Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tyr-Gly-Gly (YGG) was recently shown to be an extraneuronal metabolite of opioid peptides derived from proenkephalin A, formed in brain by the action of "enkephalinase" (membrane metalloendopeptidase, EC 3.4.24.11) and degraded by aminopeptidases. The dynamic state of YGG in mouse striatum was studied by evaluating the changes in its level elicited by inhibitors of these peptidases. Inhibition of YGG synthesis by Thiorphan or acetorphan reduced YGG levels with a t1/2 (mean +/- SEM) of 12 +/- 2 min, indicating an apparent turnover rate (mean +/- SEM) of 18 +/- 2 pmol/mg of protein per hr. An apparent turnover rate of 18 +/- 2 pmol/mg of protein per hr was derived from the rate of YGG accumulation elicited by the aminopeptidase inhibitor bestatin. In addition, accumulation of Tyr-Gly-Gly-Phe-Met (YGGFM) in an extrasynaptosomal fraction after blockade of its degradation by Thiorphan and bestatin occurred at a rate of 18 +/- 3 pmol/mg of protein per hr, which is likely to reflect the rate of enkephalin release in vivo. Hence, the three series of data suggest that striatal enkephalins rapidly turn over--e.g., with a t1/2 in the 1-hr range. Pentobarbital anesthesia reduced by about 60% the rate of YGG accumulation elicited by bestatin and the extrasynaptosomal YGGFM accumulation elicited by Thiorphan and bestatin. This suggests that the activity of striatal enkephalin neurons is depressed during anesthesia. Pentobarbital (and chloral hydrate) did not affect the steady-state level of YGGFM but rapidly reduced that of YGG. Hence, the steady-state levels of YGG seem a reliable index of changes in enkephalin release, and measuring levels of characteristic fragments might therefore provide a general means of evaluating neuropeptide release in vivo.
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PMID:Steady-state level and turnover rate of the tripeptide Tyr-Gly-Gly as indexes of striatal enkephalin release in vivo and their reduction during pentobarbital anesthesia. 352 54

Purified rat brain cathepsin B (EC 3.4.22.1) converted prodynorphins or proenkephalins to shorter active forms by the preferential removal of C-terminal dipeptides. The substrate affinities for Met-enkephalin-Arg-Phe or -Arg-Gly-Leu were Km 46 and 117 microM, and kcat/Km ratios were 67 and 115 microM-1, min-1, respectively. Met-Enkephalin was inactivated by the same mechanism (Km-450 microM; kcat/Km = 0.12 microM-1 min-1). The comparison of cathepsin B hydrolysis for pro-opioids, a synthetic hexapeptide and its fragments, C-blocked peptides (pro-opioid amides, Met-enkephalin amide, substance P), and bovine myelin basic protein, provided information on the influence of the C-terminal residues on dipeptide release, the rates as correlated to peptide length, and the optimal arrangement of residues favoring scission at the P1-P'1 sites. The brain enzyme was stereospecific and did not act on peptides with C-terminal D-amino acid substituents. Arg hindered and Pro blocked the release of C-terminal dipeptides when in the P'2 positions. The suppression of dipeptide release by agents inhibiting endopeptidase actions such as E-64 and leupeptin, and the endogenous brain factor (cerebrocystatin) point to similar catalytic mechanisms for the exopeptidase action.
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PMID:Preferential action of rat brain cathepsin B as a peptidyl dipeptidase converting pro-opioid oligopeptides. 353 Jan 35

