Aminoacyl-tRNA-synthetase activity of subcellular fractions of cerebral cortex]

The total activity of aminoacyl-tRNA-synthetases of myelin, synaptic membranes, heavy and light synaptosomes, mitochondria and soluble fractions of rat cerebral cortex was studied. It was found that the highest activity of the enzymes is localized in the fractions of synaptic membranes and heavy and light synaptosomes and is practically absent in the myelin fraction. The specific activity of the total aminoacyl-tRNA-synthetase fraction in the soluble fraction is 2 times as low as compared to the synaptic membranes and light and heavy synaptosomes.     

Subcellular distribution and activation by non-ionic detergents of guanylate cyclase in cerebral cortex of rat

Non-ionic detergents stimulated particulate guanylate cyclase activity in cerebral cortex of rat 8- to 12-fold while stimulation of soluble enzyme was 1.3- to 2.5-fold. Among various detergents, Lubrol PX was the most effective one. The subcellular distribution of guanylate cyclase activity was examined with or without 0.5% Lubrol PX. Without Lubrol PX two-thirds of the enzyme activity was detected in the soluble fraction. In the presence of Lubrol PX, however, two-thirds of guanylate cyclase activity was recovered in the crude mitochondrial fraction. Further fractionation revealed that most of the particulate guanylate cyclase activity was associated with synaptosomes. The sedimentation characteristic of the particulate guanylate cyclase activity was very close to those of choline acetyltransferase and acetylcholine esterase activities, two synaptosomal enzymes. When the crude mitochondrial fraction was subfractionated after osmotic shock, most of guanylate cyclase activity as assayed in the absence of Lubrol PX was released into the soluble fraction while the rest of the enzyme activity was tightly bound to synaptic membrane fractions. The total guanylate cyclase activity recovered in the synaptosomal soluble fraction was 6 to 7 times higher than that of the starting material. The specific enzyme activity reached more than 1000 pmol per min per mg protein, which was 35-fold higher than that of the starting material. The membrane bound guanylate cyclase activity was markedly stimulated by Lubrol PX. Guanylate cyclase activity in the synaptosomal soluble fraction, in contrast, was suppressed by the addition of Lubrol PX. The observation that most of guanylate cyclase activity was detected in synaptosomes, some of which was tightly bound to the synaptic membrane fraction upon hypoosmotic treatment, is consistent with the concept that cyclic GMP is involved in neural transmission.     

Autophosphorylation of chick brain synaptic polypeptides. Phosphorylation of proteins during incubation of intact synaptosomes with 32PO4

Chick brain synaptosomes incorporated phosphate into proteins when incubated in physiological buffer containing energy sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that three synaptosomal polypeptides were significantly phosphorylated after 15 sec incubation while at least fifteen polypeptides were active kinase substrates after 15 min incubation. Labeled synaptosomes were hypotonically lysed and separated by centrifugation into soluble, membrane, and mitochondrial fractions. Every fraction exhibited significant phosphate incorporation. Electrophoresis revealed that each fraction had several unique phosphorylated polypeptides and a distinctive phosphorylation pattern. The same polypeptides appear to be labeled whether MgATP was added to synaptic plasma membranes or synaptic plasma membranes were isolated after synaptosomal autophosphorylation.     

Preparation and properties of mitochondria derived from synaptosomes.

A method has been developed whereby a fraction of rat brain mitochondria (synaptic mitochondria) was isolated from synaptosomes. This brain mitochondrial fraction was compared with the fraction of "free" brain mitochondria (non-synaptic) isolated by the method of Clark & Nicklas (1970). (J. Biol. Chem. 245, 4724-4731). Both mitochondrial fractions are shown to be relatively pure, metabolically active and well coupled. 2. The oxidation of a number of substrates by synaptic and non-synaptic mitochondria was studied and compared. Of the substrates studied, pyruvate plus malate was oxidized most rapidly by both mitochondrial populations. However, the non-synaptic mitochondria oxidized glutamate plus malate almost twice as rapidly as the synaptic mitochondria. 3. The activities of certain tricarboxylic acid-cycle and related enzymes in synaptic and non-synaptic mitochondria were determined. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) activities were similar in both fractions, but pyruvate dehydrogenase (EC 1.2.4.1) activity in non-synaptic mitochondria was higher than in synaptic mitochondria and glutamate dehydrogenase (EC 1.4.1.3) activity in non-synaptic mitochondria was lower than that in synaptic mitochondria. 4. Comparison of synaptic and non-synaptic mitochondria by rate-zonal separation confirmed the distinct identity of the two mitochondrial populations. The non-synaptic mitochondria had higher buoyant density and evidence was obtained to suggest that the synaptic mitochondria might be heterogeneous. 5. The results are also discussed in the light of the suggested connection between the heterogeneity of brain mitochondria and metabolic compartmentation.     

