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)

An inhibitor of serine proteinases from human articular cartilage was purified to homogeneity by sequential ultrafiltration and ion exchange chromatography on CM-Sephadex C-50. The apparent molecular weight of the cationic glycoprotein (pI greater than 10) was determined to be 16.5 X 10(3) by SDS gel electrophoresis. The inhibitor blocked the activity of leukocyte elastase, cathepsin G and trypsin but not leukocyte collagenase. In kinetic studies for the interactions with leukocyte elastase a firm enzyme-inhibitor binding was obtained. Amino acid analyses did not reveal homologies with other serine proteinase inhibitors already purified from human tissues.
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PMID:Purification and characterization of a serine proteinase inhibitor from human articular cartilage. 349 75

We have used a high performance liquid chromatography assay, which detects chymotryptic cleavage of the phe8-his9 bond of angiotensin I to yield angiotensin II, in order to examine human lung mast cells for the presence of chymotryptic activity. Mast cells, purified from human lung by enzymatic dispersion, countercurrent elutriation, and Percoll gradient centrifugation, were lysed or challenged with goat anti-human IgE. In multiple experiments angiotensin II-converting activity was detected in lysates of 10-99% pure mast cell preparations. Regression analysis of net percent release values of histamine and the angiotensin I-converting activity from dose-response experiments demonstrated a correlation between the two parameters, indicating that the chymotrypsin-like enzyme is a constituent of the mast cell secretory granule. The chymotryptic activity was completely inhibited by 10(-3) M phenylmethylsulfonylfluoride but not by 10(-3) M Captopril, and the pH optimum of activity was 7.5-9.5. Gel filtration of released material separated the activity from tryptase and demonstrated an approximate molecular weight of 30-35,000. The mast cell enzyme, like a human skin chymotrypsin-like proteinase, can be distinguished from leukocyte cathepsin G by lack of susceptibility to inhibition by bovine pancreatic trypsin inhibitor. Thus, an enzyme with limited chymotryptic specificity is present in human lung mast cells. The Michaelis constant of the enzyme for angiotensin I of 6.0 X 10(-5) M is similar to that of endothelial cell angiotensin-converting enzyme and is consistent with a reaction of physiologic importance.
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PMID:A human lung mast cell chymotrypsin-like enzyme. Identification and partial characterization. 351 Oct 89

We report the isolation of the human gene encoding an inhibitor of neutrophil elastase and cathepsin G. We have sequenced the gene and a cDNA clone isolated from human parotid tissue. The protein encoded by this gene appears to contain two functional domains, one having a trypsin inhibitory site and the other an elastase inhibitory site. The two-domain structure of the protein is reflected in the organization of the gene, with each domain represented by a separate exon. We have also noted that the intervening sequence separating the trypsin-inhibitor-exon and the elastase-inhibitor-exon is flanked by eleven base-pair direct repeats, suggesting that this intron may have been generated by a transposition-type event.
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PMID:Isolation and sequence of a human gene encoding a potent inhibitor of leukocyte proteases. 364 Mar 38

The major urinary trypsin inhibitor UTI I is a proteoglycan. UTI c (Mr 26,000), produced by chrondroitin lyase digestion of UTI I, was isolated and characterized. About 90% of the glycosaminoglycan chain was removed by this treatment without proteolytic modification, as assessed by amino-acid composition and N-terminal sequence of UTI c. Its electrophoretic mobilities on alkaline and SDS-PAGE are identical with those of UTI II which occurs in urine during storage. To study the role of the glycosaminoglycan chain on the inhibitory properties of UTI I, UTI I and UTI c were compared using different proteinases as target enzymes. The inhibitory activity towards bovine trypsin and chymotrypsin as well as human granulocytic cathepsin G did not differ significantly. However, towards human granulocytic elastase, the equilibrium dissociation constant (Ki) is 5 times higher for UTI c than for UTI I. Weak inhibitory activities were measured on human plasmin, UTI c being more efficient than UTI I. The acid-stability of UTI I is not modified after chrondroitin lyase treatment. UTI I and UTI c are equally sensitive to trypsinolysis indicating that the covalently bound glycosaminoglycan chain does not play an important role for the stability of UTI I.
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PMID:The effect of the glycosaminoglycan chain removal on some properties of the human urinary trypsin inhibitor. 364 44

