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)

The G-200 flow-through fraction of the extract of sea urchin eggs contained a complex form of glutathione reductase (GR) [EC 1.6.4.2]. The complex was unstable and gradually dissociated with ain increase in GR activity. The activation was facilitated by high concentrations of EDTA, KCI or (NH4)2SO4. The rate of activation by salts was apparently dependent on the ionic strength. The complex form was also activated rather quickly by treatment with proteinases such as trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1] or subtilisin [EC 3.4.21.14]. Trypsin caused the complex to release the free form of GR. Even after trypsin treatment, little change was observed in the dependence of the GR activity on GSSG or NADPH concentration. The GR activity of the complex form was not inhibited at all by 0.2 mM N-ethylmaleimide (NEM) in the presence of GSSG, but was reduced to 3% in the presence of NADPH. When excess NEM was sequestered with GSH, the NEM-treated complex form was strikingly activated by trypsin, while no activation was detected with the free form of enzyme pretreated with NEM. These results suggest that the active site of GR in the complex form is largely masked by a polypeptide moiety of theinhbitiory component.
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PMID:Glutathione reductase in the sea urchin egg. III. Activation of the complex form by proteinases. 1 74

Thioltransferase, an enzyme which catalyzes the thiol/disulfide exchange reaction in the presence of GSH, was purified to homogeneity on 15% SDS-PAGE from human (36,000-fold purification) and bovine (23,000-fold) erythrocyte hemolysates. These enzymes had similar properties in their monomeric structures (M(r) = 11,000) and broad specificities for substrates ranging from low-molecular disulfides (S-sulfocysteine, cystamine, and cystine) to protein disulfides (trypsin and insulin). They were highly sensitive to SH-reagents (monoiodoacetic acid and mercuric chloride), but were protected from inactivation by the presence of disulfides (GSSG, cystamine, and cystine). Phosphofructokinase and pyruvate kinase that had been inactivated by disulfides were reactivated effectively by the addition of thioltransferase with GSH. In addition, disulfides in membrane proteins of human erythrocytes that have been oxidatively damaged by diamide treatment were reduced to the SH-free form more effectively by incubation with thioltransferase.
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PMID:Study on human erythrocyte thioltransferase: comparative characterization with bovine enzyme and its physiological role under oxidative stress. 163 68

Tissue glutathione (GSH) and glutathione disulfide (GSSG) contents were quantitated in the skins of female SENCAR mice following the topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA), and in the skin tumors generated by an initiation-promotion protocol. Total epidermal GSHt (GSH + GSSG) and GSSG contents were not reproducibly and significantly altered 0.5, 4 or 24 h after one or four topical applications of 1 microgram TPA, relative to the values obtained in age-matched, solvent-treated mice. Similar findings held for dermal GSHt at all times of analyses, and for dermal GSSG contents 0.5 and 4 h after TPA application. However, dermal GSSG contents were slightly elevated 24 h after TPA application. The GSHt and GSSG contents of skins initiated with 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA) and harvested 17, 29 and 37 days after the cessation of chronic treatment with acetone (14 weeks, twice a week) were comparable to the values measured in age-matched, non-treated skins. In contrast, GSHt contents of papillomas harvested 17, 29 and 37 days after the cessation of chronic treatment with 1 microgram TPA (14 weeks, twice a week) were 2- to 4-fold greater than the values measured in non-treated mice, and DMBA-initiated, acetone-promoted mice, and the non-tumorous tissue adjacent to the papillomas. Comparable changes did not occur in papilloma GSSG contents. GSHt contents in squamous cell carcinomas (SCC) were twice the values measured in papillomas and 5- to 8-fold greater than the values measured in non-treated skins, and the non-tumorous tissue adjacent to SCC. Similarly, GSSG contents in SCC were elevated multifold relative to papillomas, non-treated skin and the non-tumorous tissue adjacent to SCC. Epidermal cell suspensions prepared by the trypsin-flotation procedure retained less than 2% of their original GSHt content and had reduced GSHt/GSSG ratios. Collectively these studies suggest that (i) if promoting doses of TPA induce oxidative stress in murine epidermis, it cannot be detected by measurements of GSH/GSSG; (ii) the antioxidant capacity of epidermal cells prepared by the trypsin-flotation procedure is severely compromised; and (iii) GSHt contents progressively increase during skin tumor ontogeny.
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PMID:Assessment of the antioxidant/prooxidant status of murine skin following topical treatment with 12-O-tetradecanoylphorbol-13-acetate and throughout the ontogeny of skin cancer. Part II: Quantitation of glutathione and glutathione disulfide. 174 38