THE EFFECT OF ELECTRICAL STIMULATION ON THE TURNOVER OF PHOSPHATIDIC ACID IN SYNAPTOSOMES FROM GUINEA-PIG BRAIN

Synaptosomes prepared from guinea-pig cerebral cortex were suspended in a medium containing [³²P]orthophosphate and subjected to electrical stimulation. When the synaptosomal phospholipids were subsequently separated, the most highly labelled was phosphatidic acid and electrical stimulation over a 10 min period increased incorporation of ³²P₁ into this lipid. Stimulated synaptosomes were osmotically lysed and subsynaptosomal fractions isolated. The electrically stimulated increase in phosphatidic acid labelling was localized in a fraction enriched in synaptic vesicles. This phospholipid effect was not merely a reflection of an increased specific radioactivity of synaptosomal ATP, due to the electrically stimulated increase in respiration. The time course of the phosphatidic acid effect suggests that it is synchronous with release of transmitter.     

Pyridoxal phosphate and glutamate decarboxylase in subcellular particles of mouse brain and their relationship to convulsions

The subcellular distribution of pyridoxal phosphate (PLP) was studied in mouse brain, as well as the effect of pyridoxal phosphate-γ-glutamyl hydrazone (PLPGH—a convulsant drug which decreases both PLP levels and glutamate decarboxylase activity [GAD] in whole brain) upon both the PLP concentration and the GAD activity in subcellular fractions. An electron microscopic evaluation of the subcellular particles of control and PLPGH-treated animals was also carried out. The main findings were the following: (1) PLP was localized mainly in the supernatant and crude mitochondrial fractions; two-thirds of the amount present in the latter were located in the subfraction containing pure mitochondria, and the remainder was in the synaptosomal fraction. After osmotic disruption of synaptosomes, PLP was found in both the intrasynaptosomal mitochondria and the synaptoplasm. (2) Treatment of mice with PLPGH decreased levels of PLP in several brain fractions, this effect being much more notable in the soluble fractions than in the particulate fractions. After osmotic disruption of the synaptosomes, a specific decrease of PLP in the synaptoplasm was observed. (3) Treatment with PLPGH produced also an inhibition of GAD activity in most of the fractions studied, when this enzyme was assayed in the absence of PLP. In general, the inhibition was greater in those fractions in which levels of PLP were also affected. In synaptosomes, this correlation between the decreased levels of PLP and decreased activity of GAD occurred only in the synaptoplasm. (4) The activation of GAD by PLP added to incubation mixtures was much greater in those fractions from PLPGH-treated animals which displayed extensive inhibition of GAD, in comparison to the corresponding fractions from control animals. (5) No ultrastructural changes were detected in the subcellular fractions from treated animals. Our results show that the decreases of both the levels of PLP and the activity of GAD (as previously found in whole brain) actually occur in the synaptosomes, a finding that supports the hypothesis that the role of PLP in the mechanisms controlling excitability can be explained, at least in part, by its regulatory action on GAD activity, which in turn determines the rate of GABA synthesis at the nerve endings.     

Localization of Thiamine-Binding Protein in Synaptosomes from the Rat Brain

Using preparations of synaptosomes and subsynaptosomal fractions from the rat brain, we studied the localization of thiamine-binding protein (TBP) in the subcellular structures of the neurons. In addition, we studied the distribution in synaptosomes of two types of activity typical of TBP (thiamine triphosphatase and thiamine-binding activities), as well as the effects of factors destroying the plasma membrane of synaptosomes on binding of [¹⁴C]thiamine with the latter. We found that the thiamine-associated activity of TBP was the highest in fractions of the synaptic vesicles and plasma membranes. Hydrolysis of thiamine triphosphate was also most active in these structures. Our results allow us to conclude that TBP is localized mostly in the synaptic vesicles and plasma membranes of synaptosomes.     

