Search PubMed⌕ Search

Biomedical subjects

S Wilk

Publications and source records attributed to S Wilk.

At least 91 records · Page 5Linked to original sources
PubMed1987

Intracerebroventricular infusion of inhibitors of endopeptidase-24.11 ('enkephalinase') increases the spontaneous firing frequency of an identifiable set of cells in the substantia nigra.

The effect of inhibitors of the membrane-bound metalloendopeptidase-24.11 ('enkephalinase') on the activity of electrophysiologically identifiable neurons in the substantia nigra is described. Dopaminergic and non-dopaminergic cells were examined. Cells were classified by their responses to striatal stimulation. Only those cells in which the stimulation evoked excitation (alone or mixed with inhibition) responded to the inhibitors. Those cells in which the evoked response was only inhibition did not respond to the drugs. Infusion of 1 mumol of N-[1-(R,S)-carboxy-2-phenyl-ethyl]Phe-pAB (CPAB), 1 or 2 mumol of N-[1-(R,S)-carboxy-3-phenylpropyl]Phe-pAB (CPPAB) into the lateral ventricle produced statistically significant increases (pre- to post-drug treatment) in the spontaneous activity of cells exhibiting excitatory evoked responses: average increases were 33.3%. The increase in spontaneous activity reached an apparent maximum 20 min after the end of the infusion. The increased firing frequency was shown to result from the inhibition of the enzyme, rather than a non-specific effect, as the infusion of 2 mumol of N-[1-(R,S)-carboxy-2-phenyl-ethyl]Leu-pAB, an inhibitor structurally related to CPAB and CPPAB yet two orders of magnitude less potent, was without effect on the activity of nigral neurons. The inhibition of the enzyme by 1 mumol CPAB was verified through in vitro assay. We hypothesize that inhibition of the enzyme enhances peptide-modulated (tachykinin and/or enkephalin) excitation in select neurons of the substantia nigra.

Animals↗
PubMed1987

Regulation of a thyrotropin-releasing hormone-degrading enzyme in GH3 cells: induction of pyroglutamyl peptidase I by 3,5,3'-triiodothyronine.

The effect of exposure of GH3 cells to T3 on the TRH-degrading enzymes pyroglutamyl peptidase I (EC 3.4.19.3) and prolyl endopeptidase (EC 3.4.21.26) was studied. T3 produced a dose-dependent increase in the specific activity of pyroglutamyl peptidase I after 3 days of exposure. The EC50 for T3 was 5 X 10(-10) M. The specific activity of prolyl endopeptidase was unaffected by exposure to T3. The increase in pyroglutamyl peptidase I activity was dependent upon the time of exposure of the cells to this hormone. A maximal effect occurred at 72 h. The stimulation of pyroglutamyl peptidase I by T3 was totally blocked by cycloheximide, indicating that this enzyme is induced in GH3 cells by T3. The effect of T3 on the two TRH-degrading enzymes was also studied in the ACTH-secreting cell line AtT20. T3 had no effect on these enzymes in the AtT20 cell, suggesting that the effect of T3 on pyroglutamyl peptidase I may be cell specific. These studies indicate that the induction of pyroglutamyl peptidase I by T3 may contribute to the negative feedback regulation of T3 levels.

Aminopeptidases↗
PubMed1986

Neuropeptide-specific peptidases: does brain contain a specific TRH-degrading enzyme?

The particulate fraction of brain homogenates contains an enzyme that cleaves the pyroglutamyl-histidyl bond of thyrotropin-releasing hormone (TRH) but is clearly distinct from the more widely distributed pyroglutamyl peptidase (EC 3.4.19.3). This particulate enzyme is highly localized to brain where it is found on synaptosomal membranes. It exhibits an unusual degree of substrate specificity. For example, it does not cleave the pyroglutamyl-histidyl bond of luteinizing hormone-releasing hormone (LHRH) or the pyroglutamyl histidyl bond of the chromogenic substrate pyroglutamyl-histidyl-2-naphthylamide. Evidence is reviewed supporting the possibility that this enzyme, first detected in serum and originally referred to as "thyroliberinase", may be the first neuropeptide-specific peptidase to be characterized.

