Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.

Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi, incorporated 32P into a variety of specific proteins. Veratridine and high (60 mM) K+, which increase Ca2+ transport across membranes, through a mechanism involving membrane depolarization, as well as the calcium ionophore A23187, each markedly stimulated the incorporation of 32P into two specific proteins (80,000 and 86,000 daltons) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. All three agents failed to stimulate protein phosphorylation in calcium-free medium containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Moreover, the Ca2+-dependent protein phosphorylation could be reversed by the addition of sufficient EGTA to chelate all free extracellular Ca2+. Veratridine, high K+, and A23187 also stimulated 45Ca2+ accumulation by synaptosomes. Tetrodotoxin blocked the stimulation both of protein phosphorylation and of 45Ca2+ accumulation by veratridine but not by high K+ or A23187. Cyclic nucleotides and several putative neurotransmitters were without effect on protein phosphorylation in these intact synaptosome preparations. The absence of any endogenous protein phosphorylation in osmotically shocked synaptosome preparations incubated with 32Pi, and the inability of added [gamma-32P]ATP to serve as a substrate for veratridine-stimulated protein phosphorylation in intact preparations, indicated that the Ca2+-dependent protein phosphorylation occurred within intact subcellular organelles. Fractionation of a crude synaptosome preparation on a discontinuous Ficoll/sucrose flotation gradient indicated that these organelles were synaptosomes rather than mitochondria. The data suggest that conditions which cause an accumulation of calcium by synaptosomes lead to a calcium-dependent increase in phosphorylation of specific endogenous proteins. These phosphoproteins may be involved in the regulation of certain calcium-dependent nerve terminal functions such as neurotransmitter synthesis and release.     

Dephosphin, a 96,000 Da substrate of protein kinase C in synaptosomal cytosol, is phosphorylated in intact synaptosomes

A 96,000 dalton phosphoprotein, called dephosphin, is phosphorylated in intact synaptosomes from rat brain and is rapidly dephosphorylated upon depolarisation-dependent calcium entry. A 96,000 dalton phosphoprotein is also a substrate of protein kinase C in synaptosomal cytosol, and the aim of the study was to determine whether the two proteins may be the same. Dephosphin in intact synaptosomes and the 96,000 dalton protein kinase C substrate comigrated on polyacrylamide gels. Both phosphoproteins had identical phosphopeptide maps after digestion with V8 protease. Both phosphoproteins ran on isoelectric focussing gels with a pI of 6.3-6.7 and focussed as a series of 5-6 spots. Both proteins were phosphorylated exclusively on serine. Both proteins could be resolved into a doublet on longer polyacrylamide gels. The two subunits were of 96 and 93 kDa in both phosphorylation conditions and had dissimilar phosphopeptide maps. However, phosphopeptide maps of either the 96 or 93 kDa subunits were identical in intact synaptosomes compared with synaptosomal cytosol. These results show that a phosphoprotein phosphorylated in intact synaptosomes and a 96,000 dalton protein kinase C substrate from rat brain synaptosomal cytosol are the same, and raise the possibility that protein kinase C is the protein kinase responsible for dephosphin phosphorylation in intact synaptosomes.     

Optimal fixation of chick synaptosomes using different osmotic pressures

Chick synaptosomes were found to remain intact following fixation in the presence of 0.2 M sucrose or 0.3 M sucrose. Increasing the sucrose concentration up to 0.4 M significantly decreased the number of intact synaptosomes; however, typical intrasynaptosomal organelles such as synaptic vesicles and mitochondria were visible in all preparations. This work suggests possibly important species differences in synaptosomal structure between chick and rat synaptosomes.     

