Synaptosomal Soluble Fraction Research Articles

Postnatal Age and Protein Tyrosine Phosphorylation at Synapses in the Developing Rat Brain

The relationship between postnatal age and protein tyrosine kinase activity in synaptosomes prepared from the rat forebrain was studied. Synaptosomal particulate and soluble fractions, as well as total homogenates, the cell soluble fraction, and P3, were prepared from rats ranging in postnatal age from 5 to 60 days and analyzed for (a) tyrosine kinase activity using polyglutamyltyrosine (4:1) as the substrate, (b) the presence of endogenous substrates for tyrosine phosphorylation using polyclonal antibodies specific for phosphotyrosine, and (c) levels of pp60src. Enzyme activity, expressed per milligram of protein, in the total homogenate, P3, and both the cell and synaptosomal soluble fractions was highest in the brains of young animals (postnatal days 5-10) and decreased thereafter to adult levels. In contrast, tyrosine kinase activity in the synaptosomal particulate fraction exhibited a unique biphasic developmental profile, increasing to maxima at postnatal days 10 and 20 before decreasing to adult values. Endogenous substrates for tyrosine phosphorylation were identified by incubating subcellular fractions with 2 mM ATP in the presence of sodium orthovanadate and probing nitrocellulose blots of proteins separated by gel electrophoresis with antiphosphotyrosine antibodies. Several phosphotyrosine-containing proteins were detected in the synaptosomal particulate and P3 fractions, including proteins of Mr 180K, 145K, 120K, 100K, 77K, 68K, 62K, 54K, 52K, and 42K. In the cell soluble fraction a protein doublet of Mr 54/52K and a 120K protein were the major phosphotyrosine-containing proteins. The 54/52K doublet was the major protein tyrosine kinase substrate in the synaptosomal soluble fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibody Br4 recognizes specific neuronal celltypes

A monoclonal antibody termed as Br4 was prepared by a fusion of the myeloma P3 x 63-AG8-653 with spleen cells from BALB/c mice immunized with chick embryonic brain cells. Immunocytochemically, it reacts strongly with certain neurons in cerebral cortex, hippocampus, substantia nigra, and granular layer of cerebellum from rat brain but does not react with the white matter. A monoclonal antibody Br4 also reacts with primary cultured chick neurons, but not with cultured astrocytes. Western blots show that Br4 recognizes three proteins of 145,000, 108,000, and 97,000 molecular weight from the rat brain. The protein with molecular weight 145,000 (p145) and the protein with 97,000 molecular weight (p97) are essentially soluble; p145 is especially enriched in the synaptosomal soluble fraction. The protein with 108,000 molecular weight (p108) is found in both membrane-bound and soluble forms. Western blots show that among the tissues examined the three proteins are found most abundant in brain and especially enriched in the cerebral cortex and hippocampus. The Br4 antigens may be useful in identifying neuronal cell subpopulations in the central nervous system.

Intrasynaptosomal distribution of the ras, rho and smg-25A GTP-binding proteins in bovine brain

We have purified to near homogeneity and characterized three small molecular weight ( M r) GTP-binding proteins, the c-Ki- ras protein (c-Ki- ras p21), the rho protein ( rho p20) and a novel smg-25A protein ( smg p25A), from bovine brain crude membranes. In the present studies, the intrasynaptosomal distribution of these 3 small M r G proteins has been investigated using bovine brain. ras p21 and rho p20 are found in the synaptosomal membrane fraction but not in the synaptosomal soluble fraction. In contrast, smg p25A is found in both the synaptosomal membrane and soluble fractions. These results indicate that the intrasynaptosomal distribution of these small M r G proteins is different and suggest that they are involved in different neuronal functions.

Synaptic protein tyrosine kinase: partial characterization and identification of endogenous substrates.

The subcellular distribution of protein tyrosine kinase in rat forebrain was determined using [Val5]-angiotensin II as exogenous substrate. Enzyme activity was present in each of the fractions analyzed and was enriched in synaptic membranes (SMs) and the synaptosomal soluble fraction (2.2- and 2.5-fold over the homogenate, respectively). SMs also phosphorylated polyglutamyltyrosine (pGT; molar ratio of 4:1), the Vmax for angiotensin and pGT phosphorylation being 26.3 +/- 1.6 and 142 +/- 4 pmol/min/mg, respectively. Extraction of SMs with several different detergents resulted in enhanced enzyme activity and the solubilization of 33-37% of the angiotensin and 43-70% of the pGT-phosphorylating activity. Isolated postsynaptic densities (PSDs) contained tyrosine kinase and phosphorylated angiotensin and pGT. The Vmax values for angiotensin and pGT phosphorylation by PSDs were 17 +/- 5 and 23 +/- 1 pmol/min/mg, respectively. Six putative endogenous substrates for SM tyrosine kinase, with molecular weights of 205K, 180K, 76K, 60K, 50K, and 45K, were identified. Each of these proteins, except p76, was phosphorylated in the detergent-insoluble residue obtained following the extraction of SMs with Triton X-100 as well as in PSDs, indicating that the postsynaptic apparatus is an active site of tyrosine phosphorylation. The phosphorylation of p76 was localized to the Triton X-100 extract and also occurred in the synaptosomal soluble fraction. The results indicate that tyrosine kinase and its substrates are located in both pre- and postsynaptic compartments and suggest a role for this enzyme in synaptic function.

Thyrotrophin releasing hormone degradation by rat synaptosomal peptidases: Production of the metabolite His-Pro

The dipeptide, His-Pro, is the major product of the degradation of TRH by rat synaptic membranes in vitro. A small amount of His-Pro is also formed from TRH by the synaptosomal soluble fraction. From inhibitor studies, the main route to His-Pro appears to involve removal of the pGlu residue by membrane-bound metal-dependent pyroglutamylaminopeptidase followed by deamidation. The deamidation step is not mediated by proline endopeptidase (EC3.4.21.26) nor dipeptidylpeptidase-IV (EC3.4.14.5) since it is insensitive to bacitracin and diprotin-A, and may therefore involve a novel membrane-bound TRH metabolizing enzyme. His-Pro is degraded rapidly by the soluble synaptosomal fraction, presumably by prolidase (EC3.4.13.9) and more slowly by the synaptic membrane fraction.

Monoclonal antibody to soluble guanylate cyclase of rat brain

Guanylate cyclase (GTP pyrophosphate-lyase, cyclizing, EC 4.6 .1.2), the enzyme responsible for the synthesis of cyclic GMP, is detected mostly in soluble fraction and partly associated with particulate fraction of mammalian tissues [ l-61. In rat brain, guanylate cyclase is found mainly in synaptosomal soluble fraction suggesting its involvement in the neural transmission [6]. We have purified the soluble guanyiate cyclase from bovine brain to an apparent homogeneity and produced the antibody to the enzyme by immunizing a rabbit [7]. The antibody effectively inhibited the soluble guanylate cyclase activity from various mammalian tissues, but not the Triton-dispersed particulate guanylate cyclase from tissues. A method for preparing a monoclonal antibody has been established [8]. Because of its high specificity, a monoclonal antibody would offer a method for studying the different properties of enzyme molecules and for histochemically demonstrating the cellular as well as subcellular localization of guanylate cyclase. We report here production and properties of the monoclonal antibody to the soluble guanylate cyclase of rat brain. The purified guanylate cyclase (64OMg) was injected into BALB/c mouse intraperitoneally with 2 X lo9 heat-inactivated pertussis organisms. The booster injections were carried out twice at a 3-week interval without pertussis organisms. Spleen cells were taken from immunized mouse 3 days after the last booster injection and fused with mouse myeloma cells P3-NS I/l-Ag4-1 (NS I) as in [9]. Spleen cells (1.6 X 108) were fused with 4 X 10’ myeloma cells by the addition of 1 ml 50% polyethylene glycol4000. The cells were suspended in growth medium (RPM1 1640 supplemented with 15% fetal bovine serum, 2 mM L-glutamine and 1 mM pyruvate) and inoculated in two 96-well Nunc Microtest Plates. Two days later, 0.1 ml of HAT selective medium (growth medium supplemented with 100 PM hypoxanthine, 0.4 PM aminopterin and 16 PM thymidine) was added and half of the medium was replaced 3 times during the subsequent 10 days. Fourteen days after cell fusion, culture fluid (60 fl) was assayed for anti-guanylate cyclase antibody by measuring enzyme inhibition as described below. Monoclonal hybridoma cell lines were obtainec’ by a limiting dilution method repeated twice.

Fucose incorporation and identification of fucosylglycoproteins in synaptosomes.

A synaptosome-enriched fraction from sheep cortex was incubated with L-fucose. The uptake of the sugar into this preparation was dependent on time, temperature, and concentration. A Kmapp of 0.94 mM-L-fucose and a Vmaxapp value of 0.24 nM-L-fucose/mg synaptosomal soluble protein/20 min was determined. After incubation for 10 min at 25 degrees C with L-[3H]fucose, 70% of the radioactive label was found in the soluble fraction. DEAE-cellulose chromatography resulted in the elution of three fucosylprotein peaks which were then characterised by gel filtration and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). At least eleven 3H protein-staining bands were identified with M. W. 13,000--115,000. Control experiments involving the incubation of the hexose with heat-treated synaptosomes and myelin, mitochondria, and microsomes indicted that the tritiated material associated with the synaptosomal soluble fraction was not due to nonspecific binding or to the presence of contaminating subcellular material. A 3H glycopeptide was identified, and on analysis the carbohydrate moiety was found to be rich in sialic acid, fucose, galactose, mannose, and N-acetylglucosamine. Mild acid treatment of the glycopeptide released fucose, which implies that this carbohydrate occupies a terminal position in the oligosaccharide chain. From these results it is proposed that synthesis or the modification of soluble fucosylglycoproteins is possible in synaptosomes.