Aminopeptidases↗
PubMed1986

Delineation of a particulate thyrotropin-releasing hormone-degrading enzyme in rat brain by the use of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase.

The degradation of thyrotropin-releasing hormone in rat brain homogenates was studied in the presence of N-benzyloxycarbonyl-prolyl-prolinal and pyroglutamyl diazomethyl ketone, specific and potent active-site-directed inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase, respectively. Substantial TRH degradation was observed, suggesting the presence of another thyrotropin-releasing hormone-degrading enzyme(s). Reports of a thyrotropin-releasing hormone-degrading enzyme with narrow specificity that cleaves the pGlu-His bond of this tripeptide led us to develop a coupled assay using pGlu-His-Pro-2NA as the substrate to measure this activity. Cleavage of the pGlu-His bond of this substrate under conditions in which pyroglutamyl peptide hydrolase is not expressed occurred in the particulate fraction of a rat brain homogenate. This particulate pyroglutamyl-peptide cleaving enzyme was not inhibited by pyroglutamyl diazomethyl ketone but was inhibited by metal chelators such as EDTA and o-phenanthroline. The particulate pyroglutamyl-peptide cleaving enzyme was found predominantly in the brain. Activity in brain regions varied widely with highest levels present in cortex and hippocampus and very low levels in pituitary. The data suggest that degradation of thyrotropin-releasing hormone by the particulate fraction of a brain homogenate is catalyzed mainly by an enzyme that cleaves the pGlu-His bond of thyrotropin-releasing hormone but is distinct from pyroglutamyl peptide hydrolase.

Aminopeptidases↗
PubMed1986

Evidence for regulation of a thyrotropin-releasing hormone degradation pathway in GH3 cells.

GH3 cells, cloned from a rat anterior pituitary tumor, synthesize and secrete PRL in response to TRH. One of the pathways of TRH degradation is removal of the N-terminal pyroglutamyl residue catalyzed by pyroglutamyl peptide hydrolase (PPH; EC 3.4.11.8). We recently described the synthesis and properties of 5-oxoprolinal, a specific and potent (Ki = 26 nM) inhibitor of PPH. The effect of long term exposure of GH3 cells to 5-oxoprolinal on PPH activity was studied by incubating cells with inhibitor for 3 days, harvesting, washing to remove inhibitor, and assaying for PPH. Unexpectedly, we found a marked (300%) increase in PPH activity. This effect was dependent on the concentration of 5-oxoprolinal (EC50 = 10(-7) M) and was time dependent, with a rapid increase in enzyme activity occurring during the first 24 h. Cycloheximide did not block the increase. The results suggest that the activity of PPH in GH3 cells is subject to complex regulatory mechanisms.

Amidohydrolases↗
PubMed1985

The effect of inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase on TRH degradation in rat serum.

The identity of the enzymes catalyzing the degradation of thyrotropin releasing hormone (TRH) in rat serum was investigated by the use of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase. These inhibitors did not protect TRH from degradation, but o-phenanthroline afforded significant protection. The participation of "thyroliberinase", a metalloenzyme which cleaves TRH at the pyroglutamyl-His bond was implied. A coupled assay using the chromogenic substrate pyroglutamyl-His-Pro-2-naphthylamide and excess diaminopeptidase IV was developed to specifically quantitate "thyroliberinase" activity. Rat serum catalyzed the degradation of 67.5 nmoles substrate/ml serum/h. The data indicate that TRH is degraded in rat serum predominantly by "thyroliberinase" and that prolyl endopeptidase and pyroglutamyl peptide hydrolase do not contribute significantly to this process.

Aminopeptidases↗
PubMed1985

Pyroglutamyl diazomethyl ketone: potent inhibitor of mammalian pyroglutamyl peptide hydrolase.

