Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Microorganisms capable of producing L-pyrrolidonecarboxylate peptidase [L-pyrrolidonyl peptidase, EC 3.4.11.8] were screened and a strain of Bacillus amyloliquefaciens was chosen as one of the most potent producers of the enzyme. The enzyme was purified from lysozyme-lysate of the bacterial cells by salting out with ammonium sulfate, adsorption on DEAE-cellulose, covalent chromatography on PCMB-Sepharose and by gel filtration on Sephadex G-150. By these procedures, the enzyme was purified about 800-fold with an activity recovery of 9%, and the preparation was electrophoretically homogenous. The enzyme was most active and stable at pH 7-8. The presence of 2-mercaptoethanol and EDTA was effective for stabilizing the enzyme. The molecular weight was estimated to be 72,000 by the gel filtration method and to be 24,000 by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a subunit oligomer, presumably trimer. The enzyme was inactivated by the addition of PCMB, sodium tetrathionate, Hg2+ and Cu2+, but the activity lost was restored by the addition of 2-mercaptoethanol and EDTA. The purified enzyme split amide and ester linkages in L-pyroglutamyl derivatives of L-alanine, beta-naphthylamine, alpha-naphthol, and 4-methylumbelliferone, but was completely inert towards various peptides and esters used as substrates for usual amino- and carboxy-peptidases, and for endopeptidases such as trypsin, subtilisin and alpha-chymotrypsin.
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PMID:Purification and characterization of L-pyrrolidonecarboxylate peptidase from Bacillus amyloiliquefaciens. 2 93

p-Chloromercuribenzoate-treated hemoglobin was digested by trypsin. The hydrolysate was subjected to gel-filtration on Bio-Gel P-4 and Sephadex G-50 columns, ion-exchange chromatography on CM-Sephadex and DE 52 columns, and paper electrophoresis. Peptides obtained by this procedure were analyzed for amino acid compositions and amino-terminal amino acid sequences. The results showed that p-chloromercuribenzoate-treated hemoglobin was hydrolyzed to a limited extent by trypsin at the bonds involving the carboxyl group of a lysine or arginine residue in planes A--E in the parent hemoglobin, which represent the external region of the parent tetramer. It is concluded therefore that the slight modification of hemoglobin enhances the susceptibility of the protein to proteases and that the hydrolysis of the modified protein is limited.
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PMID:Limited proteolysis of p-chloromercuribenzoate-dissociated hemoglobin by trypsin. 45 29

Human neutrophil collagenase (HNC) has been purified from extracts of fresh and outdated buffy coats and from the exudates of phorbol myristate acetate-stimulated neutrophils. The HNC present in the starting material from such preparations can be either latent or active, or have an approximate molecular weight of 75 or 58 kDa, depending upon whether the extraction buffer contains protease inhibitors and/or antioxidants. The purification of these different forms of HNC is described and is made possible by taking appropriate precautions to stabilize the HNC. For example, a purification protocol is described that allows the purification to homogeneity of the active and PCMB-active latent 58 kDa forms of HNC in high yield with specific collagenase activities that greatly exceed that of trypsin-activated human fibroblast collagenase (HFC). The pattern of activation of the latent 58 and 75 kDa species by trypsin, organomercurials and oxidants has been investigated. HNC is shown to preferentially hydrolyze type I over types II and III collagens in solution. The specificity of HNC toward the hydrolysis of 60 octapeptides has been examined and compared with HFC. HNC is shown to be a glycoprotein that contains complex N-linked oligosaccharides.
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PMID:Human neutrophil collagenase. 148 44

Eel liver glutamate dehydrogenase (GDH) [EC 1.4.1.3] was eightfold activated by trypsin and the molecular weight of the subunit of the native GDH decreased from 54,000 to 50,000. The C-terminal amino acid of both subunits was Thr. One peptide was released after proteolysis of the native GDH by trypsin and purified by anhydrotrypsin agarose and reversed-phase HPLC. The isolated peptide consisted of 39 amino acids and its amino acid sequence was as follows: H2NS-E-A-V-E-K-E-D-D-P-N-F-F-K-M-V-E-G-F-F-D-K-G-A-A-I- V-E-N-K-L-V-E-E-D-L-K-T-R-COOH. The peptide contained the N-terminal of the native GDH and its molecular weight was calculated to be 4,413. We concluded that the trypsin-catalyzed activation was caused by release of this peptide from the native GDH. p-Chloromercuribenzoic acid inhibited the activity of the trypsin-treated GDH, but stimulated that of the native GDH. The response of trypsin-treated GDH to ADP and GTP was decreased compared with that of the native GDH.
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PMID:The trypsin-catalyzed activation of glutamate dehydrogenase purified from eel liver. 163 63

We have isolated and characterized a novel, large, multicatalytic protease from mammalian cells. This protease was designated PABI (protease accumulated by inhibitors). When baby hamster kidney (BHK) cells were grown in medium containing leupeptin, a potent serine-cysteine protease inhibitor, the trypsin-like protease activity (PABI) in the cells increased its level more than 100-fold over the control. This increase was also observed in other cultured cells such as COS, HepG2, and skin fibroblast cells. The activity was also elevated by treatment with other protease inhibitors including chymostatin or trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane. Immunoblot analysis, by employing antisera prepared against the purified PABI, also showed a concomitant increase of this protein in BHK, COS, and HepG2 cells on leupeptin treatment. PABI was purified to a homogeneous state from leupeptin-treated BHK cells. PABI is a glycoprotein of molecular weight 700,000. PABI was found to be a multimer of a major subunit of apparent Mr of 84,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopic analysis. PABI dissociates into subunits only under reducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PABI has both trypsin-like and chymotrypsin-like protease activities toward synthetic substrates. Both activities were inhibited by phenylmethanesulfonyl fluoride, aprotinin, bovine pancreas trypsin inhibitor, and chymostatin. Leupeptin inhibited only the trypsin-like activity of PABI. p-Chloromercuribenzoate had no effect on either activity. Furthermore, PABI degraded collagen type I and fibronectin. These results indicate that PABI is a novel protease which differs from any known proteases including cytosolic high molecular weight proteases. The physiological function of PABI is yet to be determined.
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PMID:Isolation and characterization of a novel large protease accumulated in mammalian cells in the presence of inhibitors. 267 25

A Co2+-dependent dipeptidase from E. coli strain AJ005, a peptidase-deficient mutant, was purified with streptomycin sulfate, ammonium sulfate and DEAE-cellulose. The purified dipeptidase increased by about 106-fold in specific activity, with dilysine as a substrate. The dipeptidase cleaved dilysine to two lysines among the lysine homopolymers, the possibility remaining that it is active toward peptides other than dilysine, since it was investigated in the present study only for activity toward lysine homopolymers. Activity was inhibited 54% by 10(-3) M KCN and completely by 10(-3) M PCMB, EDTA and benzethonium chloride, but not at all by soybean trypsin inhibitors. 78% and 95% of its activity was lost with 30 minutes' treatment at 45 degrees C and 50 degrees C, respectively. The apparent Km value was 6.7 X 10(-4) M for dilysine. It is probable that the dipeptidase differs from dipeptidase DP.
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PMID:Purification and properties of dipeptidase from Escherichia coli AJ005. 352 11

An enzyme was purified from soybean seeds mainly by repeated ion-exchange chromatography using benzoyl-L-arginine p-nitroanilide (BAPA) as a substrate. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The molecular weight was estimated as 59,000 by gel filtration. The enzyme was most active toward BAPA between pH 8 and 10. The enzyme was inactive toward protein substrates but hydrolyzed synthetic substrates and oligopeptides exclusively at the carboxyl side of L-arginine and L-lysine. Kinetic studies using synthetic substrates showed that, on the basis of Vmax/Km, the enzyme preferentially hydrolyzed amide substrates over ester substrates. Benzoyl-L-arginine 4-methylcoumaryl-7-amide (Bz-Arg-MCA) was the best substrate. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), tosyl-L-lysine chloromethyl ketone (Tos-Lys-CH2Cl), leupeptin, and antipain. p-Chloromercuribenzoate (PCMB) was only partially inhibitory. Various protein inhibitors of trypsin such as soybean trypsin inhibitor were ineffective. From the primary specificity and susceptibility to chemicals, the enzyme can be said to be a trypsin-like serine protease. Although the physiological role of the enzyme is unclear, it seems likely that it is involved in limited hydrolysis of certain physiological peptides during processing.
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PMID:Trypsin-like protease from soybean seeds. Purification and some properties. 637 96