In the present studies we report that membrane-associated proteases in Salmonella typhimurium and Escherichia coli catalyze limited proteolysis of IIIGlcSlow. We have previously reported (Meadow, N. D., and Roseman, S. (1982) J. Biol. Chem. 257, 14526-14537) the isolation of two electrophoretically distinguishable forms of IIIGlc, which is a phosphocarrier and regulatory protein of the phosphoenolpyruvate:glycose phosphotransferase system. The two species of IIIGlc were designated IIIGlcFast and IIIGlcSlow; IIIGlcSlow is 7 amino acid residues longer than IIIGlcFast at its NH2 terminus. The majority of the protease activity is located in the outer membrane fraction from both species of bacteria, with the cytoplasmic fraction being devoid of activity. The site of cleavage is at the Lys-Ser bond located at residues 7-8 of IIIGlcSlow. The enzyme is an endopeptidase which liberates the expected heptapeptide (Gly-Leu-Phe-Asp-Lys-Leu-Lys). Both the large fragment of the limited proteolytic reaction, IIIGlcFast, and the small fragment, the heptapeptide, are stable to further proteolysis by membranes for more than 17 h at 37 degrees C. The activity in E. coli membranes has an absolute requirement for divalent metal ion (Mg2+ or Ca2+) and is heat-resistant, whereas the activity in S. typhimurium membranes is stimulated by divalent metal ion and is heat-sensitive. These results suggest significant differences between the two enzymes. The physiological function of the limited proteolysis of IIIGlc is not known.
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PMID:Limited proteolysis of IIIGlc, a regulatory protein of the phosphoenolpyruvate:glycose phosphotransferase system, by membrane-associated enzymes from Salmonella typhimurium and Escherichia coli. 353 Dec 6

Using a sensitive radioimmunoassay, the tripeptide Tyr-Gly-Gly (YGG) which corresponds to the N-terminal sequence of opioid peptides was detected in rat brain and identified by HPLC. Its regional distribution paralleled that of (Met5)enkephalin (YGGFM), a marker of enkephalin neurons. Ablation of these neurons in the striato-pallidal pathway by intrastriatal kainate, induced a significant decrease in YGG levels in caudateputamen and globus pallidus (-49%), consistent with the hypothesis that YGG originates from enkephalin neurons. When pallidal slices were incubated under various conditions, YGG was mainly found in the incubation medium indicating a predominantly extracellular localization. Depolarization of these slices by a K+-stimulus elicited a release of YGGFM accompanied by a marked increase in YGG levels. Bestatin and amastatin further enhanced YGG levels, reflecting the participation of aminopeptidases in the metabolism of the tripeptide and its precursor. Captopril, an inhibitor of the angiotensin-converting enzyme (ACE) showed no effect on the recovery of YGGFM and YGG. In contrast, the formation of YGG was completely prevented by Thiorphan (IC50 value = 9 nM) and phosphoramidon, two inhibitors of "enkephalinase" (EC 3.4.24.11; membrane metallo-endopeptidase), thus identifying the latter as the YGG-forming enzyme. The K+-induced increase in YGG + YGGFM levels in medium containing bestatin exceeded by about 60% the amount of YGGFM released from tissues, suggesting that YGG was mainly formed by extracellular hydrolysis of the various opioid fragments of the proenkephalin molecule. In vivo, YGG levels of cerebral regions were also markedly reduced in rats treated with acetorphan, a parenterally active "enkephalinase" inhibitor. All data suggest that YGG levels constitute an index of opioid peptide release.
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PMID:The endogenous tripeptide Tyr-Gly-Gly as a possible metabolite of opioid peptides in rat brain: identification, regional distribution, effects of lesions and formation in depolarized slices. 353 54

The contents of [Met5]-enkephalin and its four hydrolysis products, Tyr, Tyr-Gly, Tyr-Gly-Gly, and Tyr-Gly-Gly-Phe, in the sample obtained after enkephalin was incubated with tissues in either the absence or the presence of the peptidase inhibitor were estimated by high performance liquid chromatography combined with electrochemical detection in order to elucidate the effect of the peptidase inhibitor on enkephalin hydrolysis. Free Tyr was the major degradative product in the absence of the peptidase inhibitor, while the major hydrolysis product was the Tyr-Gly-Gly fragment in the presence of amastatin in both total homogenates and membrane fractions prepared from either the myenteric plexus-longitudinal muscle strip of guinea-pig ileum or the striatum of guinea-pig brain. Additionally, captopril significantly decreased the generation of Tyr-Gly-Gly in the presence of amastatin in both ileal and striatal membrane fractions. Moreover, thiorphan significantly prevented the formation of Tyr-Gly in the presence of both amastatin and captopril in both membrane preparations. Finally, when [Met5]-enkephalin was incubated with either an ileal or a striatal membrane fraction for 60 min at 37 degrees C in the presence of three peptidase inhibitors, amastatin, captopril, and thiorphan, approximately 98 or 94%, respectively, of enkephalin remained intact. The results indicated that [Met5]-enkephalin was almost exclusively hydrolyzed by three distinct enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive peptidyl dipeptidase A, and thiorphan-sensitive endopeptidase-24.11 in both ileal and striatal membrane fractions.
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PMID:Effects of peptidase inhibitors on the [Met5]-enkephalin hydrolysis in ileal and striatal preparations of guinea-pig: almost complete protection of degradation by the combination of amastatin, captopril and thiorphan. 353 99

