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)

Trypsin, porcine pancreatic kallikrein, and several blood coagulation enzymes, including bovine thrombin, bovine factor Xa, human factor Xa, human plasma factor XIa, human plasma factor XIIa, and human plasma kallikrein, were inactivated by a number of substituted isocoumarins containing basic functional groups (aminoalkoxy, guanidino, and isothiureidoalkoxy). 3-Alkoxy-4-chloro-7-guanidinoisocoumarins were found to be the most potent inhibitors for the coagulation enzymes tested with kobsd/[I] values in the range of 10(3)-10(5) M-1 s-1. 4-Chloro-3-isothiureidoalkoxyisocoumarins show high inhibitory potency toward porcine pancreatic kallikrein, human plasma kallikrein, human factor XIa, human factor XIIa, and trypsin with kobsd/[I] values of the order of 10(4)-10(5) M-1 s-1. The inhibition of these serine proteases by the substituted isocoumarins are time dependent, and the inactivation of trypsin by 3-alkoxy-4-chloro-7-guanidinoisocoumarins and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin occured concurrently with the loss of the isocoumarin absorbance. The complex formed from inactivation of trypsin by these two types of inhibitors was very stable and regained less than 4% activity in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (pH 7.5) after 1 day at 25 degrees C and regained 8-45% activity upon addition of buffered 0.29 M hydroxylamine. Trypsin inactivated by other inhibitors regained full activity upon standing or addition of hydroxylamine. Thrombin inactivated by 3-alkoxy-4-chloro-7-guanidinoisocoumarins was also quite stable and only regained 9-15% activity under similar conditions. These results are consistent with a proposed mechanism, where serine proteases inactivated by aminoalkoxyisocoumarins or isothiureidoalkoxyisocoumarins form acyl enzymes that will deacylate upon standing or addition of hydroxylamine. However, the acyl enzymes formed from 3-alkoxy-4-chloro-7-guanidinoisocoumarins or 7-amino-4-chloro-3-(3-isothiureidopropoxy)-isocoumarin will decompose further, probably through a quinone imine methide, to give an irreversibly inactivated enzyme by reaction with an active-site nucleophile such as His-57. The quinone imine methide intermediate may also react with a solvent nucleophile to give an acyl enzyme that can be reactivated by hydroxylamine. The inhibitors 4-chloro-7-guanidino-3-methoxyisocoumarin and 4-chloro-3-ethoxy-7-guanidinoisocoumarin have been tested as anticoagulants in human plasma and were effective at prolonging the prothrombin time. However, they are unstable in plasma (t1/2 = 4-8 min), and their in vivo utility may be limited.
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PMID:Mechanism-based isocoumarin inhibitors for trypsin and blood coagulation serine proteases: new anticoagulants. 316 17

The amino acid sequence of a coagulant enzyme, named flavoxobin, isolated from the venom of Trimeresurus flavoviridis (the habu snake) was determined by sequencing the S-pyridylethylated derivative of the protein and its peptides generated by chemical (cyanogen bromide and hydroxylamine) and enzymatic (clostripain, Staphylococcus aureus V8 protease, Achromobacter protease I, and elastase) cleavages. Hydrazinolysis was also employed to determine the C-terminal amino acid. The enzyme consisted of 236 amino acids and had a calculated molecular weight of 25,744. Flavoxobin was found to be highly (69%) homologous in sequence to batroxobin, a coagulant enzyme from the venom of Bothrops atrox, and 27, 39, and 31% homologous to bovine thrombin, bovine trypsin, and human kallikrein, respectively. The sequence around the active site serine residue deduced from the homology relationship was Phe-Asp-Ser-Gly-Thr, which is different from the common sequence, Gly-Asp-Ser-Gly-Gly, for most serine proteases. Flavoxobin appears to be similar in secondary structure composition to batroxobin.
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PMID:Amino acid sequence of a coagulant enzyme, flavoxobin, from Trimeresurus flavoviridis venom. 317 May 3

