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)

Integrins are membrane receptors which mediate cell-cell or cell-matrix adhesion. Integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) acts as a fibrinogen receptor of platelets and mediates platelet aggregation. Platelet activation is required for alpha IIb beta 3 to shift from noncompetent to competent for binding soluble fibrinogen. The steps involved in this transition are poorly understood. We have studied a variant of Glanzmann thrombasthenia, a congenital bleeding disorder characterized by absence of platelet aggregation and fibrinogen binding. The patient's platelets did not bind fibrinogen after platelet activation by ADP or thrombin, though his platelets contained alpha IIb beta 3. However, isolated alpha IIb beta 3 was able to bind to an Arg-Gly-Asp-Ser affinity column, and binding of soluble fibrinogen to the patient's platelets could be triggered by modulators of alpha IIb beta 3 conformation such as the Arg-Gly-Asp-Ser peptide and alpha-chymotrypsin. These data suggested that a functional Arg-Gly-Asp binding site was present within alpha IIb beta 3 and that the patient's defect was not secondary to a blockade of alpha IIb beta 3 in a noncompetent conformational state. This was evocative of a defect in the coupling between platelet activation and alpha IIb beta 3 up-regulation. We therefore sequenced the cytoplasmic domain of beta 3, following polymerase chain reaction (PCR) on platelet RNA, and found a T-->C mutation at nucleotide 2259, corresponding to a Ser-752-->Pro substitution. This mutation is likely to be responsible for the uncoupling of alpha IIb beta 3 from cellular activation because (i) it is not a polymorphism, (ii) it is the only mutation in the entire alpha IIb beta 3 sequence, and (iii) genetic analysis of the family showed that absence of the Pro-752 beta 3 allele was associated with the normal phenotype. Our data thus identify the C-terminal portion of the cytoplasmic domain of beta 3 as an intrinsic element in the coupling between alpha IIb beta 3 and platelet activation.
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PMID:Ser-752-->Pro mutation in the cytoplasmic domain of integrin beta 3 subunit and defective activation of platelet integrin alpha IIb beta 3 (glycoprotein IIb-IIIa) in a variant of Glanzmann thrombasthenia. 143 6

To gain further insight into the mechanism responsible for rendering fibrinogen bound to stimulated platelets irreversible to dissociation by EDTA or excess unlabeled fibrinogen, the present study compared the reversibility of platelet interactions with fibrinogen and its plasmic degradation product, fragment D1. Like fibrinogen binding, the binding of fragment D1 became progressively less sensitive to dissociation by EDTA, PGE1, or excess unlabeled fibrinogen. Thus in the presence of EDTA, 70 +/- 19% and 55 +/- 24% (mean +/- S.D., n = 9) of bound fragment D1 failed to dissociate from platelets 60 min after stimulation with 0.15 U/ml thrombin or the combination of 5 microM ADP and 5 microM epinephrine, respectively, compared to 75 +/- 8% and 52 +/- 17% of platelet-bound, intact fibrinogen. In contrast, platelet stimulation with chymotrypsin or Zn+2 failed to support the development of irreversible fragment D1 or fibrinogen binding. Only 8 +/- 6% and 9 +/- 3% of bound fragment D1 remained associated with chymotrypsin- or Zn+2-treated platelets, respectively, compared to 7 +/- 11% and 15 +/- 6% (mean +/- S.D., n = 3) of platelet-associated fibrinogen. These observations suggest that irreversible fragment D1 and fibrinogen binding to platelets occurs by a similar mechanism that requires neither fibrinogen alpha chain 95-97 or 572-574 RGD sequences nor multivalent ligand-receptor interactions.
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PMID:Reversibility of fibrinogen fragment D1 binding to human platelets: comparison with native fibrinogen. 147 Oct 72

Two trypsin inhibitors (TI-1, TI-2) were isolated from guinea pig plasma and purified to homogeneity. In amino-acid composition as well as molecular masses, TI-1 (Mr 58,000) and TI-2 (Mr 57,000) are similar to each other and to human and mouse alpha 1-proteinase inhibitors, and mouse con-trapsin. The two inhibitors form equimolar complexes with proteinases. The effectiveness of the inhibitors was characterized by association rate constants under second-order rate conditions. The inhibitory action of TI-1 was rapid for bovine trypsin, porcine pancreatic elastase and guinea pig plasma kallikrein, but slow for bovine thrombin and guinea pig plasmin and not detectable for bovine chymotrypsin and porcine pancreatic kallikrein. The inhibitory action of TI-2 was rapid for trypsin and chymotrypsin, but slow for guinea pig plasma kallikrein and not detectable for other proteinases. These results show that TI-1 and TI-2 are physicochemically similar but functionally distinct from each other and from human alpha 1-proteinase inhibitor that inhibits trypsin, chymotrypsin and elastase.
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PMID:Guinea pig plasma trypsin inhibitors. Purification and characterization of two functionally distinct proteinase inhibitors. 153 92

Progressive decreases in platelet-bound fibrinogen accessibility to antibody and enzymes were recently reported to occur after adenosine diphosphate (ADP)-induced fibrinogen binding. Because previous studies also indicated that platelets that are activated but not aggregated by ADP in the presence of fibrinogen lose their ability to aggregate in a time-dependent manner despite negligible changes in fibrinogen binding, the present study examined the relationship between platelet aggregation and accessibility of platelet-bound fibrinogen to specific polyclonal antibody F(ab')2 fragments over a 60-minute time course. Although 125I-fibrinogen binding remained virtually unchanged, comparison of antifibrinogen antibody F(ab')2 binding and platelet aggregation 5 minutes and 60 minutes after platelet stimulation with ADP or thrombin showed decreases in F(ab')2 binding of 62% +/- 13% and 73% +/- 7% (mean +/- SD, n = 5), respectively, and decreases of 65% +/- 16% and 60% +/- 10% in platelet aggregation. In contrast, platelets stimulated with A23187 or chymotrypsin retained 87% +/- 16% and 76% +/- 12% of their ability to aggregate over the same time course, and lost only 39% +/- 14% and 36% +/- 12% of their ability to bind antifibrinogen antibody F(ab')2 fragments, respectively. Pretreatment of ADP-stimulated platelets with chymotrypsin largely prevented the progressive loss of platelet aggregability and the accompanying decreased recognition of bound fibrinogen by antifibrinogen F(ab')2 fragments. Preincubation of platelets with cytochalasin D (30 micrograms/mL) also inhibited the decrease in platelet aggregation after exposure of ADP-treated platelets to fibrinogen over a 60-minute time course. This was accompanied by only a 25% +/- 18% decrease in antifibrinogen antibody F(ab')2 binding. Present data support the hypothesis that qualitative changes in platelet-bound fibrinogen correlate with loss of the ability of platelets to aggregate, and implicate both the platelet cytoskeleton and chymotrypsin-sensitive surface membrane structures in modulating qualitative changes in bound fibrinogen on the platelet surface.
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PMID:Recognition of platelet-associated fibrinogen by polyclonal antibodies: correlation with platelet aggregation. 156 29

The folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topography of the membrane-bound ADP/ATP carrier assessed by enzymatic proteolysis. 156 52

The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (approximately 4.9 x 10(3) M-1.min-1) and reagent 2 (approximately 1.0 x 10(5) M-1.min-1) respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatible with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase.
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PMID:The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases. 157 91

Sodium orthovanadate (vanadate) prolonged the clotting time of normal human plasma in a dose-dependent manner. The prolongation of clotting time by vanadate linearly decreased with an increase in the concentration of amiloride. Vanadate also was completely additive to prolongation by heparin. When factor Xa or thrombin was incubated with vanadate, the amidolytic activity of each decreased in a dose-dependent manner with vanadate. Amiloride protected the decrease of amidolytic activity of both factor Xa and thrombin by vanadate. The amidolytic activity of trypsin also was inhibited by vanadate, but that of alpha-chymotrypsin was not inhibited, suggesting that vanadate preferentially inhibits the amidolytic activity of trypsin and trypsin-like enzymes. These results show that vanadate prolongs the clotting time of plasma through mechanisms involving in part the inhibition of the activity of both factor Xa and thrombin.
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PMID:Anticoagulant action of vanadate. 157 69

The stability of platelet aggregates is influenced by the extent of the release of granule contents; if release is extensive and aggregation is prolonged, deaggregation is difficult to achieve. The relative importance of the contributions of released substances to aggregate stability are not known, although stable thrombin-induced aggregates form in platelet-rich plasma from patients with barely detectable plasma or platelet fibrinogen, and ADP stabilizes thrombin-induced aggregates of platelets from patients with delta storage pool deficiency which otherwise deaggregate more readily than normal platelets. We degranulated platelets with thrombin (0.9 U/ml caused greater than 90% loss of delta and alpha granule contents) and recovered them as individual platelets in fresh medium. The degranulated platelets were reaggregated by thrombin (2 U/ml). To prevent continuing effects of thrombin, FPRCH2Cl was added when thrombin-induced aggregation of thrombin-degranulated platelets reached its maximum. EDTA (5 mM) or EGTA (5 mM) added at maximum aggregation did not deaggregate these platelets, indicating that the stability of these aggregates does not depend on Ca2+ in the medium. Whereas with control platelets a combination of PGE1 (10 microM) and chymotrypsin (10 U/ml) was required for deaggregation, with thrombin-degranulated platelets either PGE1 or chymotrypsin alone caused extensive deaggregation. The rate and extent of deaggregation of thrombin-degranulated platelets by a combination of PGE1 and chymotrypsin was greater than with control platelets.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Lack of stability of aggregates after thrombin-induced reaggregation of thrombin-degranulated platelets. 163 94

We discovered an enzyme in human platelets that deamidates substance P and other tachykinins. Because an amidated carboxyl terminus is important for biological activity, we purified and characterized this deamidase. The enzyme, released from human platelets by thrombin, was purified to homogeneity by ammonium sulfate precipitation, followed by chromatography on an octyl-Sepharose column and chromatofocusing on PBE 94. The purified enzyme exhibits esterase, peptidase, and deamidase activities. The peptidase activity (with furylacryloyl-Phe-Phe) is optimal at pH 5.0 while the esterase (benzoyl-tyrosine ethyl ester) and deamidase (D-Ala2-Leu5-enkephalinamide) activities are optimal at pH 7.0. With biologically important peptides, the enzyme acts both as a deamidase (substance P, neurokinin A, and eledoisin) and a carboxy-peptidase (with bradykinin, angiotensin I, substance P-free acid, oxytocin-free acid) at neutrality, although the carboxypeptidase action is faster at pH 5.5. Enkephalins, released upon deamidation of enkephalinamides, were not cleaved. Gly9-NH2 of oxytocin was released without deamidation. Peptides with a penultimate Arg residue were not hydrolyzed. Some properties of the deamidase are similar to those reported for cathepsin A. The deamidase is inhibited by diisopropylfluorophosphate, inhibitors of chymotrypsin-type enzymes, and mercury compounds while other inhibitors of catheptic enzymes, trypsin-like enzymes, and metalloproteases were ineffective. In gel filtration, the native enzyme has an Mr = 94,000 while in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr = 52,000 indicating it exists as a dimer. After reduction, deamidase dissociates into two chains of Mr = 33,000 and 21,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]diisopropylfluorophosphate labeled the active site serine in the Mr = 33,000 chain. The first 25 amino acids of both chains were sequenced. They are identical with the sequences of the two chains of lysosomal "protective protein" which, in turn, has sequence similarity to the KEX1 gene product and carboxypeptidase Y of yeast. This protective protein complexes with beta-galactosidase and neuraminidase in lysosomes and is vitally important in maintaining their activity and stability. A defect in this protein is the cause of galactosialidosis, a severe genetic disorder. The ability of physiological stimuli (e.g. thrombin or collagen) to release the deamidase from platelets indicates that it may also be involved in the local metabolism of bioactive peptides.
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PMID:A peptidase in human platelets that deamidates tachykinins. Probable identity with the lysosomal "protective protein". 169 76

A plasma kallikrein inhibitor in guinea pig plasma (KIP) was purified to homogeneity. KIP is a single chain protein and the apparent molecular weight is estimated to be 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In amino acid composition, KIP is similar to human and mouse alpha 1-proteinase inhibitors and mouse contrapsin. KIP forms an equimolar complex with plasma kallikrein in a dose- and time-dependent fashion. The association rate constants for the inhibition of guinea pig plasma kallikrein by KIP, alpha 2-macroglobulin, C1-inactivator and antithrombin III were 2.5 +/- 0.3.10(4), 2.4 +/- 0.4.10(4), 6.6 +/- 0.5.10(4) and 9.1 +/- 0.6.10(2), respectively. Comparison of the association rate constants and the normal plasma concentrations of the four inhibitors demonstrates that KIP is ten-times as effective as alpha 2-MG and other two inhibitors are marginally effective in the inhibition of kallikrein. KIP inhibits trypsin and elastase rapidly, and thrombin and plasmin slowly, but is inactive for chymotrypsin and gland kallikrein. These results suggest that KIP is the major kallikrein inhibitor in guinea pig plasma and the proteinase inhibitory spectrum is unique to KIP in spite of the molecular similarity to alpha 1-proteinase inhibitor.
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PMID:The major plasma kallikrein inhibitor of guinea pig plasma. 173 48


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