Vasoactive peptides contain a high proportion of proline residues which make them resistant to hydrolysis by many peptidases. However, post proline cleaving enzyme (PPCE; EC 3.4.21.26), a proline specific endopeptidase which specifically hydrolyzes internal peptide bonds on the carboxyl side of proline residues, has been shown to inactivate numerous vasoactive peptides including angiotensins, kinins, substance P, vasopressin and oxytocin. In order to determine whether PPCE could be involved in vascular metabolism of vasoactive peptides, we carried out localization and characterization studies of PPCE-like activity in hog aorta and mesenteric artery. PPCE was assayed fluorometrically at pH 7.0 using the specific PPCE substrate CBZ-Gly-Pro-4-methyl-coumarinylamide. The subcellular distribution of vascular PPCE was essentially the same as that of the cytosolic marker enzyme lactic dehydrogenase (LDH). PPCE was enriched six-fold in the cytosolic fraction (11.4 +/- 2.7 units/mg) and unlike the plasma membrane-bound proline specific exopeptidase dipeptidyl-(amino)peptidase IV (DAP IV; EC 3.4.14.5), little or no activity could be detected in the microsomal or plasma membrane fractions. Similar to PPCE characterized from other sites, vascular PPCE was stabilized and activated by dithiothreitol and EDTA, and inhibited by DFP, p-chloromercuriphenyl sulfonic acid, L-1-tosylamido-2-phenylethylchloromethyl ketone, Cu++, Ca++, and Zn++. Vascular PPCE was unaffected by inhibitors of trypsin and kallikrein (Aprotinin, ABTI), aminopeptidase M (bestatin, amastatin), neutral endopeptidase (phosphoramidon), angiotensin I converting enzyme (captopril) or carboxypeptidase N (MERGETPA). These data demonstrate that PPCE is present in vascular endothelium and/or smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Vascular, post proline cleaving enzyme: metabolism of vasoactive peptides. 354 18

Less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, the luteinizing hormone-releasing hormone, LHRH, is degraded in renal proximal tubules (PT) in vivo (rat) and in vitro (rabbit) to less than Glu-His (2), less than Glu-His-Trp (3), and less than Glu-His-Trp-Ser (4). LHRH may be cleaved by endopeptidases simultaneously at multiple bonds, or initially at Ser4-Tyr5 followed by carboxypeptidase hydrolysis of 4 to 3 and then 2. To distinguish between these mechanisms, [3H]LHRH analogues were incubated with rabbit renal brush-border membranes (BBM), microinfused into PT in vivo or in vitro, and products were analyzed by HPLC. [D-Ser4]LHRH was not cleaved at D Ser4-Tyr5 but yielded less than Glu-His-Trp-D-Ser-Tyr-Gly as the major metabolite plus 2 and 3. [D-Trp6]LHRH was cleaved by BBM and PT to 2 and 3, but not to 4. [D-Ser4, D-Trp6]LHRH was not cleaved by BBM, but was degraded to 2 by PT in vivo. Thus, D-amino acid substituents altered the expected cleavage pattern of these analogues. [3H]LHRH was cleaved by BBM or by endopeptidase-24.11 from porcine PT to metabolites 2, 4, small amounts of 3, and less than Glu-His-Trp-Ser-Tyr-Gly, but cleavage was strongly inhibited by the specific inhibitor phosphoramidon. Thus, normally LHRH may be cleaved in PT by endopeptidase-24.11 to 2 and 4, and by angiotensin I-converting enzyme to 3, its known cleavage site.
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PMID:Effects of D-amino acid substituents on degradation of LHRH analogues by proximal tubule. 354 29