Progesterone receptors are phosphoproteins, in which phosphorylation has been proposed as a control mechanism for some stages of hormone action. Progesterone administration was shown to increase phosphorylation of the receptor from both cytosol and nuclear extracts of whole cells. We have analyzed the receptor phosphopeptides generated by chemical and proteolytic cleavage to assess the number of phosphorylation sites and their approximate location in the receptor. Progesterone receptor was labeled in situ in the presence or absence of hormone in medium containing [32P] orthophosphate, isolated by immunoprecipitation, and then digested with several proteases. The resulting 32P-labeled peptides were resolved by either two-dimensional electrophoresis:chromatography or by reverse-phase high performance liquid chromatography. Multiple phosphopeptides (3-6) were detected after cleavage with trypsin, chymotrypsin, or V8 protease. Major increases in phosphorylation occurred at existing sites since after hormone treatment no new phosphopeptides were found. Individual phosphopeptides showed variable increases in phosphorylation of 1.5-5-fold. The A and B receptor forms showed identical phosphorylation patterns, indicating similar processing in vivo. The phosphopeptide pattern for receptor in nuclear extracts resembled that of cytosol receptor. Chemical cleavage was used to assess the distribution of phosphorylation sites. Cyanogen bromide produced a large 40-kDa polypeptide which contained all of the phosphorylation sites and comprised the residues 129-449. Hydroxylamine was used to cleave a unique bond, Asn-372-Gly-373, in the 40-kDa polypeptide. All of the phosphorylation sites were located on the amino-terminal side of the cleavage. Thus, all of the phosphorylation sites were localized to a specific region (Met-129 to Asn-372) of the progesterone receptor that does not include either the DNA or steroid binding domains.
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PMID:Hormone-dependent phosphorylation of the avian progesterone receptor. 317 May 62

Aphrodisin is a protein which is secreted in hamster vaginal discharge and acts via the vomeronasal organ of the accessory olfactory system to elicit copulatory behavior in male hamsters. The complete primary structure of aphrodisin was determined by sequence analysis of intact aphrodisin after unblocking the amino terminus with pyroglutamate aminopeptidase and from peptides generated by trypsin and Lys-C digests. Alignment of the peptides was obtained from sequence analysis of peptides from cyanogen bromide and hydroxylamine cleavages. The protein consists of 151 residues of Mr = 17,000. It has disulfide bonds linking cysteine residues at positions 38 and 42 and at 57 and 149. N-acetylglucosamine residues are linked to asparagines at positions 41 and 69. Based on its similarity to the major urinary proteins in rats and mice, aphrodisin is a putative member of the alpha 2u-globulin superfamily of extracellular proteins.
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PMID:The primary structure of aphrodisin. 318 9

The cDNA coding for the porcine pancreatic prophospholipase A2 (proPLA) has been cloned and expressed in E. coli. Expression of proPLA could only be obtained in the form of intracellular aggregates after fusing the 15 kDa proPLA to a large (greater than or equal to 45 kDa) bacterial peptide. The fusion protein was readily purified from cell lysates, and specifically cleaved. Cleavage of the fusion protein was achieved with either hydroxylamine (at Asn/Gly sequences in the denatured protein), or trypsin (between the pro- and the mature PLA in the renatured fusion protein). The former method releases a proPLA-like enzyme, while the latter directly yields PLA. Renaturation of the fusion protein was made possible by the use of a recently reported new S-sulphonation method. The released (pro)PLA was purified (yields of 2-3 mg/ltr of culture medium), and showed identical properties compared to native (pro)PLA.
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PMID:Expression of porcine pancreatic phospholipase A2. Generation of active enzyme by sequence-specific cleavage of a hybrid protein from Escherichia coli. 329 82

As a final step in the elucidation of the primary structure of subunit a of Panulirus interruptus hemocyanin (657 residues, Mr 75696 excluding two copper ions and carbohydrate), the amino acid sequence of the largest fragment obtained by limited trypsinolysis was determined. The elucidation of the sequence of residues 176-657, comprising domains two and three, was mainly based on two digests, with CNBr and trypsin, respectively, from both of which a complete set of peptides was obtained. Additional sequence information was obtained from a digest with Staphylococcus aureus V8 protease and from one fragment obtained by cleaving subunit a with hydroxylamine. A block during Edman degradations indicated an Asn-Gly sequence at positions 597-598, although only aspartic acid was identified at position 597.
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PMID:Panulirus interruptus hemocyanin. The elucidation of the complete amino acid sequence of subunit a. 348 Feb 18

