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:3.4.21.7 (plasmin)
9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

p-Nitrobenzyl p-toluenesulfonyl-L-arginine is hydrolyzed by thrombin, plasmin, and trypsin to p-nitrobenzyl alcohol and tosyl-L-arginine. The absorption of p-nitrobenzyl alcohol formed is measured at 271 nm (AmM 8.89). With 0.10 mM of the ester in 0.1 M Tris-HCl at pH 8.4 and 30 degrees C, the hydrolysis catalyzed by thrombin, plasmin, and trypsin is linearly proportional to time up to consumption of 60% of the substrate. Km is 14 micron and Vmax is 0.037 mumol/min/NIH unit for bovine thrombin, Km is 78 micron and Vmax is 0.31 mumol/min/CTA unit/ml for human plasmin, and Km is 12 micron and Vmax is 138 mumol/min/mg protein/ml for bovine trypsin. Samples of bovine and human thrombin ranging in specific clotting activity from 59 to 2,133 NIH units/mg protein showed esterase activities ranging from 0.15 to 0.4 mumol p-nitrobenzyl alcohol formed/10 min/NIH unit. Useful ranges for assay of enzymes were (per milliliter): 0.05-0.2 NIH units (thrombin), 0.005-0.02 CTA units (plasmin), and 0.01-0.04 microgram (trypsin).
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PMID:p-Nitrobenzyl p-toluenesulfonyl-L-arginine: a chromogenic substrate for thrombin, plasmin and trypsin. 2 27

p-Nitrobenzyl p-toluenesulfonyl-L-arginine has been synthesized. A number of trypsin-like enzymes can catalyze the hydrolysis of this ester leading to formation of p-nitrobenzyl alcohol. After separation from the ester and p-toluenesulfonylarginine by extraction into chloroform, the p-nitrobenzyl alcohol liberated can be measured spectrophotometrically at 271 nm. Under the conditions of the assay, the hydrolysis of 1 micronmol/ml of the ester is equivalent to an absorbance change of 4.45 cm-1 at 271 nm. With 0.10 mM p-nitrobenzyl p-toluenesulfonyl-L-arginine in 0.1 M Tris-HCl at pH 8.4 and 30 degrees, the enzymatic hydrolysis is linearly proportional to time up to consumption of 60% of the ester. Product formation is proportional to enzyme concentration with 0.05 to 0.2 NIH clotting units/ml for bovine or human thrombin, 0.005 to 0.02 CTA units/ml for human plasmin, and 0.01 to 0.04 microgram/ml protein for bovine pancreatic trypsin. In 0.1 M Tris-HCl at pH 8.4 and 30 degrees, Km is 14 micrometer and Vmax is 0.037 micronmol/min/NIH unit/ml for bovine thrombin, Km is 78 micrometer and Vmax is 0.31 micronmol/min/CTA unit/ml for human plasmin, and Km is 12 micrometer and Vmax is 138 micronmol/min/mg protein/ml for bovine trypsin. With bovine thrombin, activities at pH 7.3 and at pH 9.2 were 30% lower and 40-50% higher than the rate at pH 8.4. Samples of bovine and human thrombin ranging in specific clotting activity from 59 to 2133 NIH units/mg protein showed esterase activities varying from 0.15 to 0.4 micronmol p-nitrobenzyl alcohol formed/10 min/NIH unit.
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PMID:Assay of the esterase activity of thrombin, plasmin and trypsin with a chromogenic substrate p-nitrobenzyl p-toluenesulfonyl-l-arginine. 14 54

