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S Wilk

Publications and source records attributed to S Wilk.

At least 109 records · Page 6Linked to original sources
PubMed1981

Purification and specificity of a membrane-bound metalloendopeptidase from bovine pituitaries.

A metalloendopeptidase optimally active at a neutral pH was purified 10000-fold from particulate fractions of bovine pituitaries. The solubilized enzyme has an apparent molecular weight of about 90 000, as determined by gel filtration on Sephadex G-200 and G-100 columns. The enzyme is not sensitive to inhibition by SH-blocking agents, diisopropyl fluorophosphate, leupeptin, pepstatin, antipain, and chymostatin. Thiols and metal chelators such as ethylenediaminetetraacetic acid (EDTA) o-phenanthroline are inhibitory. An EDTA-treated enzyme can be reactivated by several divalent metal ions, with zinc giving reactivation at the lowest concentrations. The specificity and kinetic parameters of the enzyme were studied with a series of synthetic peptide naphthylamides. The enzyme cleaves bonds in which the amino group is provided by a hydrophobic amino acid residue (position P1'). Replacement of this residue by small neutral amino acids decreases or virtually eliminates activity. The nature of substituents in positions P1, P2, P3, and P4 greatly influences specificity. Relatively high kcat and kcat/Km ratios were obtained with substrates containing arginine residues in positions P1 and P2. In such cases the impression of a "trypsin-like" activity was created. High reaction rates were also observed with substrates containing small neutral amino acids in positions P1 and P2, provided that position P3 was occupied by the acidic (polar) glutaryl residue. Replacement of this residue with hydrophobic substituents greatly decreased the rate of reaction. When positions P1 and P2, however, were occupied by arginine residues, the unfavorable effect of hydrophobic substituents in position P3 or P4 on catalysis was eliminated.

Animals↗
PubMed1980

Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme.

A highly purified preparation of a cation-sensitive neutral endopeptidase was obtained from bovine pituitaries. The enzyme constitutes almost 0.1% of the protein in bovine pituitary homogenates. Polyacrylamide gel electrophoresis of the enzyme showed a single protein band, and in gel filtration experiments on calibrated Sepharose 6B columns the enzyme eluted slightly ahead of thyroglobulin, suggesting an apparent molecular weight of about 700,000. Polyacrylamide gel electrophoresis in SDS-containing buffers indicated the presence of three major components with molecular weights ranging from about 24,000 to 28,000. The enzyme hydrolyzes bonds between hydrophobic and small neutral amino acids in both model synthetic substrates and biologically active peptides such as substance P, LH-RH, and bradykinin. Peptide bonds in which the carbonyl group is contributed by a glutamyl or arginyl residue are also hydrolyzed, especially if they are preceded in the sequence by hydrophobic amino acids. Leupeptin exclusively inhibited enzymatic activity toward the arginine-containing substrates. This observation, together with the high molecular weight and broad specificity of the enzyme, raised the possibility that the isolated enzyme represents a proteolytic complex composed of units with distinctly different activities. Preliminary attempts to dissociate the enzyme into catalytic units of lower molecular weight were not successful and led to loss of activity.

Animals↗
PubMed1980

The differential effects of morphine, oxotremorine and antipsychotic drugs on DOPAC concentrations in rat brain.

The effects of morphine and oxotremorine on concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) in the rat striatum and tuberculum olfactorium (TO) have been compared with the effects of the antipsychotic drugs haloperidol, chlorpromazine and clozapine. All the drugs elevated DOPAC concentrations in both brain regions. While the dose-response curves for the antipsychotic drugs were parallel, had steep slopes and similar maxima, the curves for morphine and oxotremorine were irregularly shaped but the curve for morphine in the TO had some similarity to that of the antipsychotic drugs. From these findings, it is concluded that the dose-dependent increase in striatal DOPAC effected by antipsychotic drugs can be used to differentiate them from other drugs known to elevate dopamine metabolites.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1980

The effect of typical and atypical neuroleptics on binding of [3H]spiroperidol in calf caudate.

The effect of typical and atypical neuroleptics on the binding of [3H]spiroperidol to calf caudate membranes was studied. Saturable or specific binding was defined as the difference between binding in the absence and in the presence of 1 microM d-butaclamol. Scatchard analysis revealed nonhomogeneity of the saturable sites. Inhibition constants (Ki) and IC50 values for various typical and atypical neuroleptics and for two clinically inactive butyrophenones were determined. The Ki and IC50 values of typical neuroleptics paralleled their potencies in vivo. By contrast, the binding potencies of atypical neuroleptics did not correlate with their effects in vivo. For example, the clinically active drug clozapine has an IC50 value similar to the clinically inactive butyrophenone AHR-1900. U-25, 927, another clinically inactive butyrophenone that does not increase dopamine turnover is more potent in the binding assay than perlapine, a drug that increases dopamine turnover and elevates serum prolactin levels. The most striking discrepancy between binding and properties in vivo was found for the benzamide derivatives, sulpiride and metoclopramide. These clinically active agents, which increase dopamine turnover, have much higher Ki values than the clinically inactive butyrophenones. It is concluded that binding assays with [3H]spiroperidol in calf caudate cannot account for the antidopamine effects in vivo of atypical neuroleptics.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1979

gamma-Glutamyl DOPA and gamma-glutamyl dopamine: effect on plasma glucose levels.

