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: UNIPROT:P00750 (PLA)
16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The presence of a peripheral zone of (presumed intracellular) plasminogen activator in the normal rabbit cornea has suggested that activator, once released, might regulate the permeability of limbal vessels and angiogenesis, by plasmin-dependent pathways. Plasminogen activator (urokinase [UK]) in rabbit serum albumin (RSA) was injected once (20 microliter, 3.7 CTA U) into the corneal stroma, 2 mm from the limbus. Sprouts arose from the engorged circumlimbal vessels (16 of 20 corneas) beginning on the third day and grew into the cornea over the next several days. Histologically, PMNs were observed in association with growing vessels. Contralateral corneas injected with UK (in RSA) previously inactivated by 99.7% with the specific active site inhibitor, Phe-Ala-Arg-chloromethyl ketone showed minimal vessel engorgement or stromal edema and no vascularization (0 to 20 corneas). Injuries to the so-called (plasminogen activator-containing)"critical zone" of the cornea which elicit neovascularization possibly do so by causing extracellular release of endogenous plasminogen activator. Thus, in addition to initiating the destructive events of ulceration, activator might initiate increases in vessel permeability and also neovascularization, which would result in the eventual arrest of ulceration.
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PMID:Plasminogen activator (urokinase) causes vascularization of the cornea. 617 46

A chymostatin-sensitive step in the release of plasminogen activator from transformed fibroblasts has been described recently. By using synthetic peptidyl substrates, we have detected and characterized a chymostatin-sensitive peptidase activity in chicken embryo fibroblasts transformed by Rous sarcoma virus. The activity represents a neutral endopeptidase that exhibits phenylalanine specificity and is inhibited by diisopropyl fluorophosphate. A detailed inhibitor profile of the enzyme activity shows that it is distinct from other chymotrypsin-like phenylalanine-preferring peptidases. The endopeptidase activity in transformed fibroblasts is increased over that of parallel cultures of normal fibroblasts. The mechanism of enzyme inhibition by chymostatin is indicated by these studies, and the possible role of the enzyme in modulating plasminogen activator secretion is discussed.
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PMID:Detection and partial characterization of a chymostatin-sensitive endopeptidase in transformed fibroblasts. 630 Aug 32

The purification and properties of an estradiol-sensitive hydrolytic activity from mouse uterus which fits several criteria for being an induced protein are described. The activity in the uteri of immature animals can be stimulated 2--4-fold by estradiol to that approaching the adult level. Stimulation is blocked by puromycin. The enzyme which we have designated hydrolase II, was purified approx. 400-fold to apparent homogeneity by chromatography on Affigel Blue, DEAE-cellulose and octyl-Sepharose. Hydrolase II is a single chain polypeptide with an estimated mol. wt = 65,000 daltons and has an N-terminal serine residue. A variety of N-blocked L-amino acid nitrophenyl esters are cleaved by the enzyme. Km's at pH 7.2 were all approx. 40 microns. Of substrates tested, phenylalanine nitrophenyl ester had the highest Vmax. Cbz-beta-alanine nitrophenyl ester, which is not a normal protease substrate was cleaved with a Km of 145 microM. The enzyme had no detectable activity against peptide nitroanilide substrates for trypsin-, chymotrypsin- or elastase-like enzymes. It is inhibited by ZPCK and DIFP but not by TLCK and Ala-Ala-Pro-Ala chloromethyl ketone, a potent inhibitor of elastase-like enzymes. Mouse plasma protein protease inhibitors were without effect as was SBTI. Our results rule out hydrolase II being a carnosinase, non-serine esterase, plasminogen activator, collagenase or collagenase activator and suggest that it is a chymotrypsin-like protease.
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PMID:Properties of an estrogen-induced hydrolytic enzyme from mouse uterus. 635 Jul 23

