Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In halothane-anesthetized and -ventilated cynomologus macaque monkeys, the effects of administering vehicle (n = 3) or the neutral endopeptidase inhibitor N-[L-(1-carboxy-2-phenyl)ethyl]-L-phenylalanyl-beta-alanine (16 mg/kg, n = 5; or 100 mg/kg, n = 3, intravenously) was examined. Cisternal CSF aliquots were examined by radioimmunoassay: 1) for Met enkephalin; 2) after trypsin and carboxypeptidase B treatment for encrypted enkephalin (X-ENK); 3) for substance P; and 4) for unmetabolized drug. Similar measures were carried out in femoral artery and femoral venous plasma, except that substance P was not assayed. In CSF, prior to drug, low, but measurable levels of enkephalin (61 pg/ml), X-ENK (285 pg/ml) and substance P (16 pg/ml) were observed. Vehicle-injected animals showed no change from baseline levels over a 4-hr sampling period in either plasma or CSF levels. In contrast, following 16 mg/kg, in CSF, there was a significant 9-fold increase in MET and 11-fold increase in X-ENK at 30 min. CSF-substance P levels rose also by a factor of 2, with the peak effect observed at 60 min. All levels displayed a significant reduction by 4 hr. There was no statistical difference between the maximum effects observed with either the 16- or 100-mg/kg dose. Plasma peptide levels of enkephalin and X-ENK were not altered by drug. CSF displayed significant drug levels by 30 min, which were between 0.1 and 1% of levels observed concurrently in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of [N-(L-(1-carboxy-2-phenyl)ethyl]-L-phenylalanyl-beta-alanine (SCH32615), a neutral endopeptidase (enkephalinase) inhibitor, on levels of enkephalin, encrypted enkephalins and substance P in cerebrospinal fluid and plasma of primates. 170 28

The inhibitory effect of various dipeptides on the neurotensin-degrading metallopeptidase, endopeptidase 24.16, was examined. These dipeptides mimick the Pro10-Tyr11 bond of neurotensin that is hydrolyzed by endopeptidase 24.16. Among a series of Pro-Xaa dipeptides, the most potent inhibitory effect was elicited by Pro-Ile (Ki approximately 90 microM) with Pro-Ile greater than Pro-Met greater than Pro-Phe. All the Xaa-Tyr dipeptides were unable to inhibit endopeptidase 24.16. The effect of Pro-Ile on several purified peptidases was assessed by means of fluorigenic assays and HPLC analysis. A 5 mM concentration of Pro-Ile does not inhibit endopeptidase 24.11, endopeptidase 24.15, angiotensin-converting enzyme, proline endopeptidase, trypsin, leucine aminopeptidase, pyroglutamyl aminopeptidase I and carboxypeptidase B. The only enzyme that was affected by Pro-Ile was carboxypeptidase A, although it was with a 50-fold lower potency (Ki approximately 5 mM) than for endopeptidase 24.16. By means of fluorimetric substrates with a series of hydrolysing activities, we demonstrate that Pro-Ile can be used as a specific inhibitor of endopeptidase 24.16, even in a complex mixture of peptidase activities such as found in whole rat brain homogenate.
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PMID:Specific inhibition of endopeptidase 24.16 by dipeptides. 176 Oct 32

We have utilized [3H] gelatin to document high activity of a metalloproteinase present in freshly isolated rat glomeruli. [3H] gelatin degradation by glomeruli was markedly inhibited by EDTA (10 mM: -89 +/- 2.3%) and o-phenanthroline (2 mM: -72 +/- 0.1%), inhibitors of metalloproteinases. No significant inhibition of [3H]gelatin degradation was observed with inhibitors of serine or cysteine proteinases. Most (greater than 80%) of the glomerular metalloproteinase (GLOMP) activity was associated with the pellet after centrifugation of sonicated glomeruli at 100,000 g for 90 min. The pH optimum for gelatin degradation by sonicated glomeruli was approximately pH 8.5. Sodium dodecyl sulfate substrate (gelatin)-polyacrylamide gel electrophoresis revealed a single major band of EDTA-inhibitable gelatin-degrading activity with a molecular mass of approximately 116-125 kDa. The GLOMP activity was not inhibited by tissue inhibitors of metalloproteinases, did not appear to be latent, and was not activated by organomercurial activators of several latent metalloproteinases. GLOMP activity was increased 3.4-fold after incubation with trypsin (20 micrograms/ml, 25 min, 22 degrees C). These data indicate that GLOMP is distinct from the previously described matrix metalloproteinases, as well as other metalloproteinases present in the kidney, including the gelatinase secreted by cultured mesangial cells, Meprin, and endopeptidase 24.11 (enkephalinase, EC 3.4.24.11).
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PMID:A novel metalloproteinase present in freshly isolated rat glomeruli. 190 57

