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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Routine evaluation of 12 children with Cooley's anemia revealed that each one had a prolonged partial thromboplastin time. However, prothrombin time and thrombin time were within the normal range. Specific assays demonstrated low levels of the four contact factors: factors XI, XII, prekallikrein, and high molecular weight kininogen. Further investigation revealed activity against para-nitroanilide peptide substrates in unactivated plasma from all 12 patients. Following gel filtration on Sephadex G200, the activity emerged in one peak in the void volume, indicating a molecular weight of greater the 500,000. Activity was greatest against H-D-Pro-Phe-Arg-pNA, the substrate for plasma kallikrein, and was inhibited by diisopropyl fluorophosphate and trasolyl. It was unaffected by hirudin, soy bean trypsin inhibitor, and lima bean trypsin inhibitor. It was destroyed by heating at 56 degrees C. Specific antisera against human prekallikrein and human alpha-macroglobulin did not reduce the activity. It is concluded that a high molecular weight kallikrein-like protease, is present in the plasma of these patients. It is postulated that it is released into the circulation from tissue as a result of damage due to iron overload. It is further postulated that this protease brings about in vivo activation of the contrast factors, resulting in a fall in their circulating levels.
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PMID:Demonstration of kallikrein-like protease activity in nonactivated plasma of patients with Cooley's anemia. 633 56

A new type of fibrinogenase was isolated from the venom of the western diamondback rattlesnake (Crotalus atrox). Unlike thrombin, the newly isolated fibrinogenase did not cause formation of a fibrin clot. Various properties of the fibrinogenase we isolated were compared with crotalase isolated from the venom of C. adamanteus. It was found that fibrinogenase has considerable similarity to crotalase isolated by Markland and Damus in 1971. Crotalase is a thrombin-like enzyme and produces a fibrin clot from fibrinogen. The A alpha chain of fibrinogen was first split and the B beta chain was cleaved later. The fact that no fibrin clot forms indicates that the cleavage sites in A alpha and B beta chains of fibrinogen must be different from thrombin sites. The fibrinogenase also released bradykinin by interacting with plasma proteins. It hydrolyzed TAME (p-toluenesulfonyl-L-arginine methyl ester), BAEE (N-benzoyl-L-arginine ethyl ester). TLME (N-tosyllysine methyl ester) but not BAA (N-benzoylarginine amide), TAA (N-tosylarginineamide) or ATEE (N-acetyltyrosine ethyl ester). The enzyme is an acidic protein with pI of 4.6 and a mol. wt of 31,000. It consists of 272 total amino acid residues, 21% of which are acidic amino acids. Fibrinogenase is a specific form of protease. A newly liberated amino group after hydrolysis of dimethyl-casein can be detected by the reagent trinitrobenzenesulfonic acid (TNBS). Fibrinogenase differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzyme's action.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation and characterization of fibrinogenase from western diamondback rattlesnake venom and its comparison to the thrombin-like enzyme, crotalase. 636 74

This study was conducted to determine whether a kinin-generating proteinase (kininogenase) previously described in the porcine anterior pituitary exists in a latent form. Porcine anterior pituitaries were homogenized in 0.25 M sucrose (pH 7.5) and sequentially centrifuged at 1000 X g for 5 min, 1500 X g for 20 min, 10 000 X g for 20 min, and 105 000 X g for 60 min. The various fractions were assayed for their ability to generate kinins from kininogen and cleave H-D-Pro-Phe-Arg-p-nitroanilide (S-2302) before or after various activation procedures. Untreated pituitary fractions had a small amount of proteolytic activity. However, large increases in kininogenase and S-2302 hydrolytic activity were observed in the 105 000 X g pellet after dialysis, or incubation with trypsin. Repeated freezing and thawing, detergents, phospholipase A2, melittin, plasmin, thrombin, urokinase and Factor Xa failed to activate kininogenase activity in the 105 000 X g pellet. However, plasmin produced massive increases in S-2302 hydrolytic activity. The kininogenase and S-2302 hydrolytic activity was sensitive to inhibition by soybean trypsin inhibitor and aprotinin, and had a broad pH optimum between 7 and 9. The data indicate that the porcine anterior pituitary kininogenase largely exists in a latent form. Also, the porcine anterior pituitary appears to contain an additional latent proteinase which can hydrolyze S-2302.
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PMID:Activation of a latent kinin-generating proteinase in the porcine anterior pituitary. 638 52