The structure of postsynaptic densities isolated from dog cerebral cortex: I. overall morphology and protein composition

A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of methods of Davis and Bloom (21, 22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resoltuion SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nuclei acids, and very little phospholipids being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 percent that of a mitochondrial fraction) and a small amount of 5'- nucleotidase activity (of a specific activity between 6 and 7 percent that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures approximately 400nm long and approximately 40nm wide, made up of apparent particles 13-28nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter approximately 400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 or more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing (125)I-labeled myelin, synaptic vesicle, and mitochondrial fraction proteins with synaptosomes, and then isolating the density fraction from the mixture, it was concluded that a major 26,000 mol wt density fraction protein was common to both mitochondria and density, that none of the proteins of the density were contaminants from the mitochondrial fraction, that a minor approximately 150,000 band was a contaminant from the synaptic vesicle fraction, and that the moderately staining PSD fraction protein of 17,000 mol wt band was the result of contamination by the major basic protein of myelin. On the basis of the marker enzymatic assays and the mixing experiments, it is considered that the density fraction is moderately pure biochemically, and that its protein composition, aside from a few exceptions noted above, reflects its in situ character.     

EFFECTS OF ACETYLCHOLINE ON THE IN CORPORATION OF [32P]ORTHOPHOSPHATE IN VITRO INTO THE PHOSPHOLIPIDS OF SUBSYNAPTOSOMAL MEMBRANES FROM GUINEA-PIG BRAIN

-Synaptosomes prepared from guinea-pig cerebral cortex were incubated with ³²P₁ in a medium with or without 10^?4 M-acetylcholine and 10^?4 M-eserine. They were then subjected to osmotic shock and density-gradient centrifugation for the preparation of subsynaptosomal fractions and the phospholipids of each fraction were separated by two-dimensional thin-layer chromatography. The fraction containing synaptic vesicles and that containing mitochondria were the most highly labelled of the sub-synaptosomal fractions. Phosphatidic acid followed by phosphatidylinositol had the highest specific activity of the phospholipids studied. Acetylcholine caused a marked increase in the specific activity of the vesicular but not of the mitochondrial phosphatidic acid. Phosphatidylinositol specific activity also increased in the presence of acetylcholine but the increase was more reproducible in the fraction containing microsomal membranes than in the vesicle fraction. The other phospholipids were relatively poorly labelled and no effect of acetylcholine on the incorporation of ³²P₁ into these lipids could be detected. Acetylcholine also caused a decrease in the amount of phosphatidic acid in the synaptic vesicles.     

Separation of synaptic junctional complexes from thin layers of rabbit cerebral cortex

Synaptic junctional fractions were separated from rabbit brain by procedures based on combining the methods of Cotman and Taylor [4], Orosz et al. [16, 17] and Lisman et al. [13]. Thin layers of cerebral cortices were homogenized to obtain a crude mitochondrial-synaptosomal fraction. The sedimentation rates of mitochondria and mitochondria containing synaptosomes were increased by raising the density of mitochondria with an insoluble dense formazan deposit inside mitochondria after iodo-nitrotetrazolium treatment. The synaptic plasma membrane fraction isolated by this method contained no mitochondrial contamination. After Triton X-100 treatment the insoluble residues of the detergent were centrifuged through discontinuous sucrose gradients. A great enrichment of morphologically identifiable intact synaptic junctions was observed in some of the obtained interface layers.     

Subcellular distribution of GTP-binding proteins, Go and Gi2, in rat cerebral cortex

The localization of GTP-binding protein (G-protein) subunits, Go alpha, Gi2 alpha and beta, in subcellular fractions of rat cerebral cortex was determined by means of immunoassays specific for the respective subunits. High concentrations of all three subunits were observed in both crude mitochondrial and microsomal fractions. Muscarinic cholinergic receptors were also densely localized in these fractions. Then the crude mitochondrial and microsomal fractions were subfractionated by sucrose density gradient centrifugation. Each fraction obtained was evaluated morphologically by electron microscopy and biochemically by determination of membrane markers. The crude mitochondrial fraction was subfractionated into myelin, synaptic plasma membrane, and mitochondrial fractions. All the G-protein subunits examined and muscarinic receptors were exclusively localized in the synaptic plasma membrane fraction. Among the submicrosomal fractions, the heavy smooth-surfaced microsomal fraction showed the highest concentrations of all G-protein subunits and receptors, while the rough-surfaced microsomal fraction contained low amounts of them. The heavy smooth-surfaced microsomal fraction also contained high specific activity of (Na(+)-K+)-ATPase, a marker of the plasma membrane. These results indicated that the Go alpha, Gi2 alpha and beta subunits are mainly localized in the plasma membrane in the brain.     