Most reports in the literature state that human plasma kallikrein does not destroy the capacity of human high molecular weight kininogen (HMrK) to function as a cofactor in the contact phase activation of factor XII. In the present work preparations of highly purified human plasma kallikrein that showed high plasminogen activator (PGA) activities rapidly reduced the cofactor function of human HMrK. Gel electrophoresis with SDS without reduction showed that all kallikrein preparations tested contained two protein bands, one major band with a Mr of about 83,000, and one weak band with a Mr of 80,000. The main band is probably identical with kallikrein I, which Levison & Tomalin (1982b), using Ac-Pro-Phe-Arg-OMe-HCl as substrate, found to be ten times more active (in terms of kcat/Km) than kallikrein II with Mr 3000 daltons lower. The rate of HMrK destruction in our experiments varied with the kallikrein preparation used, but assays of their hydrolytic activities against benzoyl arginine ethylester (BAEe) or the plasma kallikrein selective tripeptide substrate H-D-Pro-Phe-Arg-pNA (S-2302) did not discriminate between enzyme preparations with different HMrK-destroying capacities. Assay of PGA activities demonstrated a correlation between the level of PGA measured, and the HMrK-destroying capacity.
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PMID:Reduced cofactor function of human high molecular weight kininogen induced by human plasma kallikrein. 643 52

Extrinsic (tissue-type) plasminogen activator antigen in human plasma, as measured by a two-site immunoradiometric assay, is composed of a fibrin-adsorbable and a nonadsorbable fraction. Gel filtration on Ultrogel AcA 44 in 1.6M KSCN of the fibrin-adsorbable fraction showed a peak with Mr congruent to 70,000, which contained plasminogen activator activity and was assumed to represent free extrinsic plasminogen activator. The nonadsorbable fraction showed a broad peak with Mr congruent to 140,000 without plasminogen activator activity. Overnight incubation at 37 degrees C of postexercise plasma revealed a shift of the Mr congruent to 70,000 peak to the Mr congruent to 140,000 position, suggesting that the Mr congruent to 140,000 peak consists of extrinsic plasminogen activator-protease inhibitor complex(es). alpha 2-Antiplasmin is the main inhibitor of extrinsic plasminogen activator in plasma 13 and is therefore most probably at least in part responsible for the generation of the Mr congruent to 140,000 component. A possible involvement of other plasma proteinase inhibitors was explored by incubation of 125I-labeled extrinsic plasminogen activator in alpha 2-antiplasmin-depleted plasma. A complex was formed with a t1/2 of about 1 hr, which was identified by immunoprecipitation as extrinsic plasminogen activator-alpha 1-antitrypsin complex. Additional evidence for the presence of extrinsic plasminogen activator complexes with alpha 2-antiplasmin and alpha 1-antitrypsin in plasma was obtained from two-site immunoradiometric assays, in which solid-phase anti-inhibitor antibody bound the corresponding complex, which was then detected with radiolabeled, affinospecific antibody against extrinsic plasminogen activator. It was concluded that plasma contains both free extrinsic plasminogen activator and plasminogen activator complexes with alpha 2-antiplasmin and alpha 2-antitrypsin. These complexes are also present in plasma collected on the active site inhibitor, D-Phe-Pro-Arg-CH 2Cl, at rest and after exercise and are therefore assumed to circulate in vivo.
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PMID:Complexes between tissue-type plasminogen activator and proteinase inhibitors in human plasma, identified with an immunoradiometric assay. 668 24

