients showed a significantly increased immunoglobulin binding with respect to normal control sera. Antineuronal antibody production by MND lymphocytes was studied by using Epstein-Barr virus transformation followed by fusion with a mouse myeloma cell line. Antibody-producing clones were isolated. This procedure would allow a more detailed analysis of the antineuronal antibody production in MND.

The metabolic turnover of the major proteins of the postsynaptic density

We have used the method of Austin, Lowry, Brown and Carter, to measure the steady-state metabolic half-life of tubulin (α and β individually) and actin (β and β together) in the total cytosolic (S 3), microsomal (P 3), synaptic plasma membrane (SPM) and synaptic junction (SJ) subcellular fractions from 6-day-old and adult chicken forebrain. In the SPM and SJ fractions we also measured the steady-state metabolic half-life of the major postsynaptic density protein (mPSDp). In SPM and SJ fractions from 6-day-old chickens tubulin and actin turned over approximately twice as slowly ( t 1 2 ≈ 24 days ) as tubulin and actin in the S 3 fraction ( t 1 2 ≈ 13 days ). This difference was unlikely merely to be due to association with membranes since the t 1 2 values for the proteins were the same in P 3 and S 3. The estimated t 1 2 values for mPSDp were similar to that for tubulin and actin in SPM and SJ fractions. Similar results were obtained in adult chickens except that all t 1 2 values in all fractions were approximately 30% larger. The calculated t 1 2 values did not change between labelling periods of 4 and 6.5 h suggesting that the lag phase of incorporation of newly synthesized PSD proteins is sufficiently rapid to not produce this result artefactually. When the brain from a non-labelled chicken was homogenized in the presence of the S 3 fraction from a labelled chicken and sub-fractionated the relative specific activities of the SPM and SJ fractions produced were 1–2% of those from the labelled brain. These results support the notion that tubulin and actin are intrinsic components of the PSD.

Characteristics of a Ca 2+/calmodulin-dependent binding of the Ca 2+ channel antagonist, nitrendipine, to a postsynaptic density fraction isolated from canine cerebral cortex

Synaptic membrane (SM) and postsynaptic density (PSD) fractions isolated from the cerebral cortex (CTX) and cerebellum (CL) of the canine brain were found to contain one class of specific nitrendipine binding sites. The specific binding constants were: CTX-SM, K d = 110 pM ( B max = 126 fmo1/mg protein); CTX-PSD, K d = 207 pM ( B max = 196 fmo1/mg; CL-SM, K d = 100 pM ( B max = 65 fmol/mg); CL-PSD, K d = 189 pM ( B max = 80 fmol/mg). Treatment of the CTX-SM and CTX-PSD fractions with 0.5% deoxycholate and 1.0% N-lauroyl sarcosinate removed 88–91% and 42–51% of the nitrendipine binding, respectively, indicating that the major nitrendipine binding present in the SM fractions are of non-synaptic origin. Moreover, the percentages of total protein and specific nitrendipine binding removed from PSDs by these detergents were similar, indicating no preferential dissociation of the latter, and suggesting that the receptor protein is firmly bound and is probably an intrinsic component of the PSD fraction. Both Ca 2+ and calmodulin were found to be important for the binding of nitrendipine to the CTX-SM and CTX-PSD fractions since: (a) R24571, a calmodulin antagonist, was found to inhibit nitrendipine binding to the CTX-SM and CTX-PSD fractions with IC 50 values of 1.1 μM and 0.9 μM, respectively; (b) removal of Ca 2+ from the CTX-SM and CTX-PSD fractions with 0.2 mM EGTA resulted in losses of specific nitrendipine binding of 80 and 90%, respectively; (c) Ca 2+ alone restored nitrendipine binding to EGTA-pretreated CTX-SM fractions and not to CTX-PSD fractions, with the latter needing both Ca 2+ and calmodulin to restore nitrendipine binding; (d) EGTA treatment removed 14–16% and 89–91% of nitrendipine bound to the CTX-SM and CTX-PSD fractions, respectively, suggesting that calmodulin (but not Ca 2+) is needed to maintain the nitrendipine-nitrendipine receptor-calmodulin complex; (e) Ca 2+-reconstituted EGTA-pretreated CTX-SM fractions and the Ca 2+ plus calmodulin-reconstituted EGTA-pretreated CTX-SM and CTX-PSD fractions were found to have similar binding constants to those for the corresponding native, untreated fractions; and (f) the Ca 2+/calmodulin dependency on nitrendipine binding was similar to the well-known Ca 2+/calmodulin dependency on phosphorylation in EGTA-pretreated PSD fractions. It needed much less Ca 2+ to saturate Ca 2+/calmodulin-dependent phosphorylation of the pretreated CTX-PSD fractions than the nitrendipine binding. Yet, less calmodulin was needed to saturate nitrendipine binding than the phosphorylation. Dithiothreitol was found to inhibit specific binding of nitrendipine to the CTX-SM and CTX-PSD fractions with IC 50 values of 3.6 mM and 3.4 mM, respectively. Verapamil was also inhibitory for the specific binding. However, the benzothiazepine derivatives (±)- cis-diltiazem, (+)- cis-diltiazem and (−)- cis-diltiazem, were found to have no significant effects on either binding of nitrendipine to CTX-SM and CTX-PSD fractions or dissociation by nifedipine of the nitrendipine bound to the subcellular fractions. Previous binding of nitrendipine did not significantly influence the consequent cAMP- or Ca 2+/calmodulin-dependent phosphorylations of CTX-PSD proteins whether performed directly after binding incubation or after removal of excess reagents. However, prior phosphorylation of the CTX-SM fraction in the presence of cAMP, but not of Ca 2+/calmodulin, gave a (≈)40% increase in subsequent specific nitrendipine binding. The results show, for the first time, that a nitrendipine binding site, possibly a voltage-dependent calcium channel, is present in the PSD fraction isolated from the brain.

