Studies on the turnover of mouse brain synaptosomal proteins

(l) The half-lives of the proteins of various fractions of whole mouse brain increase with increasing insolubility; the supernatant and hypotonic-extractable proteins had half-lives of about 13 days, whereas the membrane proteins solubilized with Triton X-100 and SLS had half-lives of about 18 days. The proteins of the subfractions of synaptosomes had half-lives ranging from 15 to 19 days; those in the cytoplasm had a half-life of 18·3 days, in the membranes, about 17 days and in the synaptic vesicles, 15·6 days. (2) Although the half-life of the synaptic vesicles was not significantly different from that of other synaptosomal subfractions, the vesicles exhibited a different protein pattern on acrylamide gels, an observation which implies that the proteins of the vesicles are qualitatively different from those of other synaptic membranes. (3) The uptake of labelled lysine into the cytoplasm of the synaptosomes of youg mice in vivo was very rapid. (4) The data derived from the relative specific radioactivities of synaptosomal fractions compared with their whole brain analogs support the contention that a sizeable fraction of the synaptosomal cytoplasmic protein was transported to the synapse by axoplasmic flow. The relative specific radioactivities of synaptosomal membrane and synaptic vesicle proteins rose much more quickly than the comparable activities for the cytoplasmic material, and the alternate possibility of synthesis in situ is discussed.     

On the metabolism of ornithine in synaptosomal preparations

The present results show that ornithine is metabolized to glutamate by isolated synaptosomes from mouse cerebral cortex. Under the experimental conditions used the glutamate was channelled further to the tricarboxylic acid cycle, and to a lesser degree to GABA. The possible significance of these metabolic pathways are discussed. Results of an earlier study suggest an excessive metabolism of ornithine via putrescine to GABA in synaptosomes. Those results could not be verified in the present study and a possible reason for the disagreement is demonstrated. However, the present results suggest that putrescine, which is known to be produced from ornithine elsewhere in the nervous tissue, may be metabolized to GABA in synaptosomes.     

Analysis and separation of synaptosomal membrane proteins

Synaptosomal membrane proteins solubilized with 8% CHAPS-8 M urea were analyzed with twodimensional electrophoresis (2DE). The membrane proteins were resolved up to 250 spots on a 2DE map, ranging in isoelectric points (pI) from 3.5 to 10.0 and molecular weights (MW) from 10 kDa to 200 kDa. Comparison of the mapped proteins of synaptosomal membranes with those of myelin and mitochondorial membranes revealed that synaptosomal membrane proteins were characteristic in the area of pI from 4.0 to 7.5 and MW from 20 kDa to 130 kDa, and that at least 30 spots were synaptosomal membrane-specific proteins. Most of these 30 proteins have not been previously described, named, and characterized Serial numbers (from SY1 to SY30) were assigned to the proteins on the map in order to investigate them systematically. A preliminary attempt to separate synaptosomal membrane proteins was carried out using a reversed-phase HPLC system. Several proteins could either be isolated or enriched. SY10 (pI 4.6; MW 56 kDa) was one of these proteins, and was of particular interest for its unusual behavior on the reversed-phase column, and for its binding to an immobilized protein A-gel.     

S-adenosyl-l-methionine inhibits phosphoinositide metabolism in the rat brain synaptosomal suspensions

S-adenosyl-l-methionine (AdoMet) has been reported to affect events linked to noradrenergic neurotransmission. In the present work, we studied the effect of AdoMet on norepinephrine (NE)-stimulated inositol phosphate production in³H-inositol-labelled crude synaptosomal suspensions of rat brain. AdoMet (50–1000 M) decreased both the synthesis of labelled polyphosphoinositide (30–50%) and the release of inositol mono- and bisphosphate (40–50%). The AdoMet effect was not dependent on NE concentration (10–1000 M), suggesting that the inhibition of inositol phosphate release was not the result of a modification of the norepinephrine binding to its receptor sites. S-adenosyl-L-homocysteine (AdoHcy) (1 mM) an inhibitor of methyltransferase activities, partially inhibited (70%) the AdoMet (0.1 mM) effect, indicating that the methylation processes cannot explain all the effects observed. We conclude that, in addition to previously reported effects of AdoMet on NE transport, AdoMet may reduce NE-linked intracellular signalling.     

Effect of exogenous gangliosides on synaptosomal membrane ATPase activity

Changes in the activity of (Na⁺, K⁺)-ATPase of synaptosomal membranes induced by exogenous gangliosides were studied. Depending on the ganglioside-protein ratio, the enzyme activity was finally reduced to 40% when the ratio, was about 1. By analysis of the reaction kinetics the effect was characterized as a noncompetitive inhibition. Moreover the ganglioside effect, was clearly dependent on the incubation temperature. Since exogenous gangliosides thereby caused a shifting in the optimum temperature of (Na⁺, K⁺)-ATPase, the effect is discussed in terms of changes of the membrane properties. In preincubation experiments it was revealed that the interaction of the glycolipids with synaptosomal membranes itself was temperature dependent and enhanced by ATP. It is suggested that ganglioside micelles might have been incorporated by the membranes in a way comparable to a fusion process.     

Effect of dietary lipid on synaptosomal acetylcholinesterase activity.

The effect of dietary lipid on the thermotropic properties of acetylcholinesterase activity was examined in rat synaptosomal membrane preparations after feeding diets containing soya-bean oil, sunflower oil or soya-bean phosphatidylcholine as the dietary fats. Arrhenius plots and energies of activation were altered by the duration of feeding as a function of time, as well as by the composition of diet fat fed. Animals fed sunflower oil had the highest maximal velocity for acetylcholinesterase activity. The observations of this study suggest that dietary fat is an important determinant of the physicokinetic properties of lipid-dependent functions in brain synaptosomal membranes.     

Ethanol treatment alters beta-endorphin metabolism by purified synaptosomal plasma membranes

Ethanol administration has been shown to affect beta-endorphin (beta-E) levels in most brain areas. Chronic ethanol treatment has also lead to changes in the levels of Met- and Leu-enkephalin which may be due to recent finding that enkephalin A activity is significantly altered. To determine if proteolytic enzymes responsible for beta-E metabolism at the pSPM are also altered, we studied the effect of chronic ethanol (7% v/v; 8 days) administration on in vitro central beta-E metabolism in male C57/BL mice. Purified SPM was time-course incubated with beta-E (20 microM) for 30-120 min and subjected to HPLC analyses for determination of beta-endorphin and related fragments. Chronic ethanol significantly reduced the half-life for beta-E at the pSPM (T1/2 = 50/min) versus controls (T1/2 = 100.4 min). Chronic ethanol also caused significant accumulation of the behaviorally active alpha- and gamma-type endorphins formed at the pSPM. These results suggest that chronic ethanol treatment leads to an increase in the activity of peptidases responsible for beta-E metabolism at pSPM leading to an increased formation of both alpha- and gamma-type endorphins which may affect alcohol related behaviors.     

Effect of dietary level of protein on the metabolism of mouse liver nuclear proteins.

The effect of protein depletion and refeeding on the metabolism of mouse liver nuclear proteins was studied. Five days protein depletion caused a 35% decrease in total nuclear protein. A fast recovery of the lost proteins, except histones, was induced when depleted mice were refed with a normal diet. Depletion caused a decrease in total nuclear protein synthesis, whereas refeeding quickly restored its normal value. The rates of total nuclear protein breakdown were estimated either as the difference between synthesis and protein gain or from the decay of radioactivity in protein labeled by the administration of both sodium [14C]bicarbonate and [35S]methionine. By these procedures, it was found that refeeding caused a slowdown in total nuclear protein breakdown. Hence, the recovery of the protein content observed during refeeding is due to both a restoration of synthesis and a decrease of breakdown. The [14C]bicarbonate procedure did not permit to obtain a high efficiency of label and, therefore, it was unsatisfactory for the measurement of the breakdown of fractionated nuclear proteins. A labeling procedure using [35S]methionine was designed for adequate measures of the decay of radioactivity in these proteins. This allows us to find that a slow down in breakdown affects similarly during refeeding to histones, to non histones, and to a fraction which contains ribonucleoproteins and soluble proteins.     

Effect of calcium ion on the release of gamma-aminobutyric acid from synaptosomal fraction

Brain synaptosomes released endogenous gamma-aminobutyric acid (GABA) in response to Ca2+. The release of GABA in response to 2.5 mM Ca2+ increased linearly with log[K+]0, showing that a membrane potential-dependent Ca2+ channel limits the GABA release. In the presence of Ca2+ ionophore, A23187, GABA release increased linearly with log[Ca2+]0 without altering the membrane potential of synaptosomes.     

Effect of modification of synaptosomal membrane glycoproteins on adenylate cyclase activity

The effect of the modification of synaptosomal membrane glycoproteins on the activity of adenylate cyclase was studied. It was found that the binding of concanavalin A to unmodified guinea pig cerebral cortex synaptosomal membrane did not change adenylate cyclase activity. Concanavalin A binding to synaptosomal membrane of hypoxic brain cortex resulted in no decrease of enzyme activity. The level of protein-bound sialic acid in these synaptosomal fractions was 20% lower than in the control. Treatment of synaptosomal membranes with neuraminidase resulted in a decrease of sialic acid content by about 70%, but it had no significant effect on adenylate cyclase activity. The modification with concanvalin A of sugar end groups exposed by neuraminidase treatment resulted in significant decrease of both basal and fluoride-stimulated adenylate cyclase activity. These results seem to indicate that some component of the adenylate cyclase complex of brain synaptosomal membranes is closely interacting with a carbohydrate-containing macromolecule on the cell surface.This work was supported by, the Polish Academy of Sciences within the project 10.4.     

The effects in vitro of hypoglycaemia and recovery from anoxia on synaptosomal metabolism.

Synaptosomes from several regions of the rat brain were found to exhibit half-maximal rates of 14CO2 output and [14C]acetylcholine synthesis from D-[U-14C]glucose at glucose concentrations approx. 50-fold lower than those required by the brain in situ. However, synaptosomal acetylcholine synthesis was found not to be directly proportional to substrate oxidation as measured by 14CO2 output. When synaptosomes had been exposed to anoxia in vitro, their metabolic indices (14CO2 and [14C]acetylcholine synthesis, and adenine nucleotide levels) were found not to be significantly different from control aerobic values, unless they had been subjected to veratridine depolarization. This is in accord with previous findings that neither the absolute metabolic rates nor the vulnerability to hypoxic damage exhibited by brain in situ is reflected by brain slices in vitro, unless these are stimulated by depolarization. The use of synaptosomes as a model for synaptic damage in vivo is discussed.