A plasminogen activator, previously designated as rat urinary esterase A (Nustad, K., and Pierce, J. V. (1974) Biochemistry 13, 2312-2319), was separated from kallikrein of rat urine and purified to homogeneity. In polyacrylamide slab gel electrophoresis, the purified enzyme showed three closely migrating protein bands which were labeled with [14C]diisopropylphosphorofluoridate and stained on a zymogram using the chromogenic substrate methionine-alpha-naphthyl ester. Two chains, heavy chain(s) (Mr approximately 15,800, 14,200) and light chain(s) (Mr approximately 8,850, 8,550), were separated in SDS-polyacrylamide gel under reducing conditions, while two bands (Mr approximately 24,500 and 23,000) were seen under nonreducing conditions. The active site of the enzyme was associated with the heavy chain. The purified enzyme was stained for carbohydrate by the periodic acid-Schiff reagent. Five bands were distinguished in slab gel electrofocusing with isoelectric points ranging from 5.05 to 5.45. The purified enzyme lysed fibrin clots containing plasminogen but not plasminogen-free fibrin. It hydrolyzed benzyloxylcarbonyl-Gly-Gly-Arg-amino-4-trifluoromethyl coumarin, and a Km of 53 microM and a Vmax of 63 mumol/min/mg of enzyme were obtained at pH 8.0 and 37 degrees C. The enzyme cleaved kininogen substrates to produce kinin which was measured by bioassay or radioimmunoassay. The enzyme was inhibited by soybean or lima bean trypsin inhibitor, aprotinin, alpha 1-antitrypsin, phenylmethanesulfonyl fluoride, D-Phe-Phe-ArgCH2Cl, antipain, leupeptin, benzamidine, and pentamidine. Its pH optimum was 8.5 to 9.0; it was unstable on dilution and on heating. On immunoelectrophoresis, an antiserum to the esterase formed precipitin arcs with rat plasma and this enzyme at identical positions, which in turn were different from those formed with kallikrein. This urinary enzyme belongs to the family of serine proteinases and is immunologically related to urinary kallikrein.
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PMID:Purification and characterization of rat urinary esterase A, a plasminogen activator. 668 2

A fibrinolytic agent purified from the haemolymph, hair secretion and whole body extract of Lonomia achelous (Cramer) cleaves various chromogenic peptide substrates. The best substrate were found to be pyro-Glu-Gly-Arg-pNA (S-2444) followed by D-Pro-Phe-Arg-pNA (S-2302) and Bz-Ile-Glu-(or) Gly-Arg-pNA (S-2222) designed for urokinase, plasma kallikrein and factor Xa, respectively. Using substrate S-2251 we also found a plasminogen activator.
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PMID:Studies of a fibrinolytic enzyme from the larvae of Lonomia achelous (Cramer) using chromogenic peptide substrates. 733 Aug 21

In our continuing effort to design novel thrombin inhibitors, a series of conformationally constrained amino acids (e.g. alpha-alkyl, N-alkyl cyclic, etc.) were utilized in a systematic structure-activity study of the P3, P2, and P1 positions of tripeptide arginal thrombin inhibitors. Early examples of this effort include: D-MePhe-Pro-Arg-H (15), Boc-D-Phg-Pro-Arg-H (18), D-1-Tiq-Pro-Arg-H (23, D-1-Tiq = D-1,2,3,4-tetrahydroisoquinolin-1-ylcarbonyl), and Boc-D-Phe-Pro-Arg-H (25).10a,20 The current work clarifies the contribution of each residue of the tripeptide arginals toward the potent and selective inhibition of thrombin relative to that of t-PA and plasmin. The alpha-methylarginal modification in the P1 residue resulted in analogs 30 (D-MePhe at P3) and 32 (D-1-Tiq at P3) which had lower potency toward thrombin while exhibiting improved selectivity. Analogs modified at the P2 site were found to be very sensitive to the conformational changes induced by variations in side chain ring size with the flexible pipecolinic acid 31 being 2 orders of magnitude less potent at thrombin inhibition than the conformationally constrained azetidine analog 20. Examination of the P3 binding region indicated that alpha-alkylphenylglycine residues resulted in a tendency to exhibit substantial improvements in selectivity over the nonalkylated residues. Combinations of optimal P3 and P2 changes led to compounds TFA-D-Phg(alpha Et)-Azt-Arg-H (16), TFA-D-Phg(alpha Me)-Azt-Arg-H (17), Ac-D-Phg(alpha Me)-Azt-Arg-H (21), TFA-D-Phg(alpha Me)-Pro-Arg-H (27), 30, and 32, which are clearly more selective for thrombin versus plasmin than the nonconformationally constrained compounds.
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PMID:Structure-activity study of tripeptide thrombin inhibitors using alpha-alkyl amino acids and other conformationally constrained amino acid substitutions. 747 71