Rates of hydrolysis of the newly developed peptide chromogenic substrates S-2160 (N-Bz-Phe-Val-Arg-pNA, HCl), S-2238 (H-D-Phe-Pip-Arg-pNA, 2HCl), S-2222 (N-Bz-Ile-Glu-Gly-Arg-pNA, HCl), and S-2251 (H-D-Val-Leu-Lys-pNA, 2HCl) from AB Kabi Peptide Research and Chromozym TH (Z-Gly-Pro-Arg-pNA, HCl) from Pentapharm Limited were tested against highly purified preparations of human plasmin, bovine trypsin, human alpha thrombin, and bovine factor Xa. S-2160, S-2238, and Chromozym TH are sensitive to thrombin, Chromozym TH and S-2238 exhibiting a substantially greater sensitivity than S-2160. All 3 substrates are insensitive to factor Xa but hydrolyzed to varying degrees by plasmin and trypsin. In contrast, S-2222 is sensitive to Xa and insensitive to thrombin. S-2251 is relatively plasmin-specific, being resistant to the clotting enzymes thrombin and Xa. S-2251 exhibits even greater sensitivity to the SK-plasmin complex than to plasmin. In addition, the substrate Chromozym PK (N-Bz-Pro-Phe-Arg-pNA, HCl) was evaluated and found to be relatively specific for plasma kallikrein. Assays for antithrombin III and heparin using S-2222 as the substrate and factor Xa as the enzyme, plasma plasminogen and plasmin inhibitors using S-2251 as the substrate, and plasma prekallikrein and kallikrein inhibitors using Chromozym PK as the substrate have been developed. Synthetic peptides mimicking amino acid sequences adjacent to proteolytic activation cleavage of plasma serine protease precursors appear to be sensitive and relatively specific tools applicable to kinetical and clinical studies of these enzymes and their inhibitors.
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PMID:Serine protease specificity for peptide chromogenic substrates. 14 72

Fibrin polymers formed from fibrinogen with thrombin in the presence of EDTA were suspended in a medium containing glucose, arabic gum and imidazole-HCl buffer and were sonicated at 20 kHz for 20 min to make a suspension containing fibrin particles of small size. The fibrin suspension was used as a substrate of plasmin for determining the enzymic activity of plasmin and plasminogen activated with urokinase. The kinetic study on the reaction of the fibrin particles with plasmin in the presence and the absence of fibrinogen revealed that Km value of fibrin for plasmin is 4.2 x 10(-7) M and the Ki value of fibrinogen is 1.2 x 10(-5) M.
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PMID:Fibrin suspension as a substrate fop plasmin: determination and kinetics. 16 68

Rat ovarian granulosa cells were isolated from immature female rats after stimulation with pregnant mare's serum gonadotropin and then maintained in culture. Proteoglycans were labeled using [35S]sulfate, D-[3h]glucosamine, or L-[3H]serine as precursors. 35S-labeled proteoglycans in the medium increased linearly up to 72 h after a 6- to 8-h lag period, and those in a 4 M guanidine HCl extract of the cell layer increased for about 16 h and then reached a plateau and stayed fairly constant up to 72 h. Two distinct sizes of proteoglycans were observed in the medium. The smaller (Kav = 0.60 on Sepharose CL-2B) had lower buoyant densities in dissociative gradients (rho less than 1.4 g/ml). The larger (Kav = 0.26 on Sepharose CL-2B) had high buoyant densities (recovered mainly in the bottom (D1) fraction of the dissociative gradient). More than 90% of the D1 proteoglycans contained dermatan sulfate chains (average Mr = 38,000) which yielded 84% 4-sulfated and 15% disulfated disaccharides after digestion with chondroitinase ABC. About 8% of the 35S-label in D1 was present as a heparan sulfate proteoglycan. When [3H]-glucosamine was used as a precursor, 28% of the 3H activity in the D1 proteoglycans was located in three major oligosaccharide components, two of which were similar or identical with those observed previously in D1 proteoglycans isolated from porcine follicular fluid. These results plus similar susceptibility of the labeled proteoglycans to proteolytic enzymes, especially plasmin, suggest that the granulosa cells synthesize the predominant follicular fluid proteoglycans.
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PMID:Biosynthesis of proteoglycans by rat granulosa cells cultured in vitro. 50 Jul 20

The ionization of tyrosine residues in fibrinogen was studied by a spectrophotometric method. The total of 100 tyrosine residues in the fibrinogen molecule was classified into four states: (1) 28 tyrosine residues with pK 10.1 (m = 1.0). (2) tyrosine residues with pK 11.5 (m = 1.0), (3) 20 tyrosine residues with pK 12.2 (m = 3.0) and (4) 10 tyrosine residues non-ionizable. When fibrinogen was treated with 4 M guanidine . HCl, all of the tyrosine residues became ionizable with the ionization characteristics of pK 10.1 (m = 1.0). The ionization characteristics of tyrosine residues in plasmin-digested fibrinogen were similar to those of fibrinogen, while in CNBr-treated fibrinogen they were fairly different. The value, m, stands for the number of hydroxyl ions involved in the ionization of a tyrosine residue.
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PMID:Four states of tyrosine residues in the fibrinogen molecule. 63 97

