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Biomedical subjects

N Zisapel

Publications and source records attributed to N Zisapel.

At least 91 records · Page 5Linked to original sources
PubMed1983

Metal ion effects on target sites of modification in metallocarboxypeptidase B.

Native carboxypeptidase B and its Co2+-substituted derivative were oxidized by the active-site-directed agent m-chloroperbenzoic acid. The following results were obtained a) In the cobalt enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Peptide or ester pseudo-substrates protected the cobalt enzyme but not the zinc enzyme against inactivation. b) Upon oxidation and formation of Co3+, cleavage of peptide bonds occurred in the cobalt enzyme but not in the zinc enzyme. Both enzymes retained their original metal content. c) Following oxidation of the enzymes, amino acid analysis revealed a modification of a methionyl residue in the zinc enzyme only; the cobalt enzyme, on the other hand, showed a modification of a histidyl residue. d) Peptide mapping of the enzymes after cleavage by cyanogen bromide indicated that two methionyl peptides were missing in the oxidized zinc enzyme. These peptides point to Met-64 as the site of modification. The peptide map of the oxidized cobalt enzyme was similar to that of the unmodified native (i.e., zinc) enzyme. These studies indicate that the specific metal ion present in the enzyme imposes certain structural and functional differences on the active site, leading to differing reactivities of specific amino acid residues and to a different alignment of the active-site-directed reagent in the two enzymes.

Amino Acids↗
PubMed1983

Inhibition by melatonin of dopamine release from rat hypothalamus in vitro: variations with sex and the estrous cycle.

The inhibitory effect of melatonin on evoked dopamine release from whole hypothalamus as well as from its three regions (preoptic area, median and posterior hypothalamus) was studied in male rats and in female rats during various stages of the estrous cycle. Inhibition of dopamine release by 1 microM melatonin was significantly higher at the estrous and lower at the diestrous than at the metestrous and proestrous stages. The variations in inhibition of dopamine release during the estrous cycle occurred predominantly in the preoptic area, and were nonsignificant in the other hypothalamic regions. In male rats, a constant level of inhibition was observed. In addition, the concentration of melatonin needed to produce half maximal effect in the female shifted from approximately 0.6 nM at the estrous and metestrous stages to more than 1 microM at the proestrous and diestrous stages. The corresponding concentration in male rats was of the order of 1 microM, and did not change.

Animals↗
PubMed1982

Cross-linking of synaptic vesicle proteins. Effect of ATP.

Synaptic vesicles isolated from bovine brain were subjected to cross-linking with the bifunctional amino group reagent dimethyl adipimidate. The resulting proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The reagent (10 mM) caused partial or complete disappearance from the SDS gel of most of the major polypeptides of the vesicles and the formation of new polymeric species with molecular weights greater than 500000. Using lower concentrations of the adipimidate (2-5 mM) a more selective cross-linking occurred, with the disappearance of a group of protein bands having apparent molecular weight values of 60000-68000, 40000-41000 and 25000-30000. The extent of cross-linking was independent of vesicle concentration in the range 0.3-3.0 mg protein per ml. Addition of ATP or AMP to the cross-linking reaction mixture resulted in a marked reduction in cross-linking of all of the major vesicle polypeptides (apparent molecular weight values 160000, 77000, 55000, 42000, 32000, 28000 and 26000). Several proteins were less affected by ATP or AMP; these were mostly the same vesicle proteins as those which had become cross-linked with low concentrations (2 mM) of dimethyl adipimidate. The ATP effect was markedly reduced if the vesicles were pretreated prior to the cross-linking reaction with alkaline buffer (pH 8.5) in either the presence or the absence of ATP. In the presence of 32P-labeled ATP, several of the vesicle protein bands became phosphorylated, but the extent of their cross-linking did not depend upon the state of phosphorylation of the major phoshorylated proteins. The results are consistent with the presence of aggregated protein complexes and of stabilized arrays of the major proteins within the vesicle membrane.

Adenosine Monophosphate↗
PubMed1982

Plasminogen activator is enriched in the synaptosomal plasma membranes.