Pyroglutamyl peptide hydrolase (EC 3.4.11.8), a cysteine protease, cleaves the N-terminal pyroglutamyl residue from pyroglutamyl peptides such as thyrotropin releasing hormone. Pyroglutamyl diazomethyl ketone was synthesized as an active site directed inhibitor. Preincubation of the partially purified bovine brain enzyme with nanomolar concentrations of inhibitor produced rapid inactivation. Inhibitor concentrations five orders of magnitude higher did not inactivate other exo- and endopeptidases. A dose of 0.1 mg/kg administered intraperitoneally to mice totally inactivated the enzyme in all tissues studied including brain. Pyroglutamyl diazomethyl ketone should be of value in studies on the physiological role of this enzyme in the metabolism of pyroglutamyl-containing peptides.

Aminopeptidases↗
PubMed1985

5-Oxoprolinal: transition-state aldehyde inhibitor of pyroglutamyl-peptide hydrolase.

Pyroglutamyl-peptide hydrolase (EC 3.4.11.8) removes the N-terminal pyroglutamyl residue from pyroglutamyl-containing peptides such as thyrotropin-releasing hormone (TRH), luteinizing hormone-releasing hormone (LH-RH), neurotensin, and bombesin. The aldehyde analogue of pyroglutamate, 5-oxoprolinal, was synthesized as an active site directed transition-state inhibitor of the enzyme. 5-Oxoprolinal was found to be a potent (Ki = 26 nM) and specific competitive inhibitor of pyroglutamyl-peptide hydrolase. Other aldehydes tested inhibited the enzyme only weakly or not at all. 5-Oxoprolinal blocked the degradation of LH-RH by purified pyroglutamyl-peptide hydrolase. The inhibitor, when injected into mice, inhibited the enzyme after 10 and 30 min. 5-Oxoprolinal should be of value in studies probing the biological significance of pyroglutamyl-peptide hydrolase.

Aminopeptidases↗
PubMed1984

Inhibitors of an enkephalin degrading membrane-bound metalloendopeptidase: analgesic properties and effects on striatal enkephalin levels.

Intraperitoneal administration of N-[1-(R,S)-carboxy-2-phenylethyl-Phe-p-aminobenzoate, synthesized in this laboratory as a potent inhibitor of membrane-bound metalloendopeptidase (EC 3.4.24.11) caused a prolonged but weak analgesic effect on rats as measured by the tail flick test. It also caused a transitory but significant increase in striatal [Leu5]- and [Met5]enkephalin levels 3 h, after administration. Analogs of the inhibitor in which the phenylalanyl residue was replaced by an alanyl or glycyl residue also elicited prolonged analgesic responses although their inhibitory potencies were 75 and more than 1500 times lower respectively. The glycine containing derivative did not alter striatal enkephalin levels 3 h, after administration. The data suggest that inhibition of the metalloendopeptidase decreases the rate of degradation of endogenous enkephalins, however the analgesic properties of the inhibitors do not seem to be related to their inhibitory potencies. Factors other than changes in striatal enkephalin levels may contribute to the analgesic effect of the three N-carboxyphenylethyl derivatives.

4-Aminobenzoic Acid↗
PubMed1984

Prolyl endopeptidase: inhibition in vivo by N-benzyloxycarbonyl-prolyl-prolinal.

The activity of prolyl endopeptidase in homogenates of mouse tissues was determined 30 min after intraperitoneal injection of N-benzyloxycarbonyl-prolyl-prolinal (1.25 mg/kg), a potent transition state analog inhibitor (K1 = 14 nM) of prolyl endopeptidase (EC 3.4.21.26). A more than 85% decrease of enzyme activity was obtained in all tissues. The in vivo degradation of potential prolyl endopeptidase substrates was studied by following the release of sulfamethoxazole from N-benzyloxycarbonylglycyl-prolyl-sulfamethoxazole, a model synthetic substrate of the enzyme. When this substrate was given intraperitoneally, its enzymatic degradation was blocked after administration of the inhibitor in a dose- and time-dependent manner, indicating inhibition of the enzyme in vivo. Of interest is the long duration of the inhibition. After a relatively low inhibitor dose (5 mg/kg) significant inhibition was seen in most tissues even after 6 h. The brain was particularly sensitive to the effect of the inhibitor. Since prolyl endopeptidase readily degrades many proline-containing neuropeptides, the inhibitor should be of value in studies on the role of the enzyme in neuropeptide metabolism.