Loss of Factor VIII procoagulant activity (VIII:C) following blood collection is a major problem in providing sufficient amounts for therapeutic use and biochemical analyses. We have examined the effects of inhibition of plasma proteases and maintenance of physiological calcium ion on plasma VIII:C stability. The addition of protease inhibitors such as benzamidine, phenylmethylsulfonyl fluoride (PMSF), aprotinin, or soybean trypsin inhibitor (SBTI) to CPD plasma provided no significant protection against decay of VIII:C activity. Neither the rate of decay in the first 24 hours nor the final VIII:C activity observed after storage for 48-72 hours were significantly altered. On the other hand, addition of DFP or heparin to CPD plasma resulted in a marked improvement in VIII:C stability over 24 hours. This demonstrated that these two inhibitors are effective in preventing VIII:C degradation during storage. In addition to protease inhibition, the importance of maintaining physiological calcium ion was demonstrated by 100% stabilization of VIII:C in heparin plasma. Plasma obtained from CPD plus heparin blood could also be stabilized provided free calcium ion levels were restored to physiological concentrations. The inactivation of VIII:C in CPD plus heparin plasma was completely reversible up to 4 hours after collection. Studies on the recovery of activity after recalcification of CPD plus heparin plasma provided kinetic data which support a renaturation process of VIII:C rather than one due to enzymatic activation. The use of a thrombin-specific chromogenic substrate revealed that after recalcification and during the recovery of VIII:C activity, there was no significant thrombin activity. Although the data suggest that proteolytic degradation plays some part in VIII:C decay, only the maintenance of physiologic calcium ion levels under cover of an effective non-chelating anticoagulant and protease inhibitor allows preservation of VIII:C activity.
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PMID:Stability of VIII:C in plasma: the dependence on protease activity and calcium. 640 38

Antihemophilic factor concentrates were surveyed for amidolytic activity on the chromogenic substrates S2238, S2302, S2222, and S2251, which are sensitive to thrombin, kallikrein, factor Xa, and plasmin, respectively. For antihemophilic factor concentrates from two manufacturers, the rates of amidolysis of S2238 and S2302 were approximately an order of magnitude greater than the rates of amidolysis of S2222 and S2251. The S2238 and S2302 activities were characterized by quantitating their interactions with specific substrates or inhibitors. The Km for amidolysis of S2238 was 558 mumol/L, which is 80 times higher than for thrombin but in close agreement to the reported value for activated protein C. The S2238 activity was not inhibited by the thrombin-specific inhibitor dansylarginine N-(3-ethyl-1,5-pentanediyl)amide, nor by soybean trypsin inhibitor or micromolar concentrations of antithrombin III in the presence of heparin. The S2238 activity was inhibited by D-Phe-Pro-Arg-CH2Cl, but with an estimated second-order rate constant of 3 X 10(5) mol/L-1 minute-1, approximately 1000 times less than for thrombin. These data are consistent with the identity of the S2238 activity as activated protein C. On the other hand, the S2302 activity in antihemophilic factor concentrates was most likely attributable to kallikrein. This was based on the agreement with authentic kallikrein of the Km for S2302 of 154 mumol/L as well as by the rapid inactivation by nanomolar concentrations of the kallikrein-specific inhibitor D-Phe-Phe-Arg-CH2Cl. However, the relative resistance of the S2302 activity to inhibition by soybean trypsin inhibitor or antithrombin III and the partial inhibition by aprotinin suggested that a large proportion of the kallikrein was bound to alpha 2-macroglobulin. This was confirmed by immunoprecipitation using specific anti alpha 2-macroglobulin IgG. The potential for proteolysis of factor VIII:von Willebrand protein during its purification from antihemophilic factor concentrates was demonstrated, and the proteolyzed factor VIII coagulant species was characterized. High-pressure gel permeation chromatography of purified factor VIII:von Willebrand protein at high ionic strength resulted in two sharp peaks of factor VIII procoagulant activity. The earlier eluting peak corresponded with the void volume, and the later peak eluted with an apparent molecular weight of 53,000 daltons. Immediately after separation, the 53,000-dalton factor VIII coagulant had at least a 100-fold higher specific activity than the factor VIII coagulant present in the void volume. However, the 53,000-dalton factor VIII coagulant was labile, with a half-life of 80 minutes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of proteases in AHF concentrates: effect on factor VIII:von Willebrand protein as assessed by high-pressure gel permeation chromatography. 643 16