The distribution of [3H]kainate binding sites in primate hippocampus is similar to the distribution of both Ca2+-sensitive and Ca2+-insensitive [3H]kainate binding sites in rat hippocampus.

The distribution of [3H]kainate binding sites was determined by quantitative autoradiography in three vertebrate species: rat, monkey, and human. These animals displayed a similar pattern of binding site density in the hippocampus. Highest levels were found within the stratum lucidum and moderate levels in the inner portion of the dentate gyrus molecular layer. Although the distribution is similar, there is a lower density of binding sites in the stratum lucidum of primates than in rodents. Experiments using rat brain synaptic plasma membrane fractions indicated that inclusion of Ca2+ ions results in a selective reduction in binding at the high affinity sites. The Ca2+-inhibited and Ca2+-inhibited binding sites in the high affinity sites. The Ca2+ -inhibited and Ca2+ -insensitive binding sites in the rat hippocampus exhibited a similar distribution. Together, these results suggest that in a variety of mammalian species kainate receptors exhibit similar regional distributions, and that the high and low affinity kainate binding sites also exhibit similar regional distributions.

Subcellular site of action of imipramine in rodent brain.

To determine the site of action of imipramine, the subcellular distribution of [3H]imipramine in rodents was followed after both in vivo administration and in vitro incubation with tissue slices under "physiological" conditions. Total [3H]imipramine (10-1,000 nM) binding was associated with all primary fractions, but in particular with the nuclear (P1) and mitochondrial (P2) pellets and the synaptosomal (P2B) and myelin (P2A) fractions. Using an excess of imipramine to define any nonspecific interactions, a specific association was observed mainly in those fractions containing isolated nerve terminals and to a lesser extent with the purified myelin fraction. Preparation of subsynaptosomal fractions by osmotic lysis indicated that [3H]imipramine was associated with the synaptic vesicle and microsomal fractions and also with synaptosomal membranes. The degree of binding to the vesicular and microsomal fractions was increased with the length of preparation time, whereas there was an inverse relationship between the length of preparation and the amount bound to the synaptosomal membrane fraction. There was no evidence of an intrasynaptosomal accumulation of [3H]imipramine at concentrations up to 1,000 nM. [3H]2-Nitroimipramine, a slowly dissociating imipramine derivative, was exclusively located in synaptic membrane fractions. Prior treatment of rats with a combination of 5,7-dihydroxytryptamine and desipramine reduced 5-hydroxytryptamine levels and the levels of [3H]imipramine associated with the synaptosomal fractions to the same extent. It is concluded that imipramine is associated with a binding site localised on 5-hydroxytryptaminergic nerve terminals and that there is a redistribution to other sites (vesicular and microsomal) during the isolation procedure.

Subcellular distributions of calcium/calmodulin-stimulated and guanine nucleotide-regulated adenylate cyclase activities in the cerebral cortex.