Animals↗
PubMed1984

Peptide-degrading enzymatic activities in GH3 cells and rat anterior pituitary homogenates.

The activities of a number of peptide-degrading enzymes were compared in homogenates of GH3 cells and rat anterior pituitaries. The enzymes studied were prolyl endopeptidase (EC 3.4.21.26), a soluble metalloendopeptidase, pyroglutamyl peptide hydrolase (EC 3.4.11.8), a multicatalytic protease complex, cathepsin B (EC 3.4.22.1), cathepsin D (EC 3.4.23.5), aminopeptidase (EC 3.4.11.2), and a membrane-bound neutral metalloendopeptidase (EC 3.4.24.11). Specific substrates were used to measure the activities, and active-site-directed inhibitors were used to verify the identities of the enzymes studied. Of the two lysosomal enzymes studied, cathepsin B, the enzyme with the highest activity in both preparations, had 5 times the activity in GH3 cell homogenates as in anterior pituitary homogenates. Cathespin D had a somewhat higher activity in the anterior pituitary homogenates than in the GH3 cell homogenates. Soluble metalloendopeptidase and prolyl endopeptidase, both cytoplasmic enzymes, had about twice the activity in GH3 cell homogenates as in anterior pituitary homogenates. Membrane-bound neutral metalloendopeptidase in the GH3 cell homogenates had 25% of the activity of the anterior pituitary homogenates. Of the two TRH-degrading enzymes, the activity of prolyl endopeptidase in GH3 cell homogenates was about 25 times higher than that of pyroglutamyl peptide hydrolase. Since the secretory function of the pituitary is in part controlled by neuropeptides, the knowledge of the enzyme profiles of the GH3 cells and the anterior pituitary should be of value in studying the metabolism of neuropeptides and peptide hormones in these systems.

Animals↗
PubMed1983

Prolyl endopeptidase.

Prolyl endopeptidase (E.C. 3.4.21.26) an enzyme previously called post proline cleaving enzyme, TRH-deamidase or kininase B, may play a role in neuropeptide metabolism. This enzyme, highly active in brain and other tissues, catabolizes proline-containing peptides such as substance P, neurotensin, luteinizing hormone-releasing hormone, thyrotropin releasing hormone, bradykinin and angiotensin II. The structure of beta-neo-endorphin suggests that this opioid peptide is formed by the action of prolyl endopeptidase on a precursor of higher molecular weight. Formation of two biologically active fragments of substance P also requires the action of this enzyme. This review summarizes the current knowledge of the biochemistry of this enzyme, and its potential significance for neuropeptide physiology and pharmacology.

Animals↗
PubMed1983

Evidence that pituitary cation-sensitive neutral endopeptidase is a multicatalytic protease complex.