Bovine serum albumin (BSA) was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase under general protein phosphorylation conditions. The optimal pH for this phosphorylation was 9.0. The K0.5 (the concentration required for 50% of maximal phosphorylation) for BSA at pH 7.5 was 15 microM. One mole of phosphate was incorporated per mole of BSA, and only one phosphopeptide fragment was obtained after extensive proteolysis with trypsin. BSA phosphorylation required dithiothreitol or GSH, but GSH was only one-fiftieth as effective as dithiothreitol. GSSG counteracted the effect of dithiothreitol and GSH. Phosphorylation increased in a time-dependent and dithiothreitol concentration-dependent manner when BSA was preincubated with dithiothreitol. The increase in the incorporation of 32P correlated with the appearance of up to six free sulfhydryl groups. The effect of dithiothreitol on BSA appeared reversible, since reoxidation of reduced BSA decreased its susceptibility to phosphorylation. These experiments showed that this in vitro phosphorylation is dependent on the sulfhydryl-disulfide state of BSA. The possible implications of the sulfhydryl-disulfide state of proteins in the regulation of phosphorylation are discussed.
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PMID:Effect of sulfhydryl-disulfide state on protein phosphorylation: phosphorylation of bovine serum albumin. 298 43

Protein-mixed disulfides (PSSG) were formed by interaction of glutathione disulfide (GSSG) with lens crystallins. Total water-soluble crystallins and alpha-crystallin purified on a Sephacryl S-200 column were separately incubated with 0, 2, 4, and 8 mM (final concentrations) GSSG overnight and then dialyzed to remove unbound GSSG and GSH. Either TPCK-treated trypsin or TLCK-treated alpha-chymotrypsin were added to about 200 micrograms crystallin samples and incubated for 20 min at room temperature. Reactions were terminated by boiling in SDS-mercaptoethanol-Tris (pH 6.8) solution and subjected to electrophoresis on 10% polyacrylamide slab gels. Comparison of SDS-PAGE patterns of proteolysis with or without GSSG treatment showed that GSSG at a concentration of 2 mM or higher reduced or abolished proteolysis of alpha-crystallin by trypsin but not by alpha-chymotrypsin. The protective effect of GSSG was greater with alpha-crystallin than with beta-crystallins. Addition of alpha-crystallin-mixed-disulfide to an assay system in which trypsin was hydrolyzing N-alpha-benzoyl-DL-arginine-P-anilide (BAPNA) inhibited the tryptic activity. Direct addition of GSSG or native alpha-crystallin had no significant inhibitory effect on trypsin. Based on these results, it is speculated that alpha-crystallin glutathione mixed-disulfide appears to become resistant to trypsin probably by non-competetive inhibition of the enzyme.
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PMID:Resistance of alpha-crystallin-glutathione mixed-disulfide to tryptic digestion. 301 92

GSH is an important cellular defense against oxidant injury. Its effect in the rat liver microsomal lipid peroxidation system has been examined. Incubation of fresh rat liver microsomes with ascorbic acid and ADP-chelated iron leads to the peroxidation of microsomal lipids (production of thiobarbituric acid-reactive substances and destruction of polyunsaturated fatty acids) following a 2 to 5 min lag. Addition of 0.1 mM GSH to the system lengthened the lag period by 5 to 15 min without affecting the rate or the extent of lipid peroxidation. GSH could not be replaced in prolonging the lag by cysteine, mercaptoethanol, dithiothreitol, propylthiouracil, or GSSG. The GSH effect on the lag was abolished by heating or trypsin digestion of the microsomes, indicating that microsomal protein is required for its expression. Progressively longer lags were observed as the GSH concentration was increased from 0.1 to 5 mM, but there was no evidence of GSH oxidation as a consequence of the protection against lipid peroxidation. GSH protected against heat inactivation of the microsomal protein responsible for the GSH effect. Experiments with an oxygen electrode revealed that the GSH protection did not alter the ratio of O2 consumed to thiobarbituric acid-reactive substances produced. This implicated free radical scavenging as the mechanism of protection. These results indicate the existence of a GSH-dependent rat liver microsomal protein which scavenges free radicals. This protein may be an important defense against free radical injury to the microsomal membrane.
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PMID:Glutathione-dependent protection by rat liver microsomal protein against lipid peroxidation. 683 5