Monomer proteoglycan was isolated from porcine ovarian follicular fluid by isopycnic CsCl centrifugation in the presence of 4 M guanidine HCl and protease inhibitors. The elution profile of the D1 preparation on Sepharose 2B was similar to that of monomer proteoglycan from bovine nasal cartilage, indicating a similar molecular size. Follicular fluid proteoglycans consist of about 20% protein, 50% dermatan sulfate, and 20% oligosaccharides rich in sialic acid, galactose, mannose, glucosamine, and galactosamine. The amino acid composition of this proteoglycan is significantly different from that of cartilage proteoglycans, with a higher proportion of aspartic acid, threonine, and lysine, and lower amounts of proline and glycine. Alkali-released dermatan sulfate chains are larger on Sepharose 6B (average Mr = 56,000) than chondroitin sulfate chains from cartilage proteoglycans (average Mr = 25,000), and iduronic acid accounts for 9% of total hexuronic acid. Disaccharide units released by chondroitinase ABC consists of 67% 4-sulfated, 22% 6-sulfated, 5% non-sulfated, and 5% disulfated disaccharides. After treatment with 0.05 M NaOH, 1 M NaBH4 at 45 degrees C for 24 h, two major sialic acid-containing oligosaccharides were observed on Sephadex G-25, corresponding to penta- and hexasaccharides. The pentasaccharide contained sialic acid, galactose, glucosamine, and galactosamine in the proportions 1:2:1:1. The galactosamine is O-glycosidically linked to the protein core. This oligosaccharide accounts for approximately 77% of all the sialic acid in the follicular fluid proteoglycans. The hexasaccharide fraction contained sialic acid, galactose, mannose, and glucosamine in the proportions 1:2:1:2. It also contained a small amount of fucose and galactosamine. The linkage of these oligosaccharides to the protein core remains to be determined. The follicular fluid proteoglycans, unlike those from cartilage, do not interact with hyaluronic acid. Digestion with trypsin, chymotrypsin, or plasmin released dermatan sulfate-peptides nearly as small as those released by papain or alkali; in contrast, cartilage proteoglycans were resistant to plasmin and released peptides containing an average of more than four chondroitin sulfate chains after trypsin or chymotrypsin digestion.
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PMID:Isolation and characterization of proteoglycans from porcine ovarian follicular fluid. 76

Characterization of the cyanogen bromide (CNBr) fragments of the beta chain of human haptoglobin revealed five major fragments resulting from cleavage of four methionyl residues. The fragments were isolated by gel filtration in guanidine-HCl on Sepharose 6B and Bio-Gel P10 and P60. Compositional analyses of the five cyanogen bromide fragments accounted for 248-253 amino acid residues in agreement with the number of residues determined for the intact beta chain. Most of the carbohydrate was attached to CNBr II. Automated amino-terminal sequence analysis and carboxyl-terminal hydrolysis with carboxypeptidase of the haptoglobin beta chain and cyanogen bromide fragments identified 139 residues, or about 55% of the beta-chain molecule. The placement of the fragments within the beta-chain molecule was established by sequence analysis of whole beta chain and a plasmin cleavage fragment. The position of CNBr V was confirmed by the absence of homoserine or homoserine lactone. Cyanogen bromide reaction of intact haptoglobin 1-1 resulted in the isolation of a beta-chain fragment, CNBr III, covalently attached to the intact alpha1 chain by a single disulfide bond. The beta chain was shown to have primary structural similarities to the chymotrypsin family of serin eproteases. Partial sequence analysis of CNBr V established the region which is comparable to the serine-195 active-site region: /Asp-Thr-Cys-Tyr-Gly-Asp-Ala-Gly-Ser-Ala-Phe/ (residues 189-199, chymotrypsinogen A numbering). The active-site serine-195 is replaced by alanine; however, the specificity residue of the trypsin-like enzymes, Asp-189, is preserved. Several minor cyanogen bromide cleavage products were also identified in yields of up to 15%. These minor cleavage products give evidence that tryptophanyl residues in proteins, or glycoproteins, are also susceptible to cyanogen bromide cleavage.
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PMID:Characterization of the cyanogen bromide fragments of the beta chain of human haptoglobin. 99 9