The intracellular localization of the serine protease plasminogen activator was analyzed in homogenates of bovine brain cortex using differential fractionation procedures. The distribution of the enzyme was clearly different from that of cytosol and mitochondrial markers, and was similar to that of plasma membrane proteins and of the muscarinic acetylcholine receptor, which is a specific marker for the synaptic membrane. The specific activity of plasminogen activator was increased in fractions enriched in intact synaptosomes. Most of the enzyme in intact synaptosomes was found to be firmly associated with the synaptosomal membrane, and could be solubilized by high concentrations of salt or by non-ionic detergent. Purified synaptic vesicles, however, did not contain large amounts of plasminogen activator. Bovine brain synaptosomes were shown to contain two species of the enzyme, having apparent molecular weights of 80,000 and 55,000. The presence of plasminogen activator in the synaptosomal membrane may indicate its possible involvement in the functioning of nerve terminals.

Animals↗
PubMed1982

Inhibition of dopamine release by melatonin: regional distribution in the rat brain.

Dopamine release evoked by electrical field stimulation of slices from various regions of rat brain was assessed in the presence of 10-10-10-5 M melatonin. Inhibition of dopamine release by melatonin was observed in the ventral hippocampus, medulla pons, preoptic area and median and posterior hypothalamus. No inhibitory effect of melatonin on dopamine release was observed in the cerebral cortex, cerebellum, dorsal hippocampus and striatum. Equal concentrations of melatonin were needed to produce half-maximal inhibition in all the regions affected. The results indicate that the brain sites for inhibitory effect of melatonin on dopamine neurosecretion overlap the sites reportedly involved in its modulation of neuroendocrine functions.

Animals↗
PubMed1982

Mechanistic implications of cyanide binding to carboxypeptidase B.

The putative metal coordinating ligand cyanide was used to study the effects of modifications of the metal coordination sphere on the spectral properties and catalytic activity of cobalt and zinc carboxypeptidases. The absorption spectra of Co2+-carboxypeptidase B in the presence of cyanide pointed to a direct interaction of the ligands with the metal. Gel-filtration experiments showed that the binding of one mole of ligand per mole of enzyme metal ion resulted in maximal spectral effects. Binding of cyanide to the metal ion as measured by absorption spectroscopy was inhibited by acetyl-L-arginine, a peptide pseudosubstrate, and by acetyl-D-arginine, a competitive peptide inhibitor. Addition of acetyl arginine to the enzyme-cyanide complex caused displacement of the ligand, as evidenced by the spectral parameters. Cyanide inhibited peptide hydrolysis in a partially noncompetitive manner, i.e. it did not prevent binding of the substrate to the enzyme but the enzyme-substrate-cyanide complex was hydrolyzed at a slower rate than the enzyme-substrate complex. The dissociation constant evaluated from kinetic studies for the binding of cyanide to Co2+-carboxypeptidase B was in good agreement with that obtained from spectral measurements. Hydrolysis of the ester analog of the basis peptide substrate was not affected by cyanide. Based on these data a model is proposed in which the peptide carboxyl group displaces the water molecule from the metal coordination sphere during catalysis without increasing the coordination number.

Animals↗
PubMed1982

Tyrosyl interactions at the active site of carboxypeptidase B.

The phosphorescence emission spectra of native carboxypeptidase B and of chemically modified carboxypeptidase B (at arginyl residues) was measured in the presence and absence of peptide and ester substrates (acetyl-L-arginine and its hydroxy ester analog: acetyl-L-argininic acid). Ester binding did not affect the state of the tyrosyl residue as compared with its state in the substrate-free enzyme. In the modified enzyme, which is devoid of peptidase activity, binding of the peptide pseudosubstrate did not perturb the state of the tyrosyl residue. The luminescence spectra of Zn2+- and Co2+-carboxypeptidase B in the presence of the metal coordinating ligand cyanide, used to displace the water from the metal coordination sphere, is also described. Cyanide did not affect the luminescence spectra of the active-site tyrosyl residue in either Zn2+- or Co2+-carboxypeptidase, indicating that the tyrosyl residue was not interacting directly with the metal bound water. Hence, the effect of peptide on tyrosyl phosphorescence is not caused by the displacement of the tyrosyl from the coordination sphere, but rather by direct interaction of the peptide bond. The data are consistent with the proposition that the tyrosyl residue participates as a proton donor in amide but not in ester hydrolysis.