ATP-sensitive K+ (KATP) channels are thought only to open during conditions of metabolic impairment (e.g., myocardial ischemia). However, the regulation of KATP channel opening during ischemia remains poorly understood. We tested whether thiol (SH) group oxidation, which is known to occur during ischemia, may be involved in KATP channel regulation. Inside-out membrane patches were voltage clamped at a constant potential (O mV) in asymmetrical K+ solutions. The effects of compounds that specifically modify SH groups [p-chloromercuri-phenylsulfonic acid (pCMPS), 5-5'-dithio-bis(2-nitrobenzoic acid) [DTNB], and thimerosal] were tested. The membrane-impermeable compound, pCMPS (> or = 5 microM), caused a quick and irreversible inhibition of KATP channel activity. The reducing agent, dl-dithiothreitol (DTT) (3 mM) was able to reverse this inhibition. DTNB (500 microM) caused a rapid, but spontaneously reversible, block of KATP channel activity. After DTNB, no change was observed in single channel conductance. Oxidized glutathione (GSSG, 3 mM) did not block KATP channel activity. Thimerosal (100-500 microM) induced a DTT-reversible block of partially rundown KATP channels, or channels that underwent complete rundown; these channels were reactivated with trypsin (1 mg/ml). Thimerosal did not block KATP channels that had a high degree of activity. However, the ATP sensitivity was decreased; the concentration of ATP needed to half-maximally inhibit the channel (Ki) was increased from 47 +/- 12 to 221 +/- 35 microM (n = 6, P < 0.05). This was not due to a spontaneous change with time.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of thiol-modifying agents on KATP channels in guinea pig ventricular cells. 750 58

Matrix metalloproteinases (MMPs) and neutrophil elastase (NE) may each contribute to fibrillar collagen degradation in various disease states. Little, however, is known about the activation and localization of MMP in the heart. Accordingly, we extracted MMP and examined mechanisms of proMMP activation in whole tissue extracts of the adult rat myocardium. Incubation of extracts with serine proteases (i.e., trypsin or neutrophil elastase) at 37 degrees C resulted in a time-dependent activation of proMMPs. Based on immunoblot and measurements of MMP activity by zymography, the molecular weight of active MMP was deduced to be 52 kDa. The second-order rate constant for activation of proMMP by serine protease was 5.5 +/- 0.2 x 10(5) M-1min-1 and for oxidized glutathione (GSSG) 1.5 +/- 0.1 M-1min-1. Incubation of the extract with both serine protease and GSSG increased the rate of activation 30-fold. Based on reverse zymographic analysis of collagenase inhibition, tissue inhibitors of metalloproteinases were identified. Indirect immunofluorescence localized proMMPs/MMPs to the endothelium and subendothelial space of the endocardium and throughout the interstitial space found between groups of muscle fibers. These results suggest that the mechanism of activation of MMPs by either a serine protease and by oxidizing, thiol-modifying reagents are mechanistically different and the presence of either a serine protease or GSSG synergistically increase the rate of activation of proMMPs. Our results also suggest that MMPs may be regulated by its own endogenous inhibitors. The contribution of this proteolytic enzyme to tissue remodeling and wound healing responses that occur in various diseases states remains to be established.
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PMID:Myocardial matrix metalloproteinase(s): localization and activation. 810 89

Erythrina trypsin inhibitor (ETI) from the seeds of Erythrina caffra is a high-affinity inhibitor of trypsin, chymotrypsin and tissue plasminogen activator. Its 172 amino acid polypeptide chain is stabilized in its compact, native state by two disulfide bonds. In spite of their conservation in all trypsin inhibitors of the soybean trypsin inhibitor (STI-Kunitz) family, their state of oxidation is essential only for protein stability but not for inhibitory function. Reduction/reoxidation of ETI in the presence of glutathione reshuffling buffer (GSH/GSSG; pH 8.3) not only allows the inhibitor to be restored in its native structure, but also does not interfere with its binding affinity; carboxymethylation or carboxamidomethylation of the free thiol groups does not affect K1 significantly (for trypsin (KI)ETIox = 2.3 nM, (KI)ETICM = 1.9 nM; for chymotrypsin (KI)ETIox = 30 microM, (KI)ETICM = 25 microM). The two cystine cross-bridges in the native ETI lead to enhanced stability toward pH and chaotropic agents. As taken from intrinsic protein fluorescence at acid pH and varying ionic strength (pH < 4, I = 0.01 to 0.15 M), the oxidized inhibitor retains its spectral properties, whereas reduced and carboxymethylated or carboxamidomethylated ETI undergo at least partial denaturation. At alkaline pH, the oxidized protein is stable up to pH 9.5, whereas the reduced protein undergoes structural alterations at pH > 7, reaching a final plateau at pH 10.0 to 10.5. In the case of urea (U) or guanidinium chloride (GdmCl) denaturation at pH 7.0, structural transitions of the oxidized inhibitor show "hysteresis" with half-concentrations (cU)1/2 approximately 10 M and (cGdmCl)1/2 approximately 4.5 M for denaturation, and (cU)1/2 = 4.7 M and (cGdmCl)1/2 = 1.5 M for renaturation. In contrast, the reduced (and chemically modified) inhibitors exhibit true equilibrium transitions at (cU)1/2 = 0.9 M and (cGdmCl)1/2 = 0.5 M, respectively. Reduction/reoxidation in the absence and in the presence of denaturants (GