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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ability of neutrophils to generate free radicals is a crucial component of host defense (Babior, B. M. (1978) N. Engl. J. Med. 298, 659-668, 721-725. Neutrophil oxidants, however, can cause significant host tissue destruction (Weiss, S. J. (1989) N. Engl. J. Med. 320, 365-376), and the regulation of free radical production is not well understood. We have previously shown that recombinant antichymotrypsin (rACT), a serine protease inhibitor, inhibits superoxide production in intact neutrophils (Kilpatrick, L., Johnson, J. L., Nickbarg, E. B., Wang, Z., Clifford, T. F., Banach, M., Cooperman, B. S., Douglas, S. D., and Rubin, H. (1991) J. Immunol. 146, 2388-2393). Using a cell-free NADPH oxidase preparation, we now demonstrate that rACT alone has no effect on superoxide production and that antichymotrypsin-chymotrypsin (rACT.CT) complexes are required to inhibit superoxide, suggesting that neutrophil chymotrypsin-like proteases produce conformational changes in ACT, allowing it to become active in regulating superoxide production. Additionally, we have identified NADPH oxidase itself as the target for rACT.CT and have demonstrated that rACT.CT interferes specifically with activation of the NADPH oxidase without changing the Km for NADPH or the rate constant describing the rate-limiting step in activation. These observations suggest an important role for antichymotrypsin in the regulation of NADPH-oxidase activation, which is a prerequisite for neutrophil superoxide production, and predict possible therapeutic uses for rACT in conditions where unregulated neutrophil-free radical production has been implicated in the mechanism of tissue destruction.
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PMID:Regulation of neutrophil superoxide by antichymotrypsin-chymotrypsin complexes. 131 83

We have studied an indirect role of serine and thiol proteases in the activation of human neutrophils in vitro. Stimulation was evaluated using a chemiluminescence (CL) generation system. Receptor-dependent and receptor-independent stimuli were studied, e.g. opsonized zymosan, formyl-methionyl-leucyl-phenylalanine, platelet activating factor, phorbol myristate acetate, and calcium ionophore A23187. The serine protease inhibitors TPCK and TLCK, and thiol protease inhibitor PHMB, diminished the CL with different potencies and in a dose-dependent manner after treatment of cells with the various stimuli. Non-specific serine protease inhibitor, PMSF, and trypsin substrate TAME, showed a low inhibitory potency with respect to CL generation. Synthetic substrates for chymotrypsin (BTEE, ATEE) significantly inhibited CL with the various stimuli used with some differences in susceptibility to their inhibition. Specific chymotrypsin inhibitors diminished both the resting and activator-induced CL. We suggest that cell-bound chymotrypsin-like protease(s) is involved in the activation of signal transduction in human neutrophils after both receptor-dependent and receptor-independent stimulation.
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PMID:The effect of serine and thiol protease inhibitors on the chemiluminescence of human neutrophils in investigations in vitro. 164 40

Cerebrospinal fluid (CSF) from 20 male patients with nonneurologic disease (age 64.5 +/- 2.8 SEM) was analyzed for the presence of the serpin alpha 1-antichymotrypsin (alpha 1-ACT). A chymotrypsin-specific chromogenic substrate (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) was used to examine the CSF samples. All CSF samples showed inhibitory activity ranging from 45 to 80% inhibition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the samples revealed the presence of a 68-kDa protein migrating identical to authentic human plasma alpha 1-ACT. Complex formation was performed with iodinated bovine chymotrypsin for several representative CSF samples having the highest chymotrypsin inhibitory activity. Comparison was made with complex formation performed with commercially available authentic human plasma alpha 1-ACT. These studies showed the formation of complexes at 37 degrees C, regardless of whether the sample was subsequently boiled or not. In the case of CSF, two complex bands, mass smaller than with plasma alpha 1-ACT, were formed at the lower temperature whereas a single higher Mr band was formed when the samples were boiled. To determine whether cleavage of the serpin occurred, these studies were repeated using human neutrophil cathepsin G as target protease. A complex of approximately 90 kDa was formed with human alpha 1-ACT under these same conditions. alpha 1-ACT has been detected in senile amyloid plaques in brains of Alzheimer's disease patients, the only plasma serine protease inhibitor localized to these structures. Another serpin, protease nexin I, is also found in these plaques, but this inhibitor does not circulate in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the serpin, alpha 1-antichymotrypsin, in normal human cerebrospinal fluid. 172 48