Synaptosomal Calcium Uptake Systems: Prostaglandins Are Probably Not Involved in the Regulation of Calcium Fluxes Into and Within the Nerve Endings

Abstract: ⁴⁵Ca²⁺ uptake measurements were performed on intact and osmotically lysed synaptosomes from rat brain to study the possible influence of prostaglandins (PGs) on Ca²⁺ movements into and within the nerve endings. The K⁺-induced ⁴⁵Ca²⁺ uptake of intact synaptosomes was not influenced by several inhibitors of PG synthesis. ⁴⁵Ca²⁺ uptake in lysed synaptosomal preparations was promoted by ATP and seemed to be largely attributable to mitochondria, as it was inhibited by mitochondrial poisons. This Ca²⁺ uptake was strongly reduced by PG synthesis inhibitors but also by PG precursor fatty acids. Both PG synthesis inhibitors and precursors, according to their relative efficacy in blocking Ca²⁺ uptake, were able to induce Ca²⁺ efflux from preloaded intrasynaptosomal organelles. The PGs E₂, F_2α, D₂, and thromboxane B₂ were without effect on ⁴⁵Ca²⁺ uptake in lysed synaptosomal preparations. On the basis of our results it does not seem likely that PGs influence Ca²⁺ availability by modulating Ca²⁺ fluxes into or within the nerve endings. The observed inhibitory effects of PG synthesis inhibitors and precursors on the intrasynaptosomal Ca²⁺ uptake might be due to unspecific impairment of mitochondrial functions.     

Nicotinamide cofactors (NAD and NADP) in glyoxysomes,mitochondria, and plastids isolated from castor bean endosperm

Glyoxysomes, mitochondria, and plastids were separated from the cytosol of germinating castor bean endosperm by sucrose gradient centrifugation in a vertical rotor (25 min, 50,000g_av). The amounts of nicotinamide cofactors, NAD(H) and NADP(H), retained in the isolated organelle fractions were measured by enzyme cycling techniques. The NAD(H) was equally distributed between the cytosol and the mitochondria with a small amount in the glyoxysomes. The mitochondria retained 4 pmol of NAD(H)/ μg protein, about seven times as much as the glyoxysomes. Most of the NADP(H) was in the cytosol. However, the glyoxysomes and plastids retained significant amounts, both having 0.3 pmol NADP(H)/μg protein, twice that in the mitochondria. The subcellular distribution of NADP(H) was compared to the location of dehydrogenases capable of using this cofactor. The cytosol and plastids contained 6-phosphogluconate dehydrogenase. NADP isocitrate dehydrogenase was found in the glyoxysomes, in mitochondria, and in an unidentified subcellular fraction obtained at 1.16 g/ml in the density gradients. Knowledge of the quantities of NADP(H) and NAD(H) retained in the isolated organelles should make it possible to investigate their reduction and reoxidation in intact organelles.     

Studies on the biogenesis of an enzymatically active complex III of the respiratory chain from yeast mitochondria

Complex III isolated from yeast mitochondria catalyzed an antimycin A and Diuron-sensitive coenzyme QH₂-cytochrome c reductase activity with a turnover number of 15.7 sec^?1 and contained 10 nmoles of cytochrome b and 4.6 nmoles of cytochrome c₁ per mg of protein. Electrophoresis in sodium dodecyl sulfate acrylamide gels resolved Complex III into 10 bands with apparent molecular weights of 50,000, 40,000, 30,000, 29,000, 24,000, 17,000, 16,000, 12,000, 8,400, and 5,800. Yeast cells were labeled under nongrowing conditions with (³⁵S)-methionine in the absence or presence of inhibitors of cytoplasmi? or mitochondrial protein synthesis. Labeled Complex III was isolated by immunoprecipitation from detergent-solubilized mitochondria using antiserum raised against the purified complex. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis revealed that a 30,000-dalton protein, cytochrome b, as well as 16,000-dalton protein were labeled in the presence of cycloheximide, indicating that they are products of mitochondrial protein synthesis. Immunoprecipitates from mitochondria obtained from cells labeled in the presence of chloramphenicol contained a new radioactive peak with a molecular weight of 100,000. In addition, significant decreases in the labeling of the proteins with molecular weights of 50,000, 40,000, 30,000, and 16,000 were observed. When Complex III was isolated by immunoprecipitation from intact spheroplasts after a 5-minute pulse with (³⁵S)-methionine, the 100,000-dalton protein was labeled in the immunoprecipitate whether or not chloramphenicol was present; however, after a 1-hour chase with unlabeled methionine, decreased labeling of the 100,000-dalton protein was observed concomitant with an increased labeling of the 50,000- and 40,000-dalton proteins. These results suggest that a protein with a molecular weight of 100,000 may either be a precursor or a partially assembled form of other proteins of Complex III, most probably the two largest polypeptides.     

