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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A number of novel aromatic Tris-amidines have been synthesized and investigated for their antiproteolytic property. The basic structure of the compounds is that of mesitylene where each of the methyl groups has been substituted with a 3- or 4-amidinophenoxy moiety. The compounds displayed considerable activity against trypsin (EC 3.4.21.4) and thrombin (EC 3.4.21.5), but proved most effective against porcine pancreatic kallikrein (EC 3.4.21.8). With this enzyme a Ki value of 2.43-10(-8) M was recorded for alpha,alpha',alpha''-tris(4-amidino-2-bromophenoxy)mesitylene at pH 8.1 and 37 degrees C. The most potent thrombin inhibitor, alpha,alpha',alpha''-tris(3-amidinophenoxy)mesitylene, had a Ki value of 6.51-10(-7) M and was also a strong overall anticoagulant. The inhibitors were able to interfere with the kinin release by human plasma kallikrein at concentrations as low as 1-10(-10) M. However, despite this remarkable antikallikrein effect and the known importance of plasma kallikrein in the activation of Hageman factor (factor XII), the compounds had only little influence on the early stages of blood coagulation.
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PMID:Aromatic Tris-amidines. A new class of highly active inhibitors of trypsin-like proteases. 108 99

Human urinary kallikrein [EC 3.4.21.8] (HUK) was purified about 200-fold with an overall yield of 40 percent from crude powder by DEAE-cellulose chromatography, acetone fractionation, Sephadex G-100 gel filtration and DEAE-Sephadex A-50 chromatography. Its activity was 200 kallikrein units (KU) per A280. HUK from active fractions obtained by DEAE-Sephadex A-50 chromatography was separated into three active components showing isoelectric points of 3.9 (HUK-1), 4.0 (HUK-2), and 4.2 (HUK-3) by isoelectric focusing: each HUK component was homogeneous on disc electrophoresis. The approximate molecular weights of HUK-1, -2 and -3 were estimated to be 2.7 X 10(4), 2.7 X 10(4), and 2.9 X 10(4), respectively, by gel filtration on a Sephadex G-100 column. The optimum pH's of HUK-1, -2, and -3 in esterolytic action were found to be 8.0, 8.3, and 7.5, respectively, and they were fairly heat stable in comparison with other glandular kallikreins. The three components of HUK were weakly inhibited by Trasylol, but were not affected by soybean and ovomucoid trypsin inhibitors. They were strongly resistant to treatment with urea and weakly resistant to treatment with guanidine. The activation energies of HUK-1, -2, and -3 were found to by 1.17 X 10(4), 5.1 X 10(3), and 1.45 X 10(4) cal per mole, respectively. The Km values were estimated toward N-alpha-tosyl-L-arginine methyl ester (TAME), N-alpha-benozyl-L-arginine ethyl ester (BAEE), and N-alpha-benozyl-L-arginine methyl ester (BAME).
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PMID:Studies on urinary kallikreins. I. Purification and characterization of human urinary kallikreins. 108 37

A series of amino acids, amidino acids, and amidino esters was synthesized and the compounds were evaluated for their inhibitory activity against bovine trypsin, bovine thrombin, and porcine pancreatic kallikrein and as anticoagulants. Among these compounds, ethyl 4-amidino-2-iodophenoxyacetate was found to be the most effective inhibitor of the enzymes in question, with a potency (Ki = 3.16 x 10-6 M vs. trypsin; Ki = 4.8 x 10-5 M vs. thrombin) similar to that of p-amidinophenylpyruvic acid (Ki = 6.0 x 10-6 M vs. trypsin; Ki = 2.0 x 10-5 M vs. thrombin). Ethyl 4-amidino-2-iodophenoxyacetate was also found to be the most effective in blocking the clotting activity of plasma, as indicated by significant prolongation of the partial thromboplastin time. This paper reports the synthetic methods, the enzyme inhibitory activity, and the structure-activity relationships observed.
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PMID:Synthesis of isosteres of p-amidinophenylpyruvic acid. Inhibitors of trypsin thrombin, and pancreatic kallikrein. 113 20

A series of omega-amidinophenylalkyl amidinophenyl ethers was synthesized and examined for inhibitory activity against trypsin, pancreatic kallikrein, and thrombin. Modifications of the compounds included lengthening of the alkane chain, variation in the position of the amidino groups, and substitution of halogen on the benzene rings. The compounds act as competitive reversible inhibitors, and many of them possess considerable potency. An outstanding trypsin inhibitor was found in 4-amidinophenylethyl 4 amidino-2-bromophenyl ether (compound 7) with a Ki value of 7.3 x 10(-8) M (pH 8.1, 37 degrees). A number of aromatic diamidines with a central dioxyalkane chain were similarly studied. Here, 1-(4-amidino-2-iodophenoxy)-5-(3-amidinophenoxy)pentane (compound 32) was a highly effective inhibitor of bovine thrombin (Ki = 1.1 x 10(-6) M), of human thrombin, and of the overall clotting process of human plasma.
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PMID:New aromatic diamidines with central alpha-oxyalkane or alpha, omega-dioxyalkane chains. Structure-activity relationships for the inhibition of trypsin, pancreatic kallikrein, and thrombin and for the inhibition of the overall coagulation process. 117 Dec 38