Carboxamidopeptidase, an enzyme which inactivates neurohypophyseal hormones, has been purified 3800-fold in an overall yield of 22% from toad skin, a neurohypophyseal hormone target organ, by (NH4)2SO4 fractionation, DEAE-Sephadex chromatography, and affinity chromatography on immobilized p-aminobenzamidine and concanavalin A-agrose. The purified enzyme is capable of inactivating both [8-arginine]vasopressin (AVP) and oxytocin by hydrolyzing the Arg8-Gly9-NH2 and the Leu8-Gly9-NH2 bonds, respectively, and can hydrolyze the ester substrates, benzoyl-L-arginine ethyl ester (BzArgOEt) and acetyl-L-trypsine ethyl ester, suggesting that the enzyme has both trypsin-like and chymotrypsin-like activities. Carboxamidopeptidase is maximally active at pH 7.5-8.5 for AVP and BzArgOEt and pH 7.0 for oxytocin. Carboxamidopeptidase is inhibited by ovoinhibitor, ovomucoid, Trasylol. lima bean trypsin inhibitor, concanavalin A, antipain, leupeptin, chymostatin, elastatinal, p-nitrophenyl p-guanidinobenzoate, and 4-methylumbelliferyl p-guanidinobenzoate but not by soybean trypsin inhibitor, alpha 1-antitrypsin, hirudin, pepstatin, bestatin, phosphoramidon, or cysteine. The enzyme is also inhibited by the serine protease inhibitor, diisopropyl phosphofluoridate (i-Pr2PF), and by the chloromethyl ketone derivatives of tosyllysine, tosylphenylalanine, and (benzyloxycarbonyl)phenylalanine, as well as by the sulfhydryl group reagent, p-(chloromercuri)benzoate (PCMB). Inhibition by PCMB is reversed by cysteine. The molecular weight determined by gel filtration in the presence of 1 MNaCl is approximately 100 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of two identical subunits of 48 000 daltons. Each subunit consists of a heavy chain (28 000 daltons) and a light chain (19 000 daltons) joined by a disulfide bond(s). Labeling experiments using [3H]-i-Pr2PF showed that the enzyme active site is located in the heavy chain.
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PMID:Carboxamidopeptidase: purification and characterization of a neurohypophyseal hormone inactivating peptidase from toad skin. 676 14

His397 was replaced with alanine by site-directed mutagenesis of the cloned PRC1 gene in order to confirm the role of this residue in the proton-relay system of carboxypeptidase Y (CPY). The expressed and purified H397A showed a CD spectrum almost identical to that of the wild type enzyme, but its heat stability and conformation on heating differed somewhat. Kinetic analysis showed that the kcat values of the purified H397A toward the peptide substrates, Z-Phe-Leu and Z-Gly-Phe, were reduced to approximately 4 x 10(-5)-fold, whereas the Km values remained almost unchanged. The activity of the H397A preparation with the ester substrate, Ac-Phe-OEt, was negligible. The low activity of our H397A was lost on treatment with DFP and Z-Phe-CH2Cl, site-specific inhibitors, respectively, for Ser146 and His397, and with the HgCl2 and PCMB, SH-reagents for Cys341. After treatment with these inhibitors, the kcat value for the H397A preparation toward Z-Phe-Leu decreased 1 x 10(3)-fold or more. The value was approximately 10(-8) for the wild type enzyme. This level of activity is 10(3)-fold lower than the reported value for the same mutant of CPY [Carlsberg Res. Commun. 54, 165-171 (1989)], and more than 10-fold lower than the values for the corresponding His-to-Ala mutants of trypsin [J. Am. Chem. Soc. 114, 1784-1790 (1992)] and subtilisin [Nature 332, 564-568 (1988)]. These findings, together with the pH profiles and chromatographic behavior, are evidence that the low activity of the H397A preparation is due to contamination by wild type CPY. The decreased kcat value of our H397A mutant is the lowest reported among the corresponding histidine mutants of serine proteases. We conclude that the proton-relay system composed of Ser146 and His397 is the sole catalytic center of CPY, and that its destruction leads to complete inactivation.
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PMID:Proton-relay system of carboxypeptidase Y as a sole catalytic site: studies on mutagenic replacement of his 397. 968 40

Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative molecular mass (M(r)) of 28.7 kDa, whereas protease B, with a M(r) of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1 U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate for chymotrypsin-like serine proteases. However, the K(m) values of these two proteases on SAAPF-pNa were higher than that for alpha-chymotrypsin, indicating a lower affinity of proteases A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein, and they stained poorly with the silver staining method. However, NH(2)-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease family.
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PMID:Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus. 1157 23


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