Phosphatidic acid metabolism,calcium ions and transmitter release from electrically stimulated synaptosomes

Synaptosomes isolated from guinea pig brain cortex were stimulated electrically in a medium containing [32P]-orthophosphate. The electrical stimulation caused increased labelling of phosphatidic acid in a synaptic vesicle fraction prepared by osmotic shock of the incubated synaptosomes. Electrical stimulation also provokes transmitter release from the synaptosomes. Both increased phosphatidate labelling and transmitter release required calcium ions in the medium. The effects are discussed in relation to earlier work with acetylcholine and the possible involvement of membrane phosphatidic acid in transmitter release by exocytosis.     

ISOLATION AND PARTIAL CHARACTERIZATION OF FRACTIONS ENRICHED IN SYNAPTOSOMES FROM CHICK BRAIN

–From a pool of hemispheres, optic lobes and cerebellum of chick 3 fractions containing synaptosomes have been prepared. They were obtained by subcellular fractionation of a homogenate and centrifugation of a crude mitochondrial suspension on a discontinuous Ficoll density gradient in iso-osmoticsucrose. The synaptosomal fractions were isolated from bands at the interface of 5–9, 9–12 and 12–16% Ficoll. The characterization of these fractions by marker enzymes, such as lactate dehydrogenase, acetyl-cholinesterase, monoamine oxidase, acid phosphatase and rotenone-sensitive and -insensitive NADH: cytochrome c reductase is reported. Electron microscopic analyses showed that the first fraction (AB) at the 5–9% Ficoll interface contained myelin and other membrane fragments as well as synaptosomes, the second fraction (C) at the 9–12% Ficoll interface contained mainly synaptosomes, and the third fraction (D) at the 12–16% Ficoll interface contained synaptosomes and free mitochondria. A fourth fraction (E) was obtained as a pellet, and was enriched in free mitochondria. There was fair agreement between the distribution pattern of the marker enzyme activities and the particles of the fractions seen by electron microscopy. The content of glycoprotein-bound N-acetylneuraminic acid and total phospholipid of these fractions has been determined. Relative to the mitochondrial fraction (E) the synaptosome fraction contained on basis of particulate protein, respectively, 2–3 times as much protein-bound N-acetylneuraminic acid and 10–20 per cent more total phospholipid.     

Anatomical localization of protease-activated receptor-1 and protease-mediated neuroglial crosstalk on peri-synaptic astrocytic endfeet

We studied the localization, activation and function of protease-activated receptor 1 (PAR-1) at the CNS synapse utilizing rat brain synaptosomes and slices. Confocal immunofluoresence and transmission electron microscopy in brain slices with pre-embedding diaminobenzidine (DAB) immunostaining found PAR-1 predominantly localized to the peri-synaptic astrocytic endfeet. Structural confocal immunofluorescence microscopy studies of isolated synaptosomes revealed spherical structures stained with anti-PAR-1 antibody which co-stained mainly for glial-filament acidic protein compared with the neuronal markers synaptophysin and PSD-95. Immunoblot studies of synaptosomes demonstrated an appropriate major band corresponding to PAR-1 and activation of the receptor by a specific agonist peptide (SFLLRN) significantly modulated phosphorylated extracellular signal-regulated kinase. A significant membrane potential depolarization was produced by thrombin (1 U/mL) and the PAR-1 agonist (100 μM) and depolarization by high K(+) elevated extracellular thrombin-like activity in the synaptosomes preparation. The results indicate PAR-1 localized to the peri-synaptic astrocytic endfeet is most likely activated by synaptic proteases and induces cellular signaling and modulation of synaptic electrophysiology. A protease mediated neuron-glia pathway may be important in both physiological and pathological regulation of the synapse.     