Novel fluorescent substrates for enkephalinase (neutral endopeptidase; EC 3.4.24.11) have been developed. These new assays are based on the disappearance of energy transfer between a tryptophan or a tyrosine residue and the 5-dimethylaminonaphthalene-1-sulfonyl group (dansyl) in the substrates dansyl-Gly-Trp-Gly or dansyl-Gly-Tyr-Gly upon hydrolysis of their Gly-Trp or Gly-Tyr amide bond by enkephalinase. No significant difference in Km or kcat values were found for dansyl-Gly-Trp-Gly and dansyl-Gly-Tyr-Gly, indicating that, in contrast to thermolysin, the active site of enkephalinase easily accommodates tryptophan residues. Both tryptophan and tyrosine-containing substrates can be used for continuous recording of enkephalinase activity and should prove useful for detailed study of the substrate specificity of this enzyme.
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PMID:New substrates for enkephalinase (neutral endopeptidase) based on fluorescence energy transfer. 354 27

A high-Mr neutral endopeptidase-24.5 (NE) that cleaved bradykinin at the Phe5-Ser6 bond was purified to apparent homogeneity from human lung by (NH4)2SO4 fractionation, ion-exchange chromatography and gel filtration. The final enzyme preparation produced a single enzymically active protein band after electrophoresis on a 5% polyacrylamide gel. Human lung NE had an Mr of 650,000 under non-denaturing conditions, but after denaturation and electrophoresis on an SDS/polyacrylamide gel NE dissociated into several lower-Mr components (Mr 21,000-32,000) and into two minor components (Mr approx. 66,000). The enzyme activity was routinely assayed with the artificial substrate Z-Gly-Gly-Leu-Nan (where Z- and -Nan represent benzyloxycarbonyl- and p-nitroanilide respectively). NE activity was enhanced slightly by reducing agents, greatly diminished by thiol-group inhibitors and unchanged by serine-proteinase inhibitors. Human lung NE was inhibited by the univalent cations Na+ and K+. No metal ions were essential for activity, but the heavy-metal ions Cu2+, Hg2+ and Zn2+ were potent inhibitors. With the substrate Z-Gly-Gly-Leu-Nan a broad pH optimum from pH 7.0 to pH 7.6 was observed, and a Michaelis constant value of 1.0 mM was obtained. When Z-Gly-Gly-Leu-Nap (where -Nap represents 2-naphthylamide) was substituted for the above substrate, no NE-catalysed hydrolysis occurred, but Z-Leu-Leu-Glu-Nap was readily hydrolysed by NE. In addition, NE hydrolysed Z-Gly-Gly-Arg-Nap rapidly, but at pH 9.8 rather than in the neutral range. Although human lung NE was stimulated by SDS, the extent of stimulation was not appreciable as compared with the extent of SDS stimulation of NE from other sources.
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PMID:A high-molecular-mass neutral endopeptidase-24.5 from human lung. 355 24

This and two accompanying reports describe the intrinsic binding energy derived from a single hydrogen bond between an inhibitor and an enzyme. The results were obtained by comparing matched pairs of inhibitors of the zinc endopeptidase thermolysin that bind to the enzyme in an essentially identical manner but differ in the presence or absence of a specific hydrogen bond. This report describes five phosphorus-containing analogs of the peptides carbobenzoxy-Gly-Leu-X, in which the Gly-Leu peptide linkage is replaced with a phosphonate ester (-PO2(-)-O-). Values for the inhibition constants of these inhibitors show a direct relation with those of the corresponding phosphonamidate analogs (-PO2(-)-NH- in place of the Gly-Leu peptide moiety), which have been characterized previously as transition state analogs. However, each phosphonate ester is bound about 840 times more weakly than the analogous phosphonamidate, reflecting the loss of 4.0 +/- 0.1 kilocalories per mole in binding energy. From these results and the crystallographic analysis in the next report, it can be inferred that the value of 4.0 kilocalories per mole represents the intrinsic binding energy arising from a highly specific hydrogen binding interaction.
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PMID:Evaluation of intrinsic binding energy from a hydrogen bonding group in an enzyme inhibitor. 381 Jan 55


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