In a previous study we have shown that monoclonal antibody F1 (MoAb F1), directed against an epitope on the heavy chain of factor XII distinct from the binding site for anionic surfaces, is able to activate factor XII in plasma (Nuijens JH, et al: J Biol Chem 264; 12941, 1989). Here, we studied in detail the mechanism underlying the activation of factor XII by MoAb F1 using purified proteins. Formation of factor XIIa was assessed by measuring its amidolytic activity towards the chromogenic substrate H-D-Pro-Phe-Arg-pNA (S-2302) in the presence of soybean trypsin inhibitor and by assessing cleavage on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Upon incubation with MoAb F1 alone, factor XII was auto-activated in a time-dependent fashion, activation being maximal after 30 hours. Factor XII incubated in the absence of MoAb F1 was hardly activated by kallikrein, whereas in the presence of MoAb F1, but not in that of a control MoAb, the rate of factor XII activation by kallikrein was promoted at least 60-fold. Maximal activation of factor XII with kallikrein in the presence of MoAb F1 was reached within 1 hour. This effect of kallikrein on the cleavage of factor XII bound to MoAb F1 was specific because the fibrinolytic enzymes plasmin, urokinase, and tissue-type plasminogen activator could not substitute for kallikrein. Also, trypsin could easily activate factor XII, but in contrast to kallikrein, this activation was independent of MoAb F1. SDS-PAGE analysis showed that the appearance of amidolytic activity correlated well with cleavage of factor XII. MoAb F1-induced activation of factor XII in this purified system was not dependent on the presence of high-molecular-weight kininogen (HK), in contrast to the activation of the contact system in plasma by MoAb F1. Experiments with deletion mutants revealed that the epitopic region for MoAb F1 on factor XII is located on the kringle domain. Thus, this study shows that binding of ligands to the kringle domain, which does not contribute to the proposed binding site for negatively charged surfaces, may induce activation of factor XII. Therefore, these findings point to the existence of multiple mechanisms of activation of factor XII.
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PMID:Monoclonal antibody F1 binds to the kringle domain of factor XII and induces enhanced susceptibility for cleavage by kallikrein. 749 70

Newly developed synthetic and recombinant thrombin inhibitors possess strong anticoagulant effects. Despite these effects, interactions of these agents with enzymes in the fibrinolytic network result in the modulation of such proteases as t-PA, u-PA and streptokinase. The inhibitory spectrum of several thrombin inhibitors [D-Phe-Pro-Arg-H(GYKI 14166), D-MePhe-Pro-Arg-H(GYKI 14766), Boc-D-Phe-Pro-Arg-H (GYKI 14451), Ac-D-Phe-Pro-boroArg-OH (DuP 714), recombinant hirudin (r-Hir) and unfractionated porcine mucosal heparin complexed with antithrombin III (Heparin/AT-III)] was studied towards various serine proteases such as tissue plasminogen activator (t-PA), plasmin, plasminogen/streptokinase complex, urokinase and kallikrein. Aprotinin was also studied in the same systems as the thrombin inhibitors. All four tripeptide derivatives were found to inhibit t-PA, plasmin and plasminogen/streptokinase complex at micromolar concentrations (IC50: 0.57 mM-3.3 microM). Boc-D-Phe-Pro-Arg-H and Ac-D-Phe-Pro-boroArg-OH also inhibited urokinase, while Ac-D-Phe-Pro-boroArg-OH inhibited kallikrein as well (IC50: 0.15 mM-16 microM). In contrast, r-Hir and Heparin/AT-III did not inhibit any of these enzymes at millimolar concentrations (IC50 > or = 1 mM). Aprotinin inhibited plasmin, plasminogen/streptokinase complex and kallikrein at micromolar concentrations (IC50: 3.1-0.85 microM). In a rabbit thrombolysis model, where pre-formed clots are lysed by streptokinase, simultaneous administration of D-MePhe-Pro-Arg-H or Ac-D-Phe-Pro-boroArg-OH, at concentrations approximately 1 mumol/kg, i.v. resulted in complete inhibition of the fibrinolytic process. Aprotinin at 0.1 mumol/kg, i.v. produced similar inhibition. These results demonstrate that thrombin inhibitors may exert significant antiprotease actions against various fibrinolytic enzymes.
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PMID:Fibrinolytic compromise by simultaneous administration of site-directed inhibitors of thrombin. 804 88


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