The equine Pi system, which is highly polymorphic and was considered to be controlled by a single locus, has been shown to be controlled by four loci (named Spi 1-4). This system is the equine equivalent of the major human plasma serpin (serine protease inhibitor), human alpha 1 PI. Twenty-two haplotypes of the equine Pi system have been characterized by two-dimensional electrophoresis, resulting in the assignment of pI, Mr, and bovine trypsin and chymotrypsin inhibition characteristics to 109 proteins. These proteins have been analyzed further to determine their relatedness to each other as well as to human alpha 1 PI using immunochemical, structural, and functional criteria. The amino acid sequences of the N termini and reactive-site regions have been determined on proteins from each of the four equine Spi loci. This allowed the designation of the proteins from the Spi 1 locus as being METserpins and the functional equivalents of human alpha 1 PI. The Spi 4 proteins are ARGserpins, and by alignment the Spi 2 proteins are ILEserpins, the first so far described. The P1 residue for the Spi 3 proteins was unable to be determined. The limited peptide and immunopeptide mapping revealed that proteins from all four loci were closely related, but within the four there were two pairs (Spi 1 and 2 and Spi 3 and 4) which were more related. All were probably derived from the same gene that gave rise to human alpha 1 PI.
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PMID:The equine major plasma serpin multigene family: partial characterization including sequence of the reactive-site regions. 177 2

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, is unusual in its ability to inhibit chymotrypsin, trypsin, and elastase. To address the structural basis of its broad specificity, the gene for ecotin has been cloned and its sequence determined. A promoter of the 17-base pair spacing class was identified, and the probable transcriptional start site lies 18 base pairs upstream from a ribosome binding locus. The gene is followed by a series of conserved repetitive extragenic palindromic sequences. Ecotin has a signal peptide of 20 amino acids which confirms its periplasmic localization. Sequence analyses by Edman degradation and mass spectrometry confirmed 71% of the deduced protein sequence of calculated monomeric molecular mass 16,096 Da. Comparisons of the primary structure for the 142-amino acid protein with the major classes of serine protease inhibitors suggest that ecotin is a novel inhibitor. The reactive site of ecotin was determined to be Met84 for its complexes with chymotrypsin, trypsin, and elastase. The scissile Met84-Met85 bond lies within a disulfide-bonded protein segment similar to other classes of inhibitors.
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PMID:The sequence and reactive site of ecotin. A general inhibitor of pancreatic serine proteases from Escherichia coli. 200 6

The serine protease inhibitor chymotrypsin inhibitor 2 (CI2 or BSPI2) has been expressed in Escherichia coli with the pINIIIompA3 expression vector to produce 20-40 mg/L of culture. Recombinant CI2 purified from this system has been characterized and found to be identical with CI2 from barley. Slow-binding kinetics were observed for the interaction between CI2 and subtilisin BPN', with Ki = 2.9 x 10(-12) M. Analysis of slow-binding data indicates that binding of the inhibitor follows the simplest model of E + I = EI with no kinetically detectable intermediate steps or proteolytic cleavage of the reactive site bond in CI2 (Met-59-Glu-60). This, in agreement with crystallographic data, indicates that the enzyme-inhibitor adduct is the Michaelis complex, which is not chemically processed by the enzyme. Three mutant CI2 molecules with new P1 residues have also been examined with a range of serine proteases, including a mutant subtilisin. In agreement with earlier studies, we find the P1 amino acid an important determinant of specificity. CI2 Met----Lys-59 was found to be a temporary inhibitor of subtilisin BPN' but an effective inhibitor of subtilisin Carlsberg and subtilisin BPN'(Glu----Ser-156). The structural reasons for this are discussed in relation to mechanisms of inhibition of serine proteases.
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PMID:Recombinant chymotrypsin inhibitor 2: expression, kinetic analysis of inhibition with alpha-chymotrypsin and wild-type and mutant subtilisin BPN', and protein engineering to investigate inhibitory specificity and mechanism. 220 9

A low molecular weight serine protease inhibitor (TAP) was purified from extracts of the soft tick, Ornithodoros moubata. The peptide is a slow, tight-binding inhibitor, specific for factor Xa (Ki = 0.588 +/- 0.054 nM). The inhibitor also acts as an anticoagulant in several human plasma clotting assays in vitro. Its amino acid sequence (60 residues) has limited homology to the Kunitz-type inhibitors. However, unlike other inhibitors of this class, TAP inhibits only factor Xa. It had no effect at a 300-fold molar excess on factor VIIa, kallikrein, trypsin, chymotrypsin, thrombin, urokinase, plasmin, tissue plasminogen activator, elastase, or Staphylococcus aureus V8 protease. TAP's specificity and size suggest that it may have therapeutic value as an anticoagulant.
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PMID:Tick anticoagulant peptide (TAP) is a novel inhibitor of blood coagulation factor Xa. 233 10