gamma-Glutamyl-L-3,4-dihydroxyphenylalanine (gamma-glutamyl DOPA) and gamma-glutamyldopamine (gamma-glutamyl DA) are kidney specific prodrugs. Their effect on plasma glucose levels in the rat was compared to that of L-DOPA and dopamine (DA) after a 30 min intravenous infusion. L-DOPA and DA induced hyperglycemia after 15 min of druginfusion. A more marked and protracted elevation of plasma glucose was observed after infusion of gamma-glutamyl DA. By gamma-glutamyl DOPA had no effect on plasma glucose levels in spite of the high accumulation of DA in the pancreas after this prodrug. Of the various dopamine produrgs studied only gamma-glutamyl DOPA was not hyperglycemic in doses that are known to increase renal plasma flow in the rat. A simplified new procedure for the synthesis of gamma-glutamyl DA is described.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1979

Degradation of luteinizing hormone-releasing hormone (LHRH) by brain prolyl endopeptidase with release of des-glycinamide LHRH and glycinamide.

A highly purified preparation of rabbit brain prolyl endopeptidase cleaved the decapeptide luteinizing hormone-releasing hormone (LHRH) at the ProGly . NH2 bond leading to the release within 1-3 h incubation at 37 degrees C of des-glycinamide LHRH and glycinamide. Evidence for this site of cleavage was obtained by the detection of glycinamide or glycine and groups by a microdanyslation procedure, and by separation of the breakdown products by high performance liquid chromatography (HPLC) on a revers phase C-18 column. Incubation led to the appearance of two new peaks as detected by HPLC one of which was collected and shown to have the composition consistent with des-glycinamide LHRH. The other peak ran in the position identical to that of authentic glycinamide. Results suggest that prolyl endopeptidase could play a role in the inactivation of LHRH in vivo.

Animals↗
PubMed1978

Clozapine concentrations in brain regions: relationship to dopamine metabolite increase.

Levels of clozapine in rat striatum and tuberculum olfactorium were quantitated by a gas chromatographic technique. The relationship of the increase in 3,4-dihydroxyphenylacetic acid (DOPAC) in these regions produced by clozapine to the concentration of clozapine was explored. One hour after 10, 20 or 40 mg/kg clozapine i.p. the concentration of drug increased in proportion to the dose and at each dose was similar in striatum and T.O. The percent increase in DOPAC in both areas was related to the clozapine concentration in a typical dose--response manner and was greater in the striatum than the T.O. A relatively high concentration of clozapine (40 micron) was required to produce a half-maximal elevation of DOPAC. Striatal clozapine levels were similar in acutely and chronically treated animals. The concentrations of clozapine in striatum and T.O. reflect the dose injected and do not account for its atypical properties.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1978

Dopamine metabolites in human brain.

The relationship of human brain levels of 3,4-dihydroxyphenylacetic acid (DOPAC) to cerebrospinal fluid levels of this domapine (DA) metabolite was studied. The effect of postmortem delay was evaluated in the rat. DOPAC was resistant to postmortem changes in brain kept in situ. The level of DOPAC (free and conjugated) determined in DA-rich areas of six human brains amounted to only a fraction of the homovanillic acid (HVA) found in the same regions. The DOPAC/HVA ratio in human brain was similar to that found in CSF. We conclude that HVA is the major DA metabolite in human brain and that DA metabolite levels in CSF reflect DA metabolite levels in brain.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1978

Synthesis of ophthalmic acid in liver and kidney in vivo.

The synthesis of ophthalmic acid, an analogue of glutathione, was studied in vivo in mouse liver and kidney after administration of either L-alpha-aminobutyrate or L-gamma-glutamyl-L-alpha-aminobutyrate as precursor. L-alpha-aminobutyrate accumulated to a much greater extent, and induced a much greater synthesis of ophthalmic acid in the liver than in the kidney. In contrast, L-gamma-glutamyl-L-alpha-aminobutyrate initiated a large and more rapid synthesis of ophthalmic acid in the kidney than in the liver. Experiments with L-gamma-[G(-14)C]glutamyl-L-alpha-aminobutyrate showed that, although part of the dipeptide is degraded to its constituent amino acids, a significant proportion is directly incorporated into kidney ophthalmic acid. In contrast L-gamma-glutamyl-L-alpha-aminobutyrate serves poorly as a direct precursor of liver ophthalmic acid. The present results show that kidney gamma-glutamyl tripeptide synthesis can proceed directly from an exogenous gamma-glutamyl dipeptide precursor.

Aminobutyrates↗