Animals↗
PubMed1980

A calcium-stimulated alkaline phosphatase associated with synaptic vesicles.

Synaptic vesicles isolated from bovine cerebral cortex were found to contain alkaline phosphatase activity towards p-nitrophenylphosphate and alpha-naphthyl phosphate, but not towards pyridoxal phosphate. The enzyme had an apparent molecular weight of 125,000 and co-purified with the synaptic vesicles in parallel with the specific neurotransmitter content and with the loss of contaminating components, whereas the major phosphatase which was present in the brain homogenate, with an apparent molecular weight of 140,000 purified away. The optimal pH for the enzyme activity on p-nitrophenylphosphate was 9.8. At this pH the activity was 33.4 nmol/mg protein/min, and the apparent Km value was 0.31 +/- 0.05 mM. The pH dependency of the synaptic vesicle phosphatase activity towards p-nitrophenylphosphate differed from that of the Ca2+/Mg2+-dependentt ATP hydrolysis by the synaptic vesicles. Upon mild digestion of lyzed vesicles with trypsin, phosphatase activity was reduced whereas the ATPase activity was retained suggesting that the phosphatase and the ATPase are two different enzymes. The phosphatase was reversibly inhibited by ethyleneglycol bis (aminoethyl ether) N,N'-tetracetic acid (EGTA) and activity was restored by the addition of an equimolar amount of CA2+. Magnesium ions could restore only 30% of the activity. The activity of the synaptic vesicle phosphatase was not affected by o-phenanthroline, zinc ion or by cAMP. Tetranitromethane inactivated the enzyme irreversibly, whereas phenylmethanesulfonylfluoride diisopropylfluorophosphate and p-hydroxymercurybenzoate inhibited the activity partially. The enzyme did not have a diesterase activity. Adenosine mono-, di- and triphosphate inhibited the p-nitrophenylphosphatase activity and were also hydrolyzed by the vesicle preparation. However, the different kinetic parameters obtained with the nucleotide as inhibitors or as substrates suggest that additional enzymes are involved in the hydrolysis of the adenine nucleotides in vesicle preparation.

Alkaline Phosphatase↗
PubMed1980

Tubulin: an integral protein of mammalian synaptic vesicle membranes.

The major protein in isolated synaptic vesicles from bovine cerebral cortex has been compared to tubulin by sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis, by two-dimensional gel electrophoresis, and by peptide mapping following limited proteolysis of the protein by Staphylococcus aureus protease. The results establish in purified synaptic vesicles the presence of tubulin, which is composed of the alpha and beta subunits. In the presence of ethyleneglycolbis)aminoethyl ether)-N,N'-tetraacetic acid (EGTA) or magnesium in the isolation buffers, the synaptic vesicles contained mainly the alpha-tubulin whereas the beta subunit was less abundant. Similarly, synaptosomal plasma membranes that were prepared in the presence of EGTA also contained more of alpha-tubulin than of the beta subunit. Non-ionic detergents such as Triton X-100 or Nonidet P-40 failed to solubilize the tubulin from the synaptic vesicles. Ionic detergents such as deoxycholate and sodium dodecyl sulphate solubilized all the vesicle proteins, including tubulin. The results indicate that alpha-tubulin is an integral vesicle membrane protein, whereas most of the beta subunit is peripherally attached and can be easily dissociated from the vesicle membrane with EGTA.

Animals↗
PubMed1979

Studies on synaptic vesicles in mammalian brain characterization of highly purified synaptic vesicles from bovine cerebral cortex.