The subcellular distribution of Ca2+/calmodulin-stimulated adenylate cyclase activity was studied in comparison with that of guanine nucleotide-stimulated cyclase activity. The distributions of these activities were similar among the crude fractions but differed among the purified subsynaptosomal fractions. The specific activity of Ca2+/calmodulin-stimulated cyclase was highest in a light synaptic membrane fraction, which has few, if any, postsynaptic densities, whereas that of guanine nucleotide-stimulated cyclase was highest in a heavier synaptic membrane fraction rich in postsynaptic densities. These results suggest that the Ca2+/calmodulin-stimulated cyclase has, at least in part, a different cellular or subcellular location than the guanine nucleotide-stimulated cyclase.

Distribution of proteins in different subcellular fractions of rat brain studied by two-dimensional gel electrophoresis

The subcellular distribution of proteins normally visible on two-dimension gels of rat brain tissue punches and crude brain homogenate was investigated using two-dimensional gel electrophoresis and computerized scanning densitometry. Seven enriched subcellular fractions (cytosol, mitochondria, microsomes, nucleus, crude synaptic vesicles, myelin and synaptic membrane) were generated from a crude extract of rat brain. Fifty microgram samples of the crude homogenate and each fraction were then taken and the proteins within these samples separated by two-dimensional gel electrophoresis. Proteins were stained with silver and the gels then analyzed by computerized scanning densitometry. Of 136 proteins visible on two-dimension gels of the crude homogenate that were quantitatively examined, a total of 73 (54%) were identified as being primarily located in a single subcellular fraction. The majority of these 73 proteins were found to be located primarily in either the cytosolic or mitochondrial fractions, while fewer proteins were identified as being primarily located in the microsomal, nuclear or crude synaptic vesicular subfractions. In contrast, the myelin and synaptic membrane fractions were found to be the primary location for only a single protein each that is clearly visible in the crude homogenate. In addition, gels of four of the subfractions (mitochondria, cytosol, nucleus and myelin) contained proteins that are not normally visible on gels generated using a crude extract. The subcellular location of a number of proteins found previously to be altered by specific experimental manipulations was also determined, providing further information on these proteins in brain. These results should prove useful in future experiments designed towards isolating and characterizing specific proteins of neurochemical interest.

Specificity of prostaglandin D 2 binding to synaptic membrane fraction of rat brain

The structural requirement of the prostaglandin D 2 molecule for binding to the synaptic membrane fraction of rat brain was extensively studied by using various prostaglandin D derivatives. Most strict specificity was found in the structures of the cyclo-pentane ring and the double bond in 13,14-position. The addition and deprivation of the double bond in α- and ω-chain, except on 13,14-position, moderately affected the binding. The modification in the car☐yl terminus and ω-chain terminus did not seriously influence the binding. BW 245C and 9-β-prostaglandin D 2, potent agonists for the prostaglandin D 2 receptor in the platelet membrane, were almost ineffective. [ 3H]prostaglandin D 2 binding was not affected by the addition of various neuroactive substances to the binding assay mixture. Further, prostaglandin D 2 did not affect the known neurotransmitter receptor bindings in the rat brain.

Existence of a Ca 2+-dependent K + channel in synaptic membrane and postsynaptic density fractions isolated from canine cerebral cortex and cerebellum, as determined by apamin binding