The purpose of this study was to determine the distribution and characteristics of kinin-generating proteases (kininogenases) in rat pituitary tissue. Male rat pituitaries were dissected into their various lobes, sonicated in saline containing 12 mM deoxycholic acid, and assayed for protease activity at pH 8.5 using kininogen and chromogenic peptide substrates; kinin generation was measured by RIA. The rat pituitary was found to contain kininogenase activity highly concentrated in the pars intermedia; this activity was strongly inhibited by aprotinin and was resistant to soybean trypsin inhibitor. Antiserum against rat urinary kallikrein also inhibited the intermediate pituitary kininogenase. The kininogenase product was identified as bradykinin by reversed-phase high performance liquid chromatography. Homogenization of neurointermediate pituitaries in 0.25 M sucrose buffer (pH 7.5) followed by differential centrifugation (1,000 X g, 5 min; 10,000 X g, 20 min; 105,000 X g, 70 min) demonstrated that most of the kininogenase activity was in the 10,000 X g pellet. Kinin generation by neurointermediate pituitary extracts had a pH optimum of 8.0, and such extracts also hydrolyzed chromogenic peptide substrates for kallikreins. In addition, neurointermediate pituitary extracts were found to contain a distinct protease which hydrolyzed a chromogenic peptide substrate for thrombin. The intermediate pituitary kininogenase resembles glandular kallikrein and possibly may participate in prohormone processing.
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PMID:A kininogenase resembling glandular kallikrein in the rat pituitary pars intermedia. 655 May 32

The kininogenase activity of alpha- and beta/gamma-forms of bovine thrombin with respect to the high molecular weight (HMW) and low molecular weight (LMW) human kininogens was studied. It was shown that both forms of the enzyme split of bradykinin from these kininogens. The kininogenase activity of alpha-thrombin is completely blocked by the highly specific thrombin inhibitor Nalpha-dansyl-L-arginine-p-ethylpiperidineamide, but not by the soya bean trypsin inhibitor. The alpha- and beta/gamma-forms of thrombin hydrolyze HMW (Km(app) = 4.5 and 3.3 microM, respectively) and LMW (Km(app) = 10.1 and 4.7 microM, respectively). The specific constants (kcat/Km(app) ) for thrombin with respect to the substrates differ about 7-fold, predominantly due to the high catalytic rates of HMW as compared to LMW; the kcat values are 0.18 and 0.06 min-1, respectively. alpha-Thrombin upon a long-term (over 1 hour) exposure to HMW, besides bradykinin, splits off the product inhibiting the kininogenase activity of thrombin. No differences in the specificity of the beta/gamma-form of thrombin with resect to HMW and LMW were detected.
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PMID:[Kininogenase activity of alpha and beta/gamma forms of bovine thrombin]. 656 67

Activation of bovine plasma prekallikrein was investigated with several proteinases. Highly purified bovine plasma prekallikrein was rapidly activated to kallikrein [EC 3.4.21.8] by bovine activated Hageman factor, trypsin [EC 3.4.21.4] and Pronase P (proteinases from Streptomyces griseus) and more gradually by papain [EC 3.4.22.2] and ficin [EC 3.4.22.3]. Activation of prekallikrein was also observed with bovine plasmin [EC 3.4.21.7], but not with bovine clotting factors Xa (Stuart factor) [EC 3.4.21.6] and IXa (Christmas factor) or thrombin [EC 3.4.21.5]. Urokinase [EC 3.4.99.26], Reptilase, collagenase [EC 3.4.24.3], elastase [EC 3.4.21.11], alpha-chymotrypsin [EC 3.4.21.1], Nagarse [EC 3.4.21.14], and stem bromelain [EC 3.4.22 4] did not convert prekallikrein to kallikrein. Plasma kallikrein activated to Hageman factor released kinin rapidly from bovine high molecular weight (HMW) kininogen. However, from bovine low molecular weight (LMW) kininogen, liberation of kinin was extremely slow. The kallikrein activity was inhibited by soybean trypsin inhibitor (SBTI), Trasylol, diisopropylfluorophosphate (DFP), and N-alpha-tosyl-L-lysine chloromethylketone (TLCK), but not by egg-white trypsin inhibitor (EWTI), lima bean trypsin inhibitor (LBTI), heparin or hexadimethrine bromide (Polybrene). The kallikrein formed an enzyme-inhibitor complex with SBTI and Trasylol, but not with LBTI. Prekallikrein did not react with SBTI. Prekallikrein consists of a single polypeptide chain of molecular weight about 90,000, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Activation of prekallikrein by Hageman factor was found to involve cleavage of the single peptide bond on the disulfide-bridged polypeptide chain, and no change of molecular weight was observed during the activation. The peptide bond cleaved in prekallikrein by the activation was an Arg-X peptide bond on a disulfide-bridged polypeptide chain.
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PMID:Studies on prekallikrein of bovine plasma. II. Activation of prekallikrein with proteinases and properties of kallikrein activated by bovine Hageman factor. 676 24