A 427-fold purification of rat urinary kallikrein (RUK) was achieved in three steps involving chromatography on columns of DEAE-Sepharose CL-6B, gel filtration on Sephadex G-100 and affinity chromatography on a column of benzamidine-Sepharose. Purified enzyme showed a single band on SDS-PAGE with an estimated molecular weight of 43,000. The amino-terminal sequences of the first 25 residues of RUK resemble the reported sequence for true kallikrein and share 80% identity with rat submandibular gland (RSMG) kallikrein-like serine protease. The RUK is highly reactive towards kallikrein substrates Bz-pro-phe-arg-pNA and DL-val-leu-arg-pNA, and plasmin substrate D-val-leu-lys-pNA. RSMG enzyme is more reactive towards Bz-val-gly-arg-pNA and tosyl-gly-pro-arg-pNA, preferential chromogenic substrates for trypsin-like proteases and thrombin, respectively. Both leupeptin and aprotinin inhibit RUK strongly, but soy bean trypsin inhibitor has no effect on this enzyme. RSMG enzyme is poorly inhibited by any of these inhibitors. The data suggest that although both enzymes are members of tissue kallikrein multigene family, urinary enzyme is a true kallikrein and RSMG enzyme is a kallikrein-like serine protease with different substrate specificity.
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PMID:Purification of rat urinary kallikrein: comparative studies with rat submandibular gland kallikrein-like serine protease. 128 50

A number of ligands for the selective purification by affinity chromatography of the trypsin-like protease, porcine pancreatic kallikrein, were designed de novo by computer-aided molecular design. The ligands were designed to mimic the side-chains of a number of arginyl dipeptides and included a benzamidine moiety substituted on a triazine ring. The ligands displayed inhibitory activities against pancreatic kallikrein which mirrored the specificity constants of the dipeptides they were designed to mimic. The ligand with the highest affinity for the enzyme, an analogue of a Phe-Arg dipeptide, when immobilized to Sepharose CL-4B via a hexamethylene spacer arm, purified pancreatic kallikrein 110-fold in one step from a crude pancreatic acetone extract.
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PMID:Design of novel affinity adsorbents for the purification of trypsin-like proteases. 147 81

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

A full-length cDNA encoding human salivary-gland preprokallikrein was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in transfected Spodoptera frugiperda cells and the recombinant product secreted into the culture medium. By alternating anion-exchange chromatography and gel-filtration steps, twice repeated, prokallikrein was purified to homogeneity, which was confirmed by amino acid analysis and N-terminal sequence determination. The prepropeptide was processed correctly, including the removal of the signal peptide. The resulting proenzyme was found to be glycosylated, had a molecular mass of 35 kDa and an isoelectric point of 4.6. The yield of purified recombinant protein reached a level of 5 mg/l insect cell culture. After trypsin digestion of prokallikrein, the biological activity of the released kallikrein was demonstrated by its specific amidase, esterase and kininogenase activity. The expression and purification of prokallikrein, as described here, offers the opportunity to study the proenzyme activation through protein engineering techniques in detail.
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PMID:Purification and characterization of human salivary-gland prokallikrein from recombinant baculovirus-infected insect cells. 158 72

The distribution in the nervous system of T-kininogen, the third kallikrein-resistant kininogen of the rat, was determined using bioassays and a radioimmunoassay system. In rat brain homogenates, trypsin released large amounts of a kinin-like myostimulating activity while urinary kallikrein released small amounts. The kinins released by trypsin were identified by HPLC as mostly T-kinin. Radioimmunoassays showed that a T-kininogen-like immunoreactive factor was uniformly distributed throughout the central nervous system. Higher levels were found in female rats than in male rats. Maximum levels were observed in newborn animals. A slight increase of T-kininogen content of the brain was observed after turpentine injection while T-kininogen level in liver was dramatically increased. T-kininogen plasma contamination to the nervous tissues was estimated by injecting 125I-labelled T-kininogen. The T-kininogen content of rat cultured cells and neurons was also examined. Highest levels were found in dorsal root ganglia neurons, lower levels in Schwann cells, phaeochromocytoma cells, mixed cells from spinal ganglion and in astrocytes. Immunocytochemistry showed the presence of T-kininogen in the cytoplasm of cultured dorsal root ganglia neurons and embryonic hippocampal neurons. The distribution of T-kininogen throughout the central and peripheral nervous system of the rat, the variations of its level during the life span suggest that T-kininogen would play the role of a cysteine proteinase inhibitor and not that of a T-kinin-releasing substrate in nervous tissues.
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PMID:Distribution of immunoreactive T-kininogen in rat nervous tissues. 161 79

To investigate the response of urinary active and inactive kallikrein excretion to sodium depletion in golmerulonephritic (GN) patients, we measured the excretion of urinary active and inactive kallikreins in 10 primary GN patients before and after a low sodium (17 mEq/day), constant potassium (40 mEq/day) diet. They ranged in age from 24 to 47 years with 7 men and 3 women. The etiology included 4 IgA nephropathy, 4 mesangial proliferative GN, 1 minimal change disease and 1 focal sclerosis. The active urinary kallikrein activity was measured by assay of its enzymatic activity on synthetic chromogenic substrate S-2266. The urinary inactive kallikrein excretion was determined indirectly by substracting active kallikrein activity from total kallikrein activity. The latter was measured after trypsin activation of inactive kallikrein. The results showed a significant increase in total and active urinary kallikrein excretion following a low salt diet. Yet, the inactive urinary kallikrein excretion and the ratio of active/total kallikrein excretion showed no significant change. There was no correlation between active and inactive urinary kallikrein excretion either before or after a low sodium, constant potassium diet. These findings suggest that the renal kallikrein-kinin system of GN patients responds normally to the stimulation of sodium depletion.
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PMID:Effect of sodium depletion on urinary excretion of active and inactive kallikrein in glomerulonephritic patients. 167 3


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