Human leukocyte elastase (HLE), a serine protease involved in inflammation and tissue degradation, can be irreversibly inactivated in a time- and concentration-dependent manner by ynenol lactones. Ynenol lactones that are alpha-unsubstituted do not inactivate but are alternate substrate inhibitors that are hydrolyzed by the enzyme. Ynenol lactones that are both substituted alpha to to the lactone carbonyl and unsubstituted at the acetylene terminus are rapid inactivators of HLE and inactivate pancreatic elastase and trypsin more slowly. 3-Benzyl-5(E)-(prop-2-ynylidene)tetrahydro-2-furanone inactivates HLE with biphasic kinetics and an apparent second-order rate of up to 22,000 M-1 s-1 (pH 7.8, 25 degrees C). The rate of inactivation is pH-dependent and is slowed by a competitive inhibitor. The partition ratio is 1.6 +/- 0.1. Rapid removal of ynenol lactone during the course of inactivation yields a mixture of acyl and inactivated enzyme species, which then shows a partial recovery of activity that is time- and pH-dependent. Inactivation is not reversible with hydroxylamine. The enzyme is not inactivated if the untethered allenone is added exogenously. All of these results are consistent with a mechanism involving enzyme acylation at serine-195 by the ynenol lactone, isomerization of the acyl enzyme to give a tethered allenone, and capture of a nucleophile (probably histidine-57) to inactivate the enzyme. Substitution at the acetylene terminus of ynenol lactones severely reduces their ability to inactivate HLE, because allenone formation is slowed and/or nucleophile capture is hindered. Chemical competence of each of these steps has been demonstrated [Spencer, R.W., Tam, T.F., Thomas, E.M., Robinson, V.J.,& Krantz, A. (1986) J. Am. Chem. Soc. 108, 5589-5597].
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PMID:Kinetics and mechanism of human leukocyte elastase inactivation by ynenol lactones. 354 14

The partial amino acid sequence of porcine elastase II, isolated from crude trypsin Type II, was determined. The enzyme consists of two polypeptide chains, a light chain composed of 11 residues, and a heavy chain (Mr = 23 500) with four intrachain disulfide bridges; the two chains are held together by one interchain disulfide bond. Elastase II was fragmented into several peptides by chemical cleavages with CNBr at the two methionine residues, 99 and 180 (chymotrypsinogen numbering), and with hydroxylamine at the peptide bond following DIP-Ser195. About 50% of the sequence was determined and the positions of 120 amino acids were located, including the light chain residues and most of the active site residues. The partial sequence shows 64% difference between porcine elastase II and elastase I and only 26% difference between porcine elastase II and bovine chymotrypsin B.
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PMID:Partial amino acid sequence of porcine elastase II. Active site and the activation peptide regions. 363 56

The first primary structure of a pectinesterase has been determined by analysis of the main form of this plant enzyme from tomatoes. The analysis was untraditional in the sense that few data were obtained by frequently used methods. Thus, digestion with trypsin, Glu-specific protease and several other enzymes gave limited cleavages and highly insoluble products. Instead, the determination was to a large extent based on peptides derived from chemical cleavages with CNBr at Met, N-chlorosuccinimide at Trp and hydroxylamine at Asn-Gly, plus an enzymatic cleavage at modified cysteine residues after chemical derivatizations. The structure shows the protein chain to be 305 residues long, with four half-cystine residues and five tryptophan residues. The N-terminus has a free alpha-amino group (from isoleucine). Two types of residue, tyrosine and histidine, have been functionally implied in the catalytic activity of the enzyme. Tyrosine is common in the protein (at 18 positions). However, only two histidine residues are found, and both are close to tyrosine residues in the primary structure (His-128 separated by one intervening residue from Tyr-126, and His-269 adjacent to Tyr-268), defining segment(s) of possible interest in relation to the active site.
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PMID:Pectinesterase. The primary structure of the tomato enzyme. 373 79

The complete amino acid sequence of porcine miniplasminogen (Mr 37 600), comprising 341 residues, was determined by automated Edman degradation in a liquid-phase or solid-phase sequenator. Selected fragments were produced by cleavage with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine (BNPS-skatole), cyanogen bromide, hydroxylamine, Staphylococcus aureus protease or trypsin or with combinations thereof and by activation with urokinase. The sequence obtained was compared with the known sequences of human and bovine miniplasminogen, indicating that the porcine molecule apparently contains the same structural and functional domains as the protein of the other two species. Porcine miniplasminogen has a sequence homology of 83% with human and of 79% with bovine miniplasminogen; 74% of the amino acids are identical in all three species. The results show a higher degree of evolutionary conservatism in the structurally and/or functionally vital regions of the molecule (active site residues, kringle 5).
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PMID:Determination of the complete amino-acid sequence of porcine miniplasminogen. 384 33


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