Limulus amebocyte lysate was fractionated by heparin-Sepharose chromatography into four components (fractions A, B, C and D). Major coagulation factors, i.e., proclotting enzyme, coagulogen, and proclotting enzyme activating factor precursor (proactivator) in the lysate were eluted, respectively, in fraction A, fraction B and fraction C. Clotting enzyme activity was detected only following recombination of fraction A and fraction C in the presence of endotoxin. The conversion of proactivator to its active form (activator) was an endotoxin-dependent reaction and was inhibited by polymyxin B. Either proactivator is an endotoxin-sensitive factor or another endotoxin-sensitive factor, which activates proactivator, is present in fraction C. Optimal pH for proclotting enzyme activation by activator was broad and ranged from pH 6.0 to 8.0, while that for the endotoxin-mediated activation of proactivator was pH 7.0. No initial latent period was observed during activation of the proactivator or proenzyme. The activator was inhibited by benzamidine, leupeptin, soybean trypsin inhibitor and diisopropyl fluorophosphate, suggesting that the activator is a trypsin-type serine protease. Trypsin, but not thrombin, urokinase, plasmin, papain or alpha-chymotrypsin activated the proclotting enzyme. Therefore, limited proteolysis, i.e., of an arginyl- or lysyl-X bond(s), of the proenzyme molecule is probably involved in its activation.
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PMID:Fractionation of Limulus amebocyte lysate. Characterization of activation of the proclotting enzyme by an endotoxin-mediated activator. 713 84

The molecular size and immunochemical properties of the unfractionated factor V present in plasma collected by venipuncture into a broad-spectrum anticoagulant and platelet-inhibited mixture were compared with those reported for the isolated, single-chain factor V molecular of 330 000 daltons. The anticoagulant--plasma mixture included 0.28% trisodium citrate, 2 mM benzamidine hydrochloride, 0.02% soybean trypsin inhibitor, 2.0 mM diisopropyl phosphorofluoridate, 10 microM dansylarginine N-(3-ethyl-1,5-pentanediyl)amide, and 5 microM prostaglandin E1. The Stokes radius of unfractionated factor V present in highly inhibited plasma (93 A) is virtually identical with the Stokes radius predicted from the hydrodynamic data for the highly asymmetric, single-chain factor V molecule (91 A). With an expression which relates the Stokes radius and the sedimentation coefficient to the molecular weight of hydrodynamic units, the molecular weight obtained for factor V, using gel filtration data, is 336 000, in good agreement with the molecular weight determined from the sedimentation equilibrium, 330 000. In contrast, the Stokes radius for the factor Va present in serum is significantly smaller (50.5 A) and equivalent to the Stokes radius obtained upon activation of isolated factor V with thrombin. Immunochemical comparisons of the factor V present in the inhibited plasma and isolated factor V were conducted by using burro antibovine factor V antibody and the technique of immunoelectrophoresis. The factor V antigen present in both sources is immunochemically identical, as is the electrophoretic mobility of both factor V preparations. These data serve to justify the conclusion that the factor V isolated as a single-chain 330 000-dalton molecule corresponds to the factor V circulating in plasma.
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PMID:Molecular weight of undegraded plasma factor V. 747 Apr 75


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