A series of carboxy-alkylamidated and N-acetylated amino acids and peptides were synthesized and examined for their ability to inhibit human leukocyte elastase. The Boc-amino acid alkylamides were found to be potent specific and competitive inhibitors of this enzyme. They were found not to or only poorly inhibit several other serine proteinases such as bovine trypsin, alpha-chymotrypsin, porcine pancreatic elastase and human leukocyte cathepsin G at concentrations less than 10(-4) M. Specificity and maximum inhibition of human leukocyte elastase were achieved when the N-terminus of the amino acid was protected by a t-butyloxy-carbonyl (Boc) group, the oligopeptide fragment consisted of valine residues and when the alkyl chain was between 10 and 12 carbon atoms in length and attached to the C-terminus of the peptide fragment. Highest inhibition was obtained with the compound Boc-[Val]3-NH[CH2]11--CH3 (Ki = 0.21 microM). These specific inhibitors were also found to be non-toxic after oral administration to mice and rats (LD50 greater than 3.0 g/kg body weight).
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PMID:Synthetic inhibitors of human leukocyte elastase, Part 3. Peptides with alkyl groups at the N- or C-terminus. Non-toxic competitive inhibitors of human leukocyte elastase. 364 43

The major urinary trypsin inhibitor (UTI) was found to inhibit bovine chymotrypsin and human leucocyte elastase strongly, cathepsin G weakly. No inhibition of porcine pancreatic elastase was observed. The stoichiometry of the inhibition of bovine trypsin by UTI was determined spectrophotometrically to be 1:2 (I/E molar ratio). After incubation of UTI with this enzyme in various molar ratios, two complexes (C1 and C2) could be visualized in alkaline polyacrylamide gel electrophoresis. C1 was isolated by affinity chromatography on Con-A Sepharose. In dodecyl sulfate polyacrylamide gel electrophoresis, C1 was dissociated to give an inhibitory band with the same electrophoretic mobility as native UTI. C2 released an active inhibitory fragment with Mr near 20000. A time-course study demonstrated that at a molar ratio I/E of 1.5:1, the C2 complex appears after two hours of incubation.
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PMID:Human urinary proteinase inhibitor: inhibitory properties and interaction with bovine trypsin. 384 79

Human inter-alpha-trypsin inhibitor has been found to inactivate human trypsin, chymotrypsin, neutrophil elastase and cathepsin G. The protein was cleaved into two major fragments without loss of activity by incubation with Serratia marcescens metalloproteinase, and these were separated by ion-exchange chromatography. Inhibitory activity was found in only one of the fragments, the amino-terminal sequence of which was found to be identical with that of the native protein, as well as with that reported earlier for the urinary trypsin inhibitor. It may thus be concluded that the reactive site of the inter-alpha-trypsin inhibitor is located in the amino-terminal region.
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PMID:The reactive site of human inter-alpha-trypsin inhibitor is in the amino-terminal half of the protein. 389 Aug 90

Protease La is an ATP-dependent protease that catalyzes the rapid degradation of abnormal proteins and certain normal polypeptides in Escherichia coli. In order to learn more about its specificity and the role of ATP, we tested whether small fluorogenic peptides might serve as substrates. In the presence of ATP and Mg2+, protease La hydrolyzes two oligopeptides that are also substrates for chymotrypsin, glutaryl-Ala-Ala-Phe-methoxynaphthylamine (MNA) and succinyl-Phe-Leu-Phe-MNA. Methylation or removal of the acidic blocking group prevented hydrolysis. Closely related peptides (glutaryl-Gly-Gly-Phe-MNA and glutaryl-Ala-Ala-Ala-MNA) are cleaved only slightly, and substrates of trypsin-like proteases are not hydrolyzed. Furthermore, several peptide chloromethyl ketone derivatives that inhibit chymotrypsin and cathepsin G (especially benzyloxycarbonyl-Gly-Leu-Phe-chloro-methyl ketone), inhibited protease La. Thus its active site prefers peptides containing large hydrophobic residues, and amino acids beyond the cleavage site influence rates of hydrolysis. Peptide hydrolysis resembles protein breakdown by protease La in many respects: 1) ADP inhibits this process rapidly, 2) DNA stimulates it, 3) heparin, diisopropyl fluorophosphate, and benzoyl-Arg-Gly-Phe-Phe-Leu-MNA inhibit hydrolysis, 4) the reaction is maximal at pH 9.0-9.5, 5) the protein purified from lon- E. coli or Salmonella typhymurium showed no activity against the peptide, and that from lonR9 inhibited peptide hydrolysis by the wild-type enzyme. With partially purified enzyme, peptide hydrolysis was completely dependent on ATP. The pure protease hydrolyzed the peptide slowly when only Mg2+, Ca2+, or Mn2+ were present, and ATP enhanced this activity 6-15-fold (Km = 3 microM). Since these peptides cannot undergo phosphorylation, adenylylation, modification of amino groups, or denaturation, these mechanisms cannot account for the stimulation by ATP. Most likely, ATP and Mg2+ affect the conformation of the enzyme, rather than that of the substrate.
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PMID:Protease La, the lon gene product, cleaves specific fluorogenic peptides in an ATP-dependent reaction. 390 67