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.     

Amyloid beta-peptide effects on synaptosomes from apolipoprotein E-deficient mice

Apolipoprotein E (apoE) is present in the brain and may contribute to neurophysiologic or neuropathologic events, depending on environmental and genetic influences. Recent studies indicate a role for apoE in synaptic plasticity and maintenance of synaptic membrane symmetry, suggesting that apoE may be involved in regulating synaptic homeostasis. In the present study, cerebrocortical synaptosomes were prepared from transgenic mice lacking apoE (apoE KO) to analyze the possible contribution of apoE toward maintaining homeostasis in synaptosomes. Synaptosomal preparations from apoE KO and wild-type mice exhibited similar basal levels of reactive oxygen species, mitochondrial function, and caspase activity; however, following application of amyloid beta-peptide [Abeta(1-40)], apoE KO synaptosomes displayed increased levels of oxidative stress, mitochondrial dysfunction, and caspase activation compared with synaptosomes from wild-type mice. Synaptosomal membranes from apoE KO mice were more fluid than wild-type synaptosomes and contained higher levels of thiobarbituric acid-reactive substances, consistent with elevated levels of lipid peroxidation occurring in the synapses of apoE KO mice. Together, these data are consistent with a role for apoE in maintaining homeostasis by attenuating oxidative stress, caspase activation, and mitochondrial homeostasis in synapses.     

Voltage-Dependent Anion Channel Proteins in Synaptosomes of the Torpedo Electric Organ: Immunolocalization, Purification, and Characterization

In this study, we purified and characterized the voltage-dependent anion channel (VDAC) from the Torpedo electric organ. Using immunogold labeling, VDAC was colocalized with the voltage-gated Ca²⁺ channel in the synaptic plasma membrane. By immunoblot analysis, five protein bands in synaptosomes isolated from the Torpedo electric organ cross reacted with two monoclonal anti-VDAC antibody. No more than about 7 to 10% mitochondrial contains could be detected in any synaptosomal membrane preparation tested. This was estimated by comparing the specific activity in mitochondria and synaptosomes of succinate–cytochrome-c oxidoreductase and antimycin-insensitive NADH–cytochrome-c oxidoreductase activities; mitochondrial inner and outer membrane marker enzymes, respectively. [¹⁴C]DCCD (dicyclohexylcarbodiimide), which specifically label mitochondrial VDAC, labeled four 30–35 kDa protein bands that were found to interact with the anti-VDAC antibody. The distribution of the Torpedo VDAC protein bands was different among membranes isolated from various tissues. VDAC was purified from synaptosomes and a separation between two of the proteins was obtained. The two purified proteins were characterized by their single channel activity and partial amino acid sequences. Upon reconstitution into a planar lipid bilayer, the purified VDACs showed voltage-dependent channel activity with properties similar to those of purified mitochondrial VDAC. Amino acid sequence of four peptides, derived from VDAC band II, exhibited high homology to sequences present in human VDAC1 (98%), VDAC2 (91.8%), and VDAC3 (90%), while another peptide, derived from VDAC band III, showed lower homology to either VDAC1 (88.4%) or VDAC2 (79%). Two more peptides show high homology to the sequence present in mouse brain VDAC3 (100 and 78%). In addition, we demonstrate the translocation of ATP into synaptosomes, which is inhibited by DCCD and by the anion transport inhibitor DIDS. The possible function of VDAC in the synaptic plasma membrane is discussed.     

Beneficial effects of dietary restriction on cerebral cortical synaptic terminals: preservation of glucose and glutamate transport and mitochondrial function after exposure to amyloid beta-peptide, iron, and 3-nitropropionic acid