Apamin, a 18-amino acid neurotoxin isolated from bee venom, is a specific blocker of one class of the Ca 2+-dependent K + channels. The monoiodo derivative of the toxin with high specific radioactivity (1600 Ci/mmol) has been used to study its binding to synaptic. membrane (SM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brains. The B max (30.2 fmol/mg protein) for CTX-PSD is about twice that for CTX-SM (17.3 fmol/mg protein), suggesting a concentration of the apamin receptor protein in CTX-PSD over CTX-SM fractions. The lower value of B max for CL-PSD (12.3 fmol/mg protein), and the higher K d value (51 pM) than for CTX-SM (33 pM), CTX-PSD (24 pM), and and CL-SM (39 pM), may reflect the disruptive effect of Triton X-100 on these thin structures. The values of B max and K d for CTX-SM are similar to those (22.0 fmol/mg protein and 33 pM) for rat CTX-SM 33. Both Ca 2+ and NA + inhibit apamin binding to CTX-PSD with K 0.5 values of 14 and 31 mM, respectively, while the optimum concentration of KCl for activation is 5 mM. All these values are similar to those found for rat synaptosomes 30. Covalent labeling of the apamin binding protein, using the non-cleavable cross-linker, disuccinimidyl suberate, reveals an apamin binding polypeptide of 27 kdaltons under reducing and denaturing conditions in both the CTX-SM and CTX-PSD preparations, similar to that (28 kdaltons) reported for rat CTX-SM fractions 33. Prior phosphorylation of isolated CTX-PSD had no effect on apamin binding, nor did apamin binding influence subsequent phosphorylation of CTX-PSD. Calmodulin, an intrinsic PSD protein, may not play a role in apamin binding to PSD, since addition of calmodulin, or removal of the calmodulin by EGTA treatment, resulted in no change in the binding capacity of the PSD. The apamin binding protein seems to be bound quite firmly in the CTX-PSD fraction since treatments with 0.5% deoxycholate, 1% N-lauroyl sarcosinate, 4 M guanidine-HCl, pH 7.0, 0.5 M KCl, could only remove the apamin-receptor complexes from CTX-PSD by 40, 55, 52, 12 and 15%, respectively. These results contrast with the findings that the two detergents mentioned solubilize 80–93% of the receptor from the synaptosomal or synaptic membrane fractions, indicating that a good deal of the receptor in these fractions is membrane-bound and not connected to the PSD. The results suggest that the apamin binding protein, which spans the postsynaptic membrane, is an intrinsic PSD component. This finding is the first instance of a channel protein being found in a PSD fraction. The present results, together with previous ones showing the presence of GABA, flunitrazepam, and glutamate binding sites in the isolated PSD fraction, indicate that the PSDs are involved in not only the anchoring of membrane receptor proteins, but also in the anchoring of membrane channel proteins, and suggest a mediating role for the PSD in synaptic events.

The subcellular distribution of dl-[ 3H]2-amino-4-phosphonobutyrate binding sites in the rat brain

The subcellular distribution of dl-[ 3H]2-amino-4-phosphonobutyrate binding sites in rat brain was examined. Binding sites for this radioligand, which selectively labels a chloride/calcium-dependent subpopulation of glutamate binding sites, were highly concentrated in synaptic membrane fractions. This is wholly consistent with the proposed involvement of this site in physiological responses.

Gangliosides in axolemmal and synaptic membrane fractions from developing rats: effects of maternal ethanol consumption on offspring.

Previous work from this laboratory has shown that in utero exposure to ethanol significantly alters the synthesis of glycoproteins in synaptic, axolemmal, and myelin membranes from developing rats. In an attempt to determine whether in utero exposure to ethanol similarly alters the synthesis of other glycoconjugates involved with cell-cell interactions, the present study examined the influence of chronic maternal ethanol consumption prior to parturition on the content and synthesis of gangliosides in axolemmal and synaptic plasma membranes from developing rats. The results demonstrate that, in contrast to central nervous system glycoproteins, synaptic and axolemmal glycolipids are minimally affected by in utero exposure to ethanol. At all ages examined (17 to 34 days of age), the offspring of control and ethanol-treated rats had a comparable distribution of radiolabel among synaptic and axolemmal gangliosides, a normal concentration of ganglioside sialic acid in synaptic plasma membranes, and a near-normal distribution of sialic acid among synaptic gangliosides. The present study provides evidence which indicates that the radiolabeling patterns of axolemmal and synaptic membrane gangliosides are similar. Specifically, the most heavily labeled synaptic and axolemmal gangliosides were GT1b (20-37% of the total radioactivity) and GD1a (20-32%). A smaller proportion of radioactivity was associated with GD1b (approximately 11-16%), GM1 (5-10%), and GQ1b (4-11%), as well as with GD3 and the other monosialogangliosides (less than 5%). During the age period examined the proportion of radioactive GT1b increased in both membrane fractions.

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.

Calmodulin-dependent protein phosphatase: immunocytochemical localization in chick retina.

Calmodulin-dependent protein phosphatase, previously called CaM-BP80 or calcineurin, is present in high concentrations in the central nervous system. The level of the phosphatase has been shown by radioimmunoassay to increase during development in the retinas of embryonic and hatching chicks (Tallant, E.A., and W.Y. Cheung, 1983, Biochemistry, 22:3630-3635). The aims of this study are to immunocytochemically localize the phosphatase in developing and mature retinas and to determine if the phosphatase is present in fractions of retinal synaptic membranes and synaptic junctions. Vibratome slices of fixed chick retina and Western blots of detergent-solubilized retinal fractions are both treated sequentially with rabbit primary antisera and goat anti-rabbit Fab fragments conjugated to peroxidase, and then reacted with hydrogen peroxide and diaminobenzidine. The tissue slices are further processed for electron microscopy. This paper demonstrates the presence of peroxidase reaction product in the retina just before synapse formation. In the outer plexiform layer the product is confined to photoreceptor synaptic terminals, whereas in the inner plexiform layer it is present in synaptic terminals of bipolar cells and in dendrites of ganglion cells. In this latter site the product is present postsynaptically at bipolar and amacrine synapses. The phosphatase is detected in Western blots of both synaptic plasma membrane and synaptic junction fractions.