A highly active angiotensin-producing enzyme (enzyme II) was obtained from dog serum by acid treatment and fractionation to remove angiotensinase and converting enzyme, separate an inhibitor, and convert an inactive precursor (proenzyme II) to enzyme II. Proenzyme II was found to be converted to enzyme II by an endogenous activating enzyme identified as plasmin. Conversion was also caused by the interaction of bacterial streptokinase with human proactivator, by trypsin, and by an activator formed from liver tissue extract and dog serum. Neither plasma kallikrein nor the labile, human extrinsic tissue-type plasminogen activator induced activation. The inhibitor, which normally blocks the activation of proenzyme II, was unusually stable against high temperatures and extremes of pH, and it was not identical to any of the six known protease inhibitors of serum. Enzyme II was not identical to other angiotensin-producing enzymes such as enzyme I, renin, cathepsin D, pepsin, plasmin, tonin, or cathepsin G. Enzyme II reacted maximally at pH 4.7 and produced up to 2250 ng of angiotensin I/ml serum/hr from the substrate of dog serum (i.e., amounts 3200-fold higher than that produced by endogenous renin of normal dog serum). Since at pH 7.2, angiotensin I formation is still about 30 times higher than that of renin, enzyme II may be physiologically active under some conditions.
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PMID:Angiotensin-producing serum enzyme II. Formation by inhibitor removal and proenzyme activation. 390 15

The time-dependent inactivation of several serine proteases including human leukocyte elastase, cathepsin G, rat mast cell proteases I and II, and human skin chymase by a number of 3-alkoxy-4-chloroisocoumarins, 3-alkoxy-4-chloro-7-nitroisocoumarins, and 3-alkoxy-7-amino-4-chloroisocoumarins at pH 7.5 and the inactivation of several trypsin-like enzymes including human thrombin and factor XIIa by 7-amino-4-chloro-3-ethoxyisocoumarin and 4-chloro-3-ethoxyisocoumarin are reported. The 3-alkoxy substituent of the isocoumarin is likely interacting with the S1 subsite of the enzyme since the most reactive inhibitor for a particular enzyme had a 3-substituent complementary to the enzyme's primary substrate specificity site (S1). Inactivation of several enzymes including human leukocyte elastase by the 3-alkoxy-7-amino-4-chlorisocoumarins is irreversible, and less than 3% activity is regained upon extensive dialysis of the inactivated enzyme. Addition of hydroxylamine to enzymes inactivated by the 3-alkoxy-7-amino-4-chloroisocoumarins results in a slow (t1/2 greater than 6.7 h) and incomplete (32-57%) regain in enzymatic activity at pH 7.5. Inactivation by the 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-4-chloro-7-nitroisocoumarins on the other hand is transient, and full enzyme activity is regained rapidly either upon standing, after dialysis, or upon the addition of buffered hydroxylamine. The rate of inactivation by the substituted isocoumarins is decreased when substrates or reversible inhibitors are present in the incubation mixture, which indicates active site involvement. The inactivation rates are dependent upon the pH of the reaction mixture, the isocoumarin ring system is opened concurrently with inactivation, and the reaction of 3-alkoxy-7-amino-4-chloroisocoumarins with porcine pancreatic elastase is shown to be stoichiometric. The results are consistent with a scheme where 3-alkoxy-7-amino-4-chloroisocoumarins react with the active site serine of a serine protease to give an acyl enzyme in which a reactive quinone imine methide can be released. Irreversible inactivation could then occur upon alkylation of an active site nucleophile (probably histidine-57) by the acyl quinone imine methide. The finding that hydroxylamine slowly catalyzes partial reactivation indicates that several inactivated enzyme species may exist. The 3-alkoxy-substituted 4-chloroisocoumarins and 4-chloro-7-nitroisocoumarins are simple acylating agents and do not give stable inactivated enzyme structures.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Reaction of serine proteases with substituted 3-alkoxy-4-chloroisocoumarins and 3-alkoxy-7-amino-4-chloroisocoumarins: new reactive mechanism-based inhibitors. 391 97


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