Recent studies have shown that rats and mice maintained on a dietary restriction (DR) regimen exhibit increased resistance of neurons to excitotoxic, oxidative, and metabolic insults in experimental models of Alzheimer's, Parkinson's, and Huntington's diseases and stroke. Because synaptic terminals are sites where the neurodegenerative process may begin in such neurodegenerative disorders, we determined the effects of DR on synaptic homeostasis and vulnerability to oxidative and metabolic insults. Basal levels of glucose uptake were similar in cerebral cortical synaptosomes from rats maintained on DR for 3 months compared with synaptosomes from rats fed ad libitum. Exposure of synaptosomes to oxidative insults (amyloid beta-peptide and Fe(2+)) and a metabolic insult (the mitochondrial toxin 3-nitropropionic acid) resulted in decreased levels of glucose uptake. Impairment of glucose uptake following oxidative and metabolic insults was significantly attenuated in synaptosomes from rats maintained on DR. DR was also effective in protecting synaptosomes against oxidative and metabolic impairment of glutamate uptake. Loss of mitochondrial function caused by oxidative and metabolic insults, as indicated by increased levels of reactive oxygen species and decreased transmembrane potential, was significantly attenuated in synaptosomes from rats maintained on DR. Levels of the stress proteins HSP-70 and GRP-78 were increased in synaptosomes from DR rats, consistent with previous data suggesting that the neuroprotective mechanism of DR involves a "preconditioning" effect. Collectively, our data provide the first evidence that DR can alter synaptic homeostasis in a manner that enhances the ability of synapses to withstand adversity.     

THE LABELLING OF NERVE ENDING PHOSPHOLIPIDS IN GUINEA-PIG BRAIN IN VIVO AND THE EFFECT OF ELECTRICAL STIMULATION ON PHOSPHATIDYLINOSITOL METABOLISM IN PRELABELLED SYNAPTOSOMES

The intracerebral injection of ³²P_i into guinea-pig cortex resulted in a steady rate of incorporation into all phospholipids over a 20 h period. The specific radioactivities of phosphatidate and phos-phatidylinositol in synaptosomes prepared from cortex prelabelled, in vivo, were at a maximum after 2 h and the respective activities were 3–8 times higher than in whole cortex. This peak in labelling corresponded with the maximum specific activity of the brain ATP. No similar differential labelling pattern was observed for phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine. Electrical stimulation of the prelabelled synaptosomes produced a rapid drop in the specific activity of phosphatidylinositol and phosphatidate and an increase in the specific activity of CDP-diacylglycerol. The specific activity of synaptosomal ATP was not affected. Study of the subsynaptosomal fractions obtained after osmotic rupture of the synaptosomes revealed that the most highly labelled phosphatidylinositol was in the synaptic vesicle fraction (D) and the most active phosphatidate was in a ‘microsomal’ fraction (E). Electrical stimulation caused a loss of phosphatidylinositol radioactivity from fraction D and a loss of phosphatidate radioactivity from fraction E. The specific activity of these lipids in other fractions was not affected. A possible role for presynaptic phosphatidylinositol is suggested.     

Localization and subcellular distribution of smg p25A, a ras p21-like GTP-binding protein, in rat brain

We have made a monoclonal antibody which specifically recognizes smg p25A among many ras p21/ras p21-like GTP-binding proteins thus far purified from bovine brain membranes. By use of this antibody, we have investigated the localization and subcellular distribution of smg p25A in rat brain by light and electron microscopic immunocytochemistry and by immunoblotting. By light microscopic immunocytochemistry, specific immunoreactivity is widely distributed, most abundant in neuropil, weak in neuronal somata, and absent from white matter. By electron microscopic immunocytochemistry, intense labeling is demonstrated on most of the synapses and concentrated in the presynaptic area where synaptic vesicles are observed. Presynaptic plasma membranes are weakly labeled but mitochondria, postsynaptic plasma membranes, and postsynaptic densities are unlabeled. In subcellular fractionation analysis of cerebrum, about one-fifth of smg p25A is found in the soluble cytosol fraction and the rest is found in the particulate fraction. About half of the particulate-bound smg p25A is recovered in the P2 fraction containing synaptosomes, mitochondria, and myelin, among which a major portion of smg p25A is recovered in the synaptosomal fraction. In the synaptosomal fraction, smg p25A is concentrated about 8-fold in the fraction containing synaptic vesicles and about 3-fold in the fraction containing synaptic plasma membranes compared with the original homogenate. smg p25A is present at a low level in the fraction containing synaptosomal soluble substances but almost absent from the fractions containing intrasynaptosomal mitochondria or post-synaptic densities. These results suggest that smg p25A plays important roles in the regulation of synaptic functions such as exo-endocytotic recycling of synaptic vesicles during neurotransmitter release.