Differential up-regulation of microsomal and synaptic membrane μ opioid receptors

Naltrexone was administered to rats for 7 days by osmotic minipump (5 mg/kg/day) and thereupon, forebrain μ opioid receptor levels in subcellular fractions were monitored by homologous displacement of [ 3H]D-ala 2-mePhe 4-gly-ol 5 enkephalin binding. Microsomes displayed increases in μ receptor concentrations that were twofold greater than those associated with synaptic plasma membrane fractions (92 vs. 51%). Levels in crude membranes rose 77%. Binding affinities were unchanged.

Substrate requirements and subcellular distribution of calcium transport activities in brain membranes.

Ca2+ transport activity in synaptosomal membranes has been identified as having two major components: Ca2+-stimulated ATP hydrolysis and ATP-dependent CA2+ uptake. Both processes exhibit similar affinities for Ca2+ and operate maximally under identical buffer conditions. Subcellular fractionation studies revealed the Ca2+/Mg2+ ATPase and ATP-dependent CA2+ uptake activities to be highest in synaptic plasma membrane fractions 1 and 2, with lesser activity in synaptic vesicles and mitochondria. Progressive treatment with Triton X-100 activated, then decreased Ca2+/Mg2+ ATPase, Mg2+ ATPase and Ca2+ ATPase. ATP-dependent Ca2+ uptake was progressively decreased by similar treatment with Triton X-100. These studies illustrate that Ca2+ ATPase and ATP-dependent Ca2+ uptake may provide two important mechanisms for buffering of cytosolic Ca2+ at the nerve terminal. These systems may function to rapidly sequester cytosolic Ca2+ following a rise during depolarization and then extrude Ca2+ from the terminal against a concentration gradient. This regulation of cytosolic Ca2+, represented by two processes of the type seen in other plasma membranes, may play critical roles in calcium homeostasis in nerve cells.

Comparison of proteins involved with cyclic AMP metabolism between synaptic membrane and postsynaptic density preparations isolated from canine cerebral cortex and cerebellum.

Synaptic membrane and postsynaptic density (PSD) fractions isolated from canine cerebral cortex and cerebellum were assayed for the following proteins: adenylate cyclase and phosphodiesterase (PDE) activities against cyclic AMP and cyclic GMP, the regulatory subunit of the cyclic AMP-dependent protein kinase, and the substrate proteins for this kinase. The results were expressed on the basis of both the protein content of the fractions and the number of synapses in the synaptic membrane fractions. The number of synapses on a constant protein content basis was about three times higher in the cerebral cortex synaptic membrane fraction than in the comparable cerebellar fraction. Adenylate cyclase activity was from 3.4 to 5.6 times higher in the cerebral cortex membrane fraction than in the cerebellar membrane fraction based on protein content but only slightly higher based on synapse counts. PSD fractions had no adenylate cyclase activity. The cyclic AMP-PDE activity was from 17 to 27 times higher in the cerebral cortex membrane fraction than in the cerebellar membrane fraction based on protein content, and about five times higher based on synapse counts. By doing PDE histochemistry at the electron microscopy level it was found that all the cerebral cortex PSDs in the isolated fraction contained PDE activity, none being found associated with the broken-up material in the fraction. The amount of the regulatory subunit of the cyclic AMP-dependent protein kinase was about equal in the two fractions based on protein, but about one-third lower in cerebral cortex fraction than in cerebellar fractions. In the cerebral cortex membrane fraction the primary substrate for the cyclic AMP-dependent protein kinase is synapsin I, with much lower amounts in the cerebellar membrane fraction. The PSD fraction from the two sources also showed these differences in synapsin I content. In the cerebellar membrane fraction, the primary substrate for the enzyme is a approximately 245,000 Mr protein not found in the cerebral cortex membrane fraction. The findings that the turnover of cyclic AMP is much higher in cerebral cortex synapses than in cerebellar synapses, and that differences are found between the cerebral cortex and cerebellum with regard to the substrate proteins for the cyclic AMP-dependent protein kinase indicate a divergence in the effect of cyclic AMP between cerebral cortex and cerebellar synapses.