Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have expressed the 57-amino acid Kunitz domain of the Alzheimer's beta-amyloid precursor protein (APP751) as a bacterial fusion protein. The protease inhibitory properties of the purified fusion protein, BX9, were virtually identical in all respects tested to those of purified secreted APP751. Both proteins strongly inhibited pancreatic trypsin (Kis = 0.2 and 0.3 nM) and less well epidermal growth factor-binding protein (Kis = 1 and 3.5 nM), alpha-chymotrypsin (Kis = 3 and 6 nM), and the gamma-subunit of nerve growth factor (Kis = 8 and 9 M). Neither protein appreciably inhibited plasma and pancreatic kallikreins, thrombin, lung tryptase, neutrophil elastase, or cathepsin G. The remarkable similarity of the protease inhibitory profile of BX9 to that of secreted APP751 suggests that proper intramolecular disulfide bond formation has occurred in the bacterial fusion protein and leads to the conclusion that the amyloid precursor protein Kunitz domain is a relatively specific inhibitor of only a few trypsin-like arginine esterases.
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PMID:The protease inhibitory properties of the Alzheimer's beta-amyloid precursor protein. 211 13

Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4'-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4'-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes.
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PMID:Benzyl p-guanidinothiobenzoate hydrochloride, a new active-site titrant for trypsin and trypsin-like enzymes. 636 Jan 55

Human skin tryptase, a serine proteinase stored within mast cell secretory granules, rapidly loses enzymatic activity in solutions of physiological salt concentration, pH, and temperature. The inactivation of tryptase can be slowed and even reversed by addition of heparin, a highly sulfated glycosaminoglycan also found in the secretory granules. These properties may be relevant to tryptase regulation after secretion from mast cells. To further characterize the molecular changes underlying the functional instability of tryptase, circular dichroism (CD) and analytical ultracentrifugation were used to investigate structural changes during spontaneous inactivation. The CD spectra of active and spontaneously inactivated tryptase are different, particularly in the region around 230 nm where active tryptase displays a distinct negative peak. This peak is also observed in the CD spectrum of bovine chymotrypsin but not in trypsin, elastase, or chymotrypsinogen. Loss of activity resulting from spontaneous inactivation was accompanied by a diminution of the 230-nm signal. The kinetics for the signal loss appeared to be first-order and closely paralleled the rate of enzymatic activity loss. Dextran sulfate, a highly sulfated polysaccharide, was capable of reactivating tryptase and restoring the CD signal. After 2 h of decay (> 90% loss of activity), addition of dextran sulfate resulted in an almost immediate return of the CD signal to that of active tryptase. The return of the CD signal appeared to be more rapid than the return of enzymatic activity, thereby suggesting the presence of an unidentified step which is rate-limiting for activity return (and loss) and subsequent (prior) to the CD change accompanying activity loss. Ultracentrifugation analysis of tryptase showed a marked change in its association state upon inactivation. Sedimentation equilibrium under stabilizing conditions demonstrated the presence of a single species with the molecular weight of a tetramer. After spontaneous inactivation, a mixture of species was evident, which was characterized as monomers and tetramers in equilibrium. These results demonstrate that spontaneous inactivation of tryptase is associated with reversible conformational changes and that a consequence of inactivation is the formation of a destabilized tetrameric form. Although the molecular mechanism initiating these changes remains unclear, possible insights into the process are discussed on the basis of the similarity between the CD spectra of tryptase and chymotrypsin.
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PMID:Structural changes associated with the spontaneous inactivation of the serine proteinase human tryptase. 765 17

The x-ray crystal structure of recombinant leech-derived tryptase inhibitor (rLDTI) has been solved to a resolution of 1.9 A in complex with porcine trypsin. The nonclassical Kazal-type inhibitor exhibits the same overall architecture as that observed in solution and in rhodniin. The complex reveals structural aspects of the mast cell proteinase tryptase. The conformation of the binding region of rLDTI suggests that tryptase has a restricted active site cleft. The basic amino terminus of rLDTI, apparently flexible from previous NMR measurements, approaches the 148-loop of trypsin. This loop has an acidic equivalent in tryptase, suggesting that the basic amino terminus could make favorable electrostatic interactions with the tryptase molecule. A series of rLDTI variants constructed to probe this hypothesis confirmed that the amino-terminal Lys-Lys sequence plays a role in inhibition of human lung tryptase but not of trypsin or chymotrypsin. The location of such an acidic surface patch is in accordance with the known low molecular weight inhibitors of tryptase.
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PMID:The three-dimensional structure of recombinant leech-derived tryptase inhibitor in complex with trypsin. Implications for the structure of human mast cell tryptase and its inhibition. 924 60

Recently we have described a novel secreted protein (the WFIKKN protein) that consists of multiple types of protease inhibitory modules, including two tandem Kunitz-type protease inhibitor-domains. On the basis of its homologies we have suggested that the WFIKKN protein is a multivalent protease inhibitor that may control the action of different proteases. In the present work we have expressed the second Kunitz-type protease inhibitor domain of the human protein WFIKKN in Escherichia coli, purified it by affinity chromatography on trypsin-Sepharose and its structure was characterized by CD spectroscopy. The recombinant protein was found to inhibit trypsin (Ki = 9.6 nm), but chymotrypsin, elastase, plasmin, pancreatic kallikrein, lung tryptase, plasma kallikrein, thrombin, urokinase or tissue plasminogen activator were not inhibited by the recombinant protein even at 1 microm concentration. In view of the marked trypsin-specificity of the inhibitor it is suggested that its physiological target may be trypsin.
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PMID:Expression, purification and characterization of the second Kunitz-type protease inhibitor domain of the human WFIKKN protein. 1270 70

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that primarily inhibits the extrinsic pathway of blood coagulation. It is synthesized by various cells and its expression level increases in inflammatory environments. Mast cells and neutrophils accumulate at sites of inflammation and vascular disease where they release proteinases as well as chemical mediators of these conditions. In this study, the interactions between TFPI and serine proteinases secreted from human mast cells and neutrophils were examined. TFPI inactivated human lung tryptase, and its inhibitory activity was stronger than that of antithrombin. In contrast, mast cell chymase rapidly cleaved TFPI even at an enzyme to substrate molar ratio of 1:500, resulting in markedly decreased TFPI anticoagulant and anti-(factor Xa) activities. N-terminal amino-acid sequencing and MS analyses of the proteolytic fragments revealed that chymase preferentially cleaved TFPI at Tyr159-Gly160, Phe181-Glu182, Leu89-Gln90, and Tyr268-Glu269, in that order, resulting in the separation of the three individual Kunitz domains. Neutrophil-derived proteinase 3 also cleaved TFPI, but the reaction was much slower than the chymase reaction. In contrast, alpha-chymotrypsin, which shows similar substrate specificities to those of chymase, resulted in a markedly lower level of TFPI degradation. These data indicate that TFPI is a novel and highly susceptible substrate of chymase. We propose that chymase-mediated proteolysis of TFPI may induce a thrombosis-prone state at inflammatory sites.
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PMID:Tissue factor pathway inhibitor is highly susceptible to chymase-mediated proteolysis. 1750 77