Isolation of mitochondria from rat brain using Percoll density gradient centrifugation

We have developed procedures that combine differential centrifugation and discontinuous Percoll density gradient centrifugation to isolate mitochondria from rat forebrains and brain subregions. The use of Percoll density gradient centrifugation is central to obtaining preparations that contain little contamination with synaptosomes and myelin. Protocols are presented for three variations of this procedure that differ in their suitability for dealing with large or small samples, in the proportion of total mitochondria isolated and in the total preparation time. One variation uses digitonin to disrupt synaptosomes before mitochondrial isolation. This method is well suited for preparing mitochondria from small tissue samples, but the isolated organelles are not appropriate for all studies. Each of the procedures produces mitochondria that are well coupled and exhibit high rates of respiratory activity. The procedures require an initial setup time of 45-75 min and between 1 and 3 h for the mitochondrial isolation.     

Localization of nitric-oxide synthase in plant peroxisomes

The presence of nitric-oxide synthase (NOS) in peroxisomes from leaves of pea plants (Pisum sativum L.) was studied. Plant organelles were purified by differential and sucrose density gradient centrifugation. In purified intact peroxisomes a Ca(2+)-dependent NOS activity of 5.61 nmol of L-[(3)H]citrulline mg(-1) protein min(-1) was measured while no activity was detected in mitochondria. The peroxisomal NOS activity was clearly inhibited (60-90%) by different well characterized inhibitors of mammalian NO synthases. The immunoblot analysis of peroxisomes with a polyclonal antibody against the C terminus region of murine iNOS revealed an immunoreactive protein of 130 kDa. Electron microscopy immunogold-labeling confirmed the subcellular localization of NOS in the matrix of peroxisomes as well as in chloroplasts. The presence of NOS in peroxisomes suggests that these oxidative organelles are a cellular source of nitric oxide (NO) and implies new roles for peroxisomes in the cellular signal transduction mechanisms.     

Protein synthesis by brain-cortex mitochondria. Characterization of a 55S mitochondrial ribosome as the functional unit in protein synthesis by cortex mitochondria and its distinction from a contaminant cytoplasmic protein-synthesizing system

Homogenates of rat brain cortex were fractionated by conventional methods of velocity sedimentation and separated into a microsomal and a washed mitochondrial fraction. By electron microscopy the mitochondrial fraction was shown to be rich in synaptosomes. The mitochondria-synaptosome fraction synthesized protein in vitro by a route that was partially inhibited by cycloheximide and partly by chloramphenicol. The relative effectiveness of the two inhibitors varied greatly with the medium used. In the mitochondria-synaptosome fraction active 80S cytoplasmic ribosomes and active 55S mitochondrial ribosomes were detected; these were also seen in the electron microscope. Mild osmotic shock of the mitochondria-synaptosome fraction followed by velocity sedimentation in sucrose-EDTA allowed isolation of a mitochondrial fraction free of synaptosomes. Protein synthesis in this fraction was entirely inhibited by chloramphenicol, but was completely resistant to cycloheximide both in a medium promoting oxidative phosphorylation and in ATP-generating medium. Ouabain had no inhibitory effect on protein synthesis in a purified mitochondrial preparation. It is concluded that brain-cortex mitochondria synthesize protein entirely on 55S mitochondrial ribosomes.     

Oxidative Stress Induces a Form of Programmed Cell Death with Characteristics of Both Apoptosis and Necrosis in Neuronal Cells

Abstract: Oxidative stress is implicated in a number of neurological disorders including stroke, Parkinson's disease, and Alzheimer's disease. To study the effects of oxidative stress on neuronal cells, we have used an immortalized mouse hippocampal cell line (HT-22) that is particularly sensitive to glutamate. In these cells, glutamate competes for cystine uptake, leading to a reduction in glutathione and, ultimately, cell death. As it has been reported that protein kinase C activation inhibits glutamate toxicity in these cells and is also associated with the inhibition of apoptosis in other cell types, we asked if glutamate toxicity was via apoptosis. Morphologically, glutamate-treated cells underwent plasma membrane blebbing and cell shrinkage, but no DNA fragmentation was observed. At the ultrastructural level, there was damage to mitochondria and other organelles although the nuclei remained intact. Protein and RNA synthesis inhibitors as well as certain protease inhibitors protected the cells from glutamate toxicity. Both the macromolecular synthesis inhibitors and the protease inhibitors had to be added relatively soon after the addition of glutamate, suggesting that protein synthesis and protease activation are early and distinct steps in the cell death pathway. Thus, the oxidative stress brought about by treatment with glutamate initiates a series of events that lead to a form of cell death distinct from either necrosis or apoptosis.     

Metabolism of phosphatidylcholine, phosphatidylinositol and palmityl carnitine in synaptosomes from rat brain

Abstract— Synthesis of phosphatidylcholine, phosphatidylinositol and palmityl carnitine in synaptosomes isolated from rat brain was investigated and compared with the synthesis of these compounds in microsomes and mitochondria. Electron microscopic and marker enzyme studies showed the contaminants in the synaptosomal preparation to consist of a few microsomes and almost no free mitochondria. In synaptosomes, addition of 1,2-diglyceride exerted no effect on the incorporation of [¹⁴C]choline into phosphatidylcholine or on the incorporation of [³H]myo-inositol into phosphatidylinositol, but it stimulated the incorporation of CDP[1,2-¹⁴C]choline into phosphatidylcholine by more than 50 per cent. The incorporation of the latter in intact synaptosomes, lysed synaptosomes and purified mitochondria was 15-6, 27 and 9-9 per cent, respectively, of that in the microsomes. The incorporation of [³H]myo-inositol into the phosphatidylinositol of synaptosomes and purified mitochondria was 15-8 and 11-1 per cent, respectively, of that in the microsomes. Maximal incorporation of [³H]myo-inositol occurred at pH 7–5 in a medium containing Mg²⁺ and CTP; it was linear with time and protein concentration and was inhibited by 1 mM Ca^{2 +} but unaffected by the presence of ATP. This incorporation of myo-inositol appeared to occur through the reversal of the CDP-diglyceride: inositol transferase reaction. The demonstration of carnitine palmityl transferase in synaptosomes indicated that, as in mitochondrial and erythrocyte membranes, fatty acids can be transported across the synaptosomal membrane. In contrast to mitochondria where maximal incorporation of [¹⁴C]carnitine into palmityl carnitine was observed after 20 min of incubation, the incorporation in synaptosomes increased as a function of time up to 60 min of incubation. We conclude that synaptosomes can carry on de novo synthesis of lipids, although at a limited rate. From the present data we cannot state with certainty how much of this synthesis is attributable to membranes originating from the endoplasmic reticulum.     

Caffeine-sensitive calcium stores in presynaptic nerve endings: a physiological role?

Ca2+-sensitive minielectrodes and the fluorescent cytosolic calcium probes, quin2 and fura2, were used to study some aspects of calcium homeostasis in intact and permeabilized synaptosomes from whole rat brain. Experiments in permeabilized synaptosomes revealed the existence of a vesicular, non-mitochondrial, ATP-dependent calcium uptake system with a vanadate sensitivity similar to that of brain microsomes, or endoplasmic reticulum-type calcium sequestering organelles. By using the fluorescent probes it was possible to show that caffeine mobilizes calcium from these internal stores in intact synaptosomes. Our results indicate a role of the caffeine sensitive calcium stores in the buffering of calcium loads elicited by plasma membrane depolarization.     