Guanylate cyclase in synaptosomal soluble fraction from rat brain: Possible involvement of free radical acceptor in endogenous activation mechanisms

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing); EC 4.6.1.2), in the crude synaptosomal soluble (P 2-soluble) fraction obtained by hypo-osmotic treatment and subsequent centrifugation of crude synaptosomal (P 2) fraction from the rat brain, showed a significantly high activity when assayed in the presence of high concentration of Mn 2+. The basal activity was markedly inhibited by various compounds reacting with free radicals. Guanylate cyclase in the P 2-soluble fraction treated with acetone exhibited a low activity and was not activated by nitrosoguanidine. The responsiveness to nitrosoguanidine was restored, however, by the addition of low concentration hemin. On the other hand, the preincubation (37°C, 10min) of P 2-soluble fraction with membranous fraction of P 2 similarly induced a marked decrease of guanylate cyclase activity, but this enzyme was strongly stimulated by nitrosoguanidine. The responsiveness of the enzyme to nitrosoguanidine was also eliminated by acetone-treatment or gel-filtration on Sepharose 6B, but was partially restored by the addition of hemin. These results suggest that guanylate cyclase in the P 2-soluble fraction from rat brain may be activated endogenously by free radical(s) through a free radical acceptor which is replaceable by hemin.

Endogenous activation of guanylate cyclase in synaptosomal soluble fraction from rat brain.

Guanylate cyclase in crude mitochrondrial (P2) soluble fraction prepared from rat brain, obtained by a hypo-osmotic treatment of P2, showed extremely higher activity than that in the same fraction from other organs. In addition the soluble fraction obtained from synaptosomes (P2 - B) contained the highest enzyme activity among other subfractions of the cerebral P2 examined. Guanylate cyclase activity in the synaptosomal soluble fraction was, however, markedly suppressed by various compounds reacting with free radicals. These results suggest that guanylate cyclase in the synaptosomal soluble fraction may be activated endogenously be a free radical and involved in the regulatory mechanisms for cyclic guanosine monophosphate (cyclic GMP) at presynaptic terminals.

Superoxide dismutase catalyzes activation of synaptosomal soluble guanylate cyclase from rat brain

Guanylate cyclase in the crude synaptosomal soluble (p 2-soluble) fraction obtained by hypo-osmotic treatment of crude synaptosomal (P 2) fraction from rat brain showed a significantly high activity. The P 2-soluble fraction prepared after incubating (37 °C, 10 min) with membranous fraction of P 2 (P 2-membrane) had a low activity of guanylate cyclase, which was markedly stimulated by superoxide dismutase. The activation by superoxide dismutase was completely inhibited by KCN, β-carotene, Tiron and hemoglobin. The enzyme obtained using gel-filtration chromatography showed no response to superoxide dismutase, but the addition of hemin induced a marked restoration of the responsiveness.

Sulphated glycoproteins in synaptosomes

Synaptosomes from sheep brain were incubated with Na 2 35SO 4. After lysis at least twelve 35S-containing proteins were identified in the synaptosomal soluble fraction with molecular weights ranging between 13,000 and 150,000. Extraction of the particulate fraction with SDS/urea revealed a further eight [ 35S]proteins with 27,000–140,000 molecular weights. A glycopeptide rich in fucose, sialic acid, N-acetylglucosamine and galactose was isolated from papain treated synaptosomal material. Over half of the hexosamine and neutral sugar residues were found to contain 35S. These findings suggest that sulphation of glycoproteins can occur in isolated nerve terminals.

Purification and properties of guanylate cyclase from the synaptosomal soluble fraction of rat brain

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) was purified 2250-fold from the synaptosomal soluble fraction of rat brain. The specific activity of the purified enzyme reached 41 nmol cyclic GMP formed per min per mg protein at 37°C. In the purified preparation, GTPase activity was not detected and cyclic GMP phosphodiesterase activity was less than 4% of guanylate cyclase activity. The molecular weight was approx. 480000. Lubrol PX, hydroxylamine, or NaN 3 activated the guanylate cyclase in crude preparations, but had no effect on the purified enzyme. In contrast, NaN 3 plus catalase, N- methyl-N′- nitro-N- nitrosoguanidine or sodium nitroprusside activated the purified enzyme. The purified enzyme required Mn 2+ for its activity; the maximum activity was observed at 3–5 mM. Cyclic GMP activated guanylate cyclase activity 1.4-fold at 2mM, whereas inorganic pyrophosphate inhibited it by about 50% at 0.2 mM. Guanylyl-(β,γ-methylene)-diphosphonate and guanylyl-imidodiphosphate, analogues of GTP, served as substrates of guanylate cyclase in the purified enzyme preparation. NaN 3 plus catalase or N- methyl-N′- nitro-N- nitrosoguanidine also remarkably activated guanylate cyclase activity when the analogues of GTP were used as substrates.