Carboxypeptidase M, a plasma membrane-bound enzyme, is present in many human organs and differs from other carboxypeptidase that cleave basic COOH-terminal amino acids. Cultured Madin-Darby canine kidney (MDCK) distal tubular cells contain a kininase I-type enzyme that inactivates bradykinin by releasing Arg9. We found the properties of this kininase to be identical with carboxypeptidase M. In fractionated cells, carboxypeptidase activity sediments with membranes; and detergents, trypsin, and phosphatidylinositol-specific phospholipase C solubilize it, similar to results with human placental carboxypeptidase M. Ten microM 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and 1 mM o-phenanthroline inhibit, whereas 1.0 mM CoCl2 activates the enzyme. It has a neutral pH optimum and cleaves COOH-terminal Arg or Lys in bradykinin and in shorter peptides. The relative hydrolysis rates of peptides in the presence or absence of 1 mM CoCl2 were similar to those obtained with human carboxypeptidase M. The carboxypeptidase in MDCK cells (54 kDa) cross-reacts with antibodies to human carboxypeptidase M in Western blotting, but not with antibodies to plasma carboxypeptidase N. The enzyme is a glycoprotein; chemical deglycosylation reduced the size to 48 kDa. The presence of the enzyme on the cell membrane of MDCK cells was also shown with transmission electron microscopy using immunogold, which indicated that the enzyme is on the apical side. In addition, MDCK cells contain neutral endopeptidase 24.11 (enkephalinase) and prolylcarboxypeptidase (angiotensinase C) activities. Partitioning of solubilized carboxypeptidase M into Triton X-114 and water indicates that trypsin and phospholipase C remove a hydrophobic tail, while detergent solubilization leaves the hydrophobic moiety intact. Labeling of MDCK cells with [3H]ethanolamine resulted in the synthesis of radiolabeled carboxypeptidase M as determined by immunoprecipitation and fluorography. Thus, MDCK cells contain membrane-bound carboxypeptidase M, which is anchored to the plasma membrane via phosphatidylinositol-glycan. As a major kininase of the distal tubules, it may regulate salt and water excretion.
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PMID:Carboxypeptidase M in Madin-Darby canine kidney cells. Evidence that carboxypeptidase M has a phosphatidylinositol glycan anchor. 239 13

A kinin-potentiating peptide (KPP) generated from human plasma proteins on trypsin incubation was partially purified by ultrafiltration and ion-exchange chromatography and was characterized through some of its pharmacological properties. KPP itself was devoid of any action but it potentiated the guinea-pig ileum contractions elicited by several kinins, including an analog resistant to angiotensin-converting enzyme (ACE). In contrast, contractions induced by angiotensin II, histamine, acetylcholine, barium chloride and substance P were not potentiated. Not only did KPP have high specificity towards kinins, but its action started immediately and induced kinin potentiation in a dose-dependent and reversible manner. Furthermore KPP potentiated the bradykinin contracting effects on the rat uterus, a preparation with very poor ACE activity, and on guinea-pig ileum previously incubated with 1.10-phenanthroline, a metal chelator able to inhibit ACE and kininase I activities and with phosphoramidon, a specific inhibitor of neutral endopeptidase (NEP). The results suggest that the potentiating effect of KPP is due to a mechanism different from the inhibition of kinin metabolism by ACE, NEP and kininase I.
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PMID:Pharmacological properties of a new kinin-potentiating peptide generated from human serum proteins. 260 51

Neutral endopeptidase 24.11 contains an active-site arginine residue involved in binding the free carboxylate of substrate peptides and inhibitors. This arginine reacts rapidly with [14C]phenylglyoxal, and its reaction is selectively blocked by the presence of either the substrate Met5-enkephalin, the competitive inhibitor phenylalanylalanine, or the transition state analog phosphoramidon. The phenylglyoxal-modified peptide was isolated by a procedure involving limited digestion by trypsin, separation of the tryptic peptides by high pressure liquid chromatography (HPLC), further digestion of the modified peptide by pepsin, and a final purification by HPLC. By this procedure arginine 102 was identified as the active-site arginine. Verification of this finding came from the use of site-directed mutagenesis in which this arginine was replaced by glutamine. Both the mutant and wild-type enzyme reacted equally well with an amide containing substrate, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. However, reaction of the mutant enzyme with a substrate containing a free COOH-terminal carboxylate, 5-dimethylaminonaphthalene-1-sulfonyl-D-Ala-Gly-(NO2)Phe-Gly, was barely detectable with the mutant enzyme. Similarly the mutant enzyme showed a loss of selectivity in inhibition by D-Ala2-Met5-enkephalin compared to the corresponding amide but exhibited no difference in the maximal velocity for hydrolysis of D-Ala2-Met5-enkephalin and its amide.
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PMID:Identification of the active-site arginine in rat neutral endopeptidase 24.11 (enkephalinase) as arginine 102 and analysis of a glutamine 102 mutant. 270 83