alpha-N-acetyl-L-lysine methyl ester (NALME) is a lysine analogue that reportedly binds to low-affinity lysine binding sites in plasmin(ogen) and miniplasmin(ogen). In the studies presented here, we show that NALME has antifibrinolytic activity; however, unlike the therapeutic agents epsilon-amino-n-caproic acid (epsilon ACA) and tranexamic acid (TEA), the activity of NALME is based on inhibition of the plasmin active site. NALME (0.1-10 mM) significantly inhibited the amidase activity of plasmin, miniplasmin, and streptokinase-plasmin complex without affecting alpha-thrombin or tissue plasminogen activator. epsilon ACA and TEA (0.1-10 mM) did not affect the amidase activity of plasmin or miniplasmin. A kinetic analysis showed that NALME is a competitive inhibitor of D-Val-L-Lys-p-nitroanilide HCl (S-2251) hydrolysis by plasmin; NALME binding to plasmin completely prevented S-2251 binding. The Kl for the plasmin-NALME interaction was 0.4 mM. epsilon ACA and TEA inhibited fibrin monomer digestion by plasmin and miniplasmin without binding to the active site of either enzyme. This result suggests that epsilon ACA and TEA function as antifibrinolytics by disrupting the noncovalent association of fibrin monomer with a domain common to both plasmin and miniplasmin (probably kringle 5). NALME inhibited fibrin monomer digestion principally by decreasing amidase activity. NALME was the only lysine analogue that prevented fragment X formation; TEA and epsilon ACA primarily inhibited the formation of fragments Y and D. When plasmin was incubated simultaneously with alpha 2-antiplasmin and alpha 2-macroglobulin, epsilon ACA increased the fraction of plasmin reacting with alpha 2-macroglobulin; NALME had no effect on the plasmin distribution.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Antifibrinolytic activities of alpha-N-acetyl-L-lysine methyl ester, epsilon-aminocaproic acid, and tranexamic acid. Importance of kringle interactions and active site inhibition. 137 8

Plasmin activity in the tear fluid of the rabbit eye was examined during the wearing of soft contact lenses (SCL) and compared with the occurrence of corneal disturbances assessed in cryostat sections. Plasmin activity was determined with a semiquantitative method using dry punches of filter paper previously soaked in 0.1 M Tris-HCl buffer solution containing mmol/l D-Val-Leu-Lys-FCA (trifluoromethylaminocoumarine), pH 7.2. Punches were applied to the corneal surface for 5 s (tear collection) and incubated in wet chamber. The time of appearance of the bright yellow fluorescence in UV light was recorded and taken as a measure of plasmin activity. For calibration punches soaked in solutions containing plasmin in various concentrations, and processed in the same manner were used. Changes in the cornea were examined histochemically using methods of choice for acid glycosidases, proteases, dehydrogenases, and Na(+)-K(+)-ATPase. SCL with high and low water content were worn in rabbits in 1, 2, 4, 7, 14, 21 and 28 days. Decreased activity of Na(+)-K(+)-ATPase, GGT, and SDH in the corneal endothelium and epithelium were not accompanied by detectable plasmin activity in the tear fluid. Pronounced damage of the corneal epithelium (increased activities of acid glycosidases, acid proteases, LDH, markedly decreased activity of SDH) was accompanied by low concentration of plasmin (0.4-1.0 micrograms/ml) in the tear fluid. Middle activity of plasmin (1.0-2.0 micrograms/ml) was detectable when PMNs were present in the corneal stroma. High plasmin activity (2.0-3.0 micrograms/ml) correlated with corneal ulceration and vascularization.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Histochemical changes in the rabbit cornea and plasmin activity in the tear fluid during contact lens wear. Favourable influence of protease inhibitors (aprotinin, PC5, elastatinal). 137 62


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