Analysis of polyadenylated RNA from brain synaptosomes and mitochondria

We have isolated RNA from sheep brain synaptosomes and mitochondria separated by an aqueous two-phase system composed of dextran and poly(ethylene glycol). RNA was fractionated through oligo(dT)-cellulose columns and analyzed by electrophoresis through agarose slab gels containing methylmercuric hydroxide and stained with ethidium bromide. The electrophoretic patterns of the poly(A)-containing RNA fraction from synaptosomes and mitochondria are very similar although some high molecular weight RNA species, clearly visible in the synaptosomal fraction, are scarcely detected in the mitochondrial preparations. The electrophoretic analysis of a cleaner RNA preparation from digitonin-treated free mitochondria (mitoplasts) showed that all the poly (A)-RNA species of the synaptosomal preparation are also present in mitoplast. These results strongly suggest that all the discrete poly(A)-RNA species identified in brain synaptosomes are of mitochondrial origin.     

Threshold Effects in Synaptosomal and Nonsynaptic Mitochondria from Hippocampal CA1 and Paramedian Neocortex Brain Regions

Abstract: After a brief period of global ischemia, the hippocampal CA1 region is more susceptible to irreversible damage than the paramedian neocortex. To test whether primary differences in bioenergetic parameters may be present between these regions, respiration rates and respiratory control activities were measured. In synaptosomal and nonsynaptic mitochondria isolated from the hippocampal CA1 region, state 3 respiration rates and complex IV activities were significantly lower than those present in synaptosomal and nonsynaptic mitochondria from the paramedian neocortex. These results suggest that mitochondria from the CA1 hippocampal area differ in some properties of metabolism compared with the neocortex area, which may render them more susceptible to a toxic insult such as that of ischemia. In addition, when complex I and IV activities were titrated with specific inhibitors, thresholds in ATP synthesis and oxygen respiration became apparent. Complex I and IV activities were decreased by 60% in nonsynaptic mitochondria from the hippocampal CA1 region and paramedian neocortex before oxidative phosphorylation was severely compromised; however, in synaptosomes from these regions, complex I activities had a threshold of 25%, indicating heterogenous behaviour for brain mitochondria. Reduced complex I thresholds in mitochondria, in association with other constitutive defects in energy metabolism, may induce a decreased ATP supply in the synaptic region. The implications of these findings are discussed in relation to delayed neuronal death and processes of neurodegeneration.     

Calcium binding to the cytosol and calcium extrusion mechanisms in intact synaptosomes and their alterations with aging.

A simple method to measure cytosolic calcium binding in intact presynaptic nerve terminals (synaptosomes) from rat brain, which is based on the simultaneous determination of [Ca2+]i and total [45Ca2+] in quin2-loaded synaptosomes undergoing a switch from high- to low-calcium containing medium, is presented. Binding to the cytosolic compartment alone was obtained following depletion of calcium storing organelles in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone/oligomycin plus caffeine. Synaptosomes, as compared to various cells types, have a high calcium binding capacity to the cytosolic compartment; maximum binding, Ca.Bmax, was 4.76 mM and calculated s0.5 was 218 nM. Calcium binding to the cytosolic compartment as a function of aging was also determined; Ca.Bmax was reduced to 1.84 mM and s0.5 increased to 492 nM in 30-month-old rats, indicating that the buffering of high calcium loads is impaired in old animals. The results obtained for binding of calcium to mitochondria and caffeine-sensitive calcium stores are consistent with an age-dependent reduction in calcium bound to mitochondria, whereas caffeine-sensitive calcium stores were unaffected. Finally, we have estimated the net rates of calcium extrusion in intact synaptosomes, and found that calcium efflux through the Na/Ca exchanger and Ca(2+)-ATPase was markedly reduced in old rats.     

A rapid Percoll gradient procedure for preparation of synaptosomes

Homogenization of fresh brain tissue in isotonic medium shears plasma membranes causing nerve terminals to become separated from their axons and postsynaptic connections. The nerve terminal membranes then reseal to form synaptosomes. The discontinuous Percoll gradient procedure described here is designed to isolate synaptosomes from brain homogenates in the minimum time to allow functional experiments to be performed. Synaptosomes are isolated using a medium-speed centrifuge, while maintaining isotonic conditions and minimizing mechanically damaging resuspension steps. This protocol has advantages over other procedures in terms of speed and by producing relatively homogeneous synaptosomes, minimizing the presence of synaptic and glial plasma membranes and extrasynaptosomal mitochondria. The purified synaptosomes are viable and take up and release neurotransmitters very efficiently. A typical yield of synaptosomes is between 2.5 and 4 mg of synaptosomal protein per gram rat brain. The procedure takes approximately 1 h from homogenization of the brain until collection of the synaptosomal suspension from the Percoll gradient.     

Subcellular fractionation of postmortem brain

Abstract— Procedures used to separate subcellular organelles from fresh brain were applied to brains which had been removed from guinea pigs (1) immediately after death; (2) after the dead animal was maintained at room temperature for 3 h, followed by 16–17 h at 4°C; or (3) after the dead animal was maintained for 19–20 h at room temperature. Subcellular fractionation of the brains in 0.32 M sucrose was followed by discontinuous density gradient centrifugation of the crude mitochondrial fraction. After overnight storage of brains at room temperature, there was a moderate shift in succinate dehydrogenase activity from sub-fraction C (mitochondria) to subfraction B (synaptosomes). There was little change in the distribution of galactolipid among particulate subfractions. There was little change in distributions of monoamine oxidase or acetylcholinesterase activities. Under the less extreme postmortem conditions, there were no shifts in the subcellular distributions of brain enzymes. Ultrastructural changes were much more profound and consisted of losses of identifiable mitochondria and synaptosomes and a progressive increase in very dense bodies. Our results suggest that in spite of the marked morphological changes, meaningful separation of subcellular organelles can be achieved with postmortem tissue.     

Intracellular Localization of Lunularic Acid and Prelunularic Acid in Suspension Cultured Cells of Marchantia polymorpha

Intracellular localization of lunularic acid and prelunularic acid in suspension cultured cells of Marchantia polymorpha L. was studied. The sum of both compounds was determined as lunularic acid group (LNAs) because of the instability of prelunularic acid to convert into lunularic acid.

Mechanical disruption of the cells followed by differential centrifugation showed that LNAs was associated with the supernatant of 100,000g centrifugation. Protoplasts isolated from the cells were osmotically ruptured and the distribution of LNAs among the organelles was examined by discontinuous density gradient centrifugation of the protoplast contents. Successful isolation of intact chloroplasts, mitochondria and peroxisomes free from cytoplasm indicated that LNAs was not accumulated in these organelles. Flotation techniques resulted in an efficient isolation of pure vacuoles and revealed that LNAs was distributed almost equally in the vacuoles and cytoplasm.

     

Identification and Topography of Substrates for Protein Carboxyl Methyltransferase in Synaptic Membrane and Myelin-Enriched Fractions of Bovine and Rat Brain

The major components of crude brain synaptosomes (synaptic membranes, mitochondria, and myelin) have been separated and analyzed by polyacrylamide gel electrophoresis for the presence of proteins that serve as substrates for protein carboxyl methyltransferase. Of the three fractions, synaptic membranes contain the largest number of individual methyl acceptors (at least seven), while mitochondria contain no well-defined methyl acceptors. Undisrupted myelin contains a single major methyl acceptor with a very low apparent molecular weight. The patterns of protein methylation in synaptic membranes prepared from cerebral cortex, hippocampus, striatum, thalamus, and tectum showed marked differences; however, these differences could largely be explained by differential degrees of myelin contamination in synaptic membranes from the different regions. The effect of trypsin pretreatment on the carboxyl methylation of intact and lysed synaptosomes was studied to estimate the sidedness of the major methylation sites on synaptic membranes. One of the methyl acceptors (Mr 48K) appears to be facing the intracellular surface of the synaptosome, but most sites appear to be outward facing.     

Antibiotic inhibition of protein synthesis by mitochondria of higher plants and animals

The antibiotic sensitivity of protein synthesis by mitochondria isolated from chick brain, trout liver, potato tuber and corn root has been investigated. Like mitochondria isolated from rat liver, organelles from these sources show a much reduced antibiotic sensitivity spectrum when compared to mitochondria derived from yeast, being sensitive to chloramphenicol, carbomycin, spiramycin and mikamycin but resistant to erythromycin, lincomycin, paromomycin, neomycin C and viridiogrisein. In addition spectinomycin inhibited protein synthesis by both types of plant mitochondria but not those from chick brain, rat liver or yeast.