Activation of guanylate cyclase in cerebral cortex of rat by hydroxylamine.

Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral cortex of rat. Activation was most remarkable in crude mitochondrial fraction. When the crude mitochondrial fraction was subjected to osmotic shock and fractionated, guanylate cyclase activity recovered in the subfractions as assayed with hydroxylamine was only one-third of the starting material. Recombination of the soluble and the particulate fractions, however, restored guanylate cyclase activity to the same level as that of the starting material. When varying quantities of the particulate and soluble fractions were combined, enzyme activity was proportional to the quantity of the soluble fraction. Heating of the soluble or particulate fraction at 55 degrees for 5 min inactivated guanylate cyclase. The heated particulate fraction markedly activated guanylate cyclase activity in the native soluble fraction, while the heated soluble fraction did not stimulate enzyme activity in the particulate. The particulate fraction preincubated with hydroxylamine at 37 degrees for 5 min followed by washing activated guanylate cyclase activity in the soluble fraction in the absence of hydroxylamine. Further fractionation of the crude mitochondrial fraction revealed that the factor(s) needed for the activation by hydroxylamine is associated with the mitochondria. The mitochondrial fraction of cerebral cortex activated guanylate cyclase in supernatant of brain, liver, or kidney in the presence of hydroxylamine. The mitochondrial fraction prepared from liver or kidney, in turn, activated soluble guanylate cyclase in brain. Activation of guanylate cyclase by hydroxylamine was compared with that of sodium azide. Azide activated guanylate cyclase in the synaptosomal soluble fraction, while hydroxylamine inhibited it. The particulate fraction preincubated with azide followed by washing did not stimulate guanylate cyclase activity in the absence of azide. The activation of guanylate cyclase by hydroxylamine is not due to a change in the concentration of the substrate GTP, Addition of hydroxylamine did not alter the apparent Km value of guanylate cyclase for GTP. Guanylate cyclase became less dependent on manganese in the presence of hydroxylamine. Thus the activation of guanylate cyclase by hydroxylamine is due to the change in the Vmax of the reaction.

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.

Distribution of LH-RH in subcellular fractions of the basomedial hypothalamus

Subcellular fractionation of the mediobasal hypothalamus (MBH) and other brain structures was achieved by differential and sucrose gradient centrifugation. The fractions were monitored by measuring lactate dehydrogenase (LDH) activity (a marker for the soluble cytoplasmic fraction) and by electron microscopic examination. The luteinizing-hormone-releasing-hormone (LH-RH) content of the fractions was evaluated both by bioassay and radioimmunoassay. Significant amounts of LH-RH were found only in the MBH and in an anterobasal location corresponding to the organum vasculosum of the lamina terminalis. Within these areas, LH-RH activity was present in the first supernatant (homogenate with the exclusion of the nuclear pellet). Seventy percent of the LH-RH activity was recovered in the crude mitochondria! fraction. After further fractionation on a sucrose gradient, the distribution of LH-RH was parallel with that of LDH activity. Since LDH is predominantly located in the synaptosomal soluble fraction, it is concluded that the vast majority of LH-RH is contained within nerve endings. This finding is consistent with cyto-immunological data on the distribution of the neuropeptide in the rat hypothalamus.

Synaptosomal protein synthesis in a cell-free system.

Abstract—Synaptosomes were isolated from cerebral cortex of young rats and incubated with 14C‐labelled l‐leucine in vitro. Amino acid incorporation into proteins of the synaptosomal cytoplasm, mitochondria and membrane components was observed. There was no incorporation into proteins of the vesicles. The protein‐synthesizing system was not stimulated by the addition of either ATP or an ATP‐generating system. ATP at all concentrations was inhibitory.Two different protein‐synthesizing systems operate in the synaptosome. One, sensitive to inhibition by chloramphenicol and related antibiotics, is found in the mitochondrial subfraction and the other, inhibited by cycloheximide, is located either in the membrane components or the synaptosomal cytoplasm. This second system resembles the eukaryotic ribosomal system in its sensitivity to cycloheximide. Both the synaptosomal soluble fraction and the synaptosomal membrane fractions were shown previously to contain RNA. This RNA could function in protein‐synthesizing mechanisms in the synaptosome. These results deomonstrate that protein is synthesized in axonal components and show that it is unnecessary to postulate that all axonal protein is supplied by somato‐axonal flow.