Synaptic vesicles have been isolated from bovine cerebral cortex by sequential differential and density gradient centrifugations followed by chromatography on a Sepharose 6B column. We have studied the morphology, enzymatic markers, neurotransmitter and ATP contents and protein composition of the vesicles. The specific contents of acetylcholine, gamma-aminobutyric acid, aspartate, glutamate and catecholamines were 4--8-fold higher in the vesicle fraction compared to the crude synaptosomal pellet. Electron micrographs of the vesicle preparation showed enrichment of vesicular material with an average diameter of 50 nm. The purity of the preparation was assessed by the very low activities of enzymatic markers of cellular membranes and cytosol components. Some Ca--Mg-activated ATPase activity was detected in the vesicle preparations, but its content relative to the neurotransmitters fell on chromatography, suggesting that this activity may be partially contributed by non-synaptic vesicle components, such as small microsomes. The isolated synaptic vesicles were solubilized with 1% sodium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis. The major Coomassie blue stained bands observed with apparent molecular weights of 160,000 and 55,000 were enriched in parallel to the increase in purity of the preparation.

Acetylcholinesterase↗
PubMed1979

Expression of external-surface membrane proteins in differentiated and undifferentiated mouse neuroblastoma cells.

Murine neuroblastoma cultures were labeled externally with the cationic reagent N,N,N-[3H]-trimethylamino-beta-alanyl-N-hydroxy-succinimide ester ([3H]Me3N-beta Ala-NSuc) or with 125I/lactoperoxidase. The cells were labeled in the logarithmic and confluent growth phases as well as in a highly differentiated state following treatment with 2% dimethylsulfoxide. The labeled exterior membrane proteins were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Major changes in the exterior membrane proteins were observed during maturation and differentiation of the cells. Most of these changes were clonal-specific, while others were common to several clones. Two proteins of Mr 55,000 and 65,000 were labeled by both 125I/lactoperoxidase and Me3N-[3H]-beta Ala-NSuc. The level of labeling was dependent on the clonal lines used and the state of the cell maturation. A group of proteins displaying a molecular weight between 150,000 and 200,000 was found to be related to the transition of a culture from logarithmic to confluent growth phases. An additional protein, with an apparent molecular weight of 95,000, was common to differentiated cells of the two inducible clones used. In general the maturation of logarithmic phase cells into confluent cells resulted in a less complex electrophoretic distribution of the pattern of labeling. After dimethyl-sulfoxide treatment, further reduction in the complexity of the externally labeled proteins was observed.

Animals↗
PubMed1978

The reactivity of a functional tyrosyl residue in carboxypeptidase B. Nitration of the cadmium enzyme.

Cadmium-carboxypeptidase B was nitrated with tetranitromethane. The enzyme polymerized extensively during nitration. In the monomer nitrated Cd-carboxypeptidase B, 70% of the activity of Cd-carboxypeptidase B was retained. In order to identify the tyrosyl residues nitrated, the enzyme was digested with chymotrypsin and subtilisin and the nitrotyrosyl peptides were purified by affinity chromatography on antityrosyl-antibody-Sepharose conjugate followed by two-dimensional thin-layer chromatography. The major nitropeptides, representing 65% of the nitrotyrosyl label, were compatible with the segment of the sequence containing Tyr-240 and Tyr-259. Only 10% of the nitrotyrosyl label was found in the segment of Tyr-248. This indicates that the state of Tyr-248 in Cd-carboxypeptidase B differs from that in zinc-carboxypeptidase B where it shows chemical hyperreactivity due to its proximity to the metal ion.

Animals↗
PubMed1978

A cationic hydroxysuccinimide ester. A reagent for labeling exterior membrane proteins.

3/-labeled N,N,N,trimethylamino-beta-alanyl-N-hydroxysuccinimido ester ([3H]TMAS), a new cationic membrane reagent, was synthesized. TMAS was shown to be impermeant through human erythrocyte membranes. Under mild physiological conditions TMAS reacted primarily with amino groups of the membrane proteins and lipids. The pattern of erythrocyte proteins labeled with [3H]TMAS was examined by polyacrylamide gel electrophoresis under denaturing conditions. Externally oriented labeling of intact erythrocytes revealed a major radioactive protein with an apparent molecular weight of 90,000. By labeling ghost preparations with [3H]TMAS the radioactivity incorporated into all the major Coomassie Brilliant Blue bands resolved by gel electrophoresis. The agreement between the results obtained with anionic and cationic amino reactive probes indicates that the ionic character of the reagent has a minor effect on the pattern of labeled exterior polypeptides observed in erythrocytes.

Affinity Labels↗