Pituitary cation-sensitive neutral endopeptidase splits peptide bonds on the carboxyl side of hydrophobic amino acids (chymotrypsin-like activity), basic amino acids (trypsin-like activity), and acidic amino acids (peptidyl-glutamyl-peptide bond hydrolyzing activity). All three activities copurify, are inhibited by cations, and reside in a single high-molecular weight soluble protein complex. Treatment with sodium dodecylsulfate and 2-mercaptoethanol dissociates this complex into five low-molecular weight components. Incubation of the complex at 37 degrees C in buffers of high ionic strength produces aggregation and progressive loss of all three activities. Experiments with inhibitors and activators indicate that the three activities are catalyzed by distinct components. Benzyloxycarbonyl-glycyl-glycyl-leucinal, a peptide aldehyde transition state analog of the substrate used to measure the chymotrypsin-like activity, exclusively inhibits that activity (Ki = 2.5 x 10(-4) M), while markedly activating the trypsin-like activity. The trypsin-like activity is inhibited by leupeptin (Ki = 1.2 x 10(-6) M) and by sulfhydryl blocking agents, and activated by thiols, suggesting that this activity is due to a thiol protease. The peptidylglutamyl-peptide hydrolyzing activity is activated almost 10-fold by low concentrations of sodium dodecylsulfate, inhibited by bovine serum albumin, and suppressed at high enzyme concentrations, suggesting that this component readily interacts with other proteins, including the complex itself. The results indicate that cation-sensitive neutral endopeptidase is a multicatalytic protease complex whose distinct proteolytic activities are associated with separate components of this high-molecular weight protein.

Animals↗
PubMed1983

Inhibition of rabbit brain prolyl endopeptidase by n-benzyloxycarbonyl-prolyl-prolinal, a transition state aldehyde inhibitor.

Prolyl endopeptidase cleaves peptide bonds on the carboxyl side of proline residues within a peptide chain. The enzyme readily degrades a number of neuropeptides including substance P, neurotensin, thyrotropin-releasing hormone, and luteinizing hormone-releasing hormone. The finding that the enzyme is inhibited by benzyloxycarbonyl-prolyl-proline, with a Ki of 50 microM, prompted the synthesis of benzyloxycarbonyl-prolyl-prolinal as a potential transition state analog inhibitor. Rabbit brain prolyl endopeptidase was purified to homogeneity for these studies. The aldehyde was found to be a remarkably potent inhibitor of prolyl endopeptidase with a Ki of 14 nM. This Ki is more than 3000 times lower than that of the corresponding acid or alcohol. By analogy with other transition state inhibitors, it can be assumed that binding of the prolinal residue to the S1 subsite and the formation of a hemiacetal with the active serine of the enzyme greatly contribute to the potency of inhibition. The specificity of the inhibitor is indicated by the finding that a variety of proteases were not affected at concentrations 150 times greater than the Ki for prolyl endopeptidase. The data indicate that benzyloxycarbonyl-prolyl-prolinal is a specific and potent inhibitor of prolyl endopeptidase and that consequently it should be of value in in vivo studies on the physiological role of the enzyme.

Animals↗
PubMed1982

Determination of specificity of endopeptidases by combined high-performance liquid chromatography and amino acid analysis.

The specificity of three neutral endopeptidases toward several biologically active peptides was determined by combined high-performance liquid chromatography and amino acid analysis of the degradation products. Incubation mixtures were chromatographed on a reversed-phase column equilibrated with a mixture of acetonitrile and potassium phosphate buffer (0.05 M; pH 2.0). Reaction products were eluted with a linear gradient of acetonitrile and the absorbance of the effluent monitored at 210 nm. Fractions corresponding to discrete peaks were subjected to quantitative amino acid analysis. The peptide bond undergoing cleavage is readily assigned from the knowledge of the primary structure of the peptide and the amino acid composition of the reaction products.

Amino Acids↗
PubMed1982

Subcellular distribution of prolyl endopeptidase and cation-sensitive neutral endopeptidase in rabbit brain.

The subcellular distribution of prolyl endopeptidase, and of cation-sensitive neutral endopeptidase, two enzymes actively metabolizing many neuropeptides, was determined in homogenates of rabbit brain. The subcellular distribution of both enzymes was more similar to lactate dehydrogenase, a cytoplasmic enzyme marker, than to choline acetyltransferase, a synaptosomal marker. Only 35% of the activity of these two neutral endopeptidases was found in the crude mitochondrial fraction (P2), the bulk of the remaining activity being associated with the high-speed supernatant. Prolyl endopeptidase and cation-sensitive neutral endopeptidase thus can be regarded as mainly cytoplasmic enzymes in the rabbit brain.

Animals↗