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

Three distinct peptidyldipeptidases (exopeptidases releasing carboxyl terminal dipeptide residues) can be solubilized from nerve terminal membrane fractions from whole rat brain or striatum, and separated by ion exchange chromatography. Brain angiotensin-converting enzyme (PDP-1) cleaves Hip-His-Leu, but not 80 nM [3H-Tyr1, Leu5]-enkephalin, and is markedly inhibited by several specific inhibitors such as captopril, teprotide, and MK-422. Enkephalinase (PDP-2) cleaves 80 nM [3H-Tyr1, Leu5]-enkephalin, but not Hip-His-Leu; it is not inhibited by any of the standard competitive inhibitors of angiotensin-converting enzyme (all analogs of carboxyl-terminal peptide sequences Phe-Ala-Pro or Ala-Pro), but is strongly inhibited by captopril analogs such as thiorphan (Phe-Gly analog). A third peptidyldipeptidase (PDP-3) cleaves Hip-His-Leu, but not 80 nM [3H-Tyr1, Leu5]-enkephalin; it is inhibited by dipeptide analog inhibitors such as captopril and thiorphan, but not by longer peptides such as teprotide or tripeptide analog inhibitors such as MK-422. Both PDP-2 (enkephalinase) and PDP-3 are apparently present in nerve terminal membranes predominantly as inactive proenzyme precursors, which elute from DEAE-cellulose at high salt concentration, and are activated very slowly by a process involving one or more trypsin-like enzymes. Rechromatography of activated PDP-2 and PDP-3 achieves a nearly complete separation of the two enzymes, both markedly purified, since each is much less acidic than its proenzyme precursor. Purified enkephalinase does not appear to have any significant endopeptidase activity. It cleaves Hip-Phe-Arg 200 times more effectively than Hip-Phe-Arg-NH2, and appears to be quite selective for cleaving the terminal dipeptide residue, Phe-Arg, from bradykinin, with no release of the second dipeptide and no cleavage of the Gly4-Phe5 interior peptide bond.
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PMID:Purification and characterization of enkephalinase, angiotensin converting enzyme, and a third peptidyldipeptidase from rat brain. 631 70

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.
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PMID:Evidence that pituitary cation-sensitive neutral endopeptidase is a multicatalytic protease complex. 633 56

C6 rat glioblastoma cells are able to attach to and to spread on culture dishes which are coated with purified central nervous system myelin, in contrast to normal astrocytes, fibroblasts or neurons which adhere poorly and are unable to spread on this substrate. The metalloprotease blockers o-phenanthroline and a newly developed oligopeptide could specifically inhibit C6 cell spreading on central nervous system myelin, suggesting a crucial role for a metalloprotease. Here we characterize this metalloproteolytic activity of C6 cells using a peptide degradation assay with the iodinated tetrapeptide carbobenzoxy-Phe-Ala-Phe-125I-Tyr-amide as a substrate. Purified, salt-washed C6 plasma membranes cleaved the peptide between alanine and phenylalanine, an effect which is strongly inhibited by o-phenanthroline, but not by thiol-blocking agents or aspartic and serine protease inhibitors. The metalloendoprotease is highly sensitive to phosphoramidon but insensitive to thiorphan. The enzyme is tightly bound to the plasma membrane but not G protein-phosphatidylinositol linked. It can be solubilized in part by the detergents 3-(3-cholamidopropyldimethylamino)-1-propanesulfonate or Triton X-114. Gel filtration chromatography using the Triton X-114-solubilized proteins or the proteins removed by a short trypsin treatment revealed a molecular weight range for the C6 enzyme of 60,000-100,000. Polymerase chain reaction with primers corresponding to endopeptidase 24.11 or to the highly conserved motif of the "astacin family" showed that both enzymes were not detectable in the C6 glioblastoma cells.
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PMID:Characterization of a membrane-bound metalloendoprotease of rat C6 glioblastoma cells. 803 33


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