In the current study, the ability of four protease inhibitors to suppress radiation-induced transformation of C3H/10T1/2 cells was investigated. The inhibitors tested included: (i) aprotinin (a serine protease inhibitor), (ii) N-acetyl-L-tyrosine ethyl ester (a chymotrypsin substrate and competitive inhibitor of protein degradation), (iii) carboxypeptidase inhibitor (a metallo-exopeptidase inhibitor) and (iv) Inhibitor II (a chymotrypsin/trypsin inhibitor). While none of the inhibitors were toxic to the cells at the concentrations employed, only carboxypeptidase inhibitor and inhibitor II are internalized radiation-induced transformation in a statistically significant manner. Utilizing fluorescent labeled inhibitors, we found that carboxypeptidase inhibitors and Inhibitor II are internalized by the cells. Fluorescent-labeled inhibitor could be observed in the cells within 15 min of incubation and is present in distinct intercellular vacuoles within 1 h. These results indicate that carboxypeptidase inhibitor and Inhibitor II are internalized by C3H/10T1/2 cells and thus would be able to inhibit intracellular proteases (or other enzymes) involved in the conversion of a cell to the malignant state.
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PMID:Inhibition of radiation-induced transformation of C3H/10T1/2 cells by carboxypeptidase inhibitor 1 and inhibitor II from potatoes. 246 57

Probable participation of sperm protease in the acrosome reaction was investigated using several inhibitors and substrates. Among those examined, L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) and chymostatin, chymotrypsin inhibitors, p-nitrophenyl-p'-guanidinobenzoate (NPGB), a serine protease inhibitor, and N-benzoyl-L-tyrosine ethyl ester (BTEE), a chymotrypsin substrate, inhibited the egg jelly-induced acrosome reaction of Strongylocentrotus intermedius. TPCK and BTEE, however, did not inhibit the reaction caused by ionophores, A23187, or nigericin. To know the mechanism of inhibition by chymotrypsin inhibitors and substrates of the egg jelly-induced acrosome reaction, intracellular Ca2+ concentration [( Ca2+]i) and pH (pHi) were measured with fura-2 and 2',7'-bis (carboxy-ethyl)carboxyfluorescein (BCECF), respectively. Egg jelly caused increase of [Ca2+]i, which was depressed by BTEE. Egg jelly also caused a transient rise of pHi, which was not depressed by BTEE. In the presence of verapamil, the acrosome reaction by egg jelly was significantly inhibited concomitant with depressed increase of [Ca2+]i. The rise of pHi was not depressed by verapamil. Thus, modes of action of BTEE and of verapamil are similar to each other. Bringing these findings together, the authors present a view that a chymotrypsin-like protease of sea urchin sperm activates verapamil-sensitive Ca2+ channels, which take part in the acrosome reaction.
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PMID:Activation of Ca2+ channels during the acrosome reaction of sea urchin sperm is inhibited by inhibitors of chymotrypsin-like proteases. 255 Mar 37

Granules that are potently cytolytic in vitro can be obtained from cytotoxic lymphocytes that kill virally infected cells and tumor cells. These granules contain pore-forming proteins and several serine proteases. Here we indicate that at least two different proteases participate in the lysis mediated by granule proteins from RNK-16 rat leukemia cells. We report twelve different mechanism-based or "suicide" isocoumarin serine protease inhibitors which have different 3- and 7-substituents that confer selectivity and reactivity towards either the chymotrypsin- ("chymase") or trypsin-like ("tryptase") protease activities of RNK-16 cells. Second order inhibition rates of inactivation (kobsd/[I]) for the RNK-16 granule proteases ranged between 164 and 22,640 M-1s-1. These new, specific and highly reactive isocoumarin serine protease inhibitors also abrogated the cytolysis mediated by lymphocytes granule proteins. The eight inhibitors with large hydrophobic or basic substituents that conferred chymase or tryptase specificities were more effective at inactivating lytic function than the four elastase-directed inhibitors with smaller substituents. All twelve new isocoumarin inhibitors blocked cytolysis at lower concentrations than 3,4-dichloroisocoumarin, a potent general mechanism-based serine protease inhibitor that also blocks RNK-16 granule protease activities and lysis.
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PMID:Selective isocoumarin serine protease inhibitors block RNK-16 lymphocyte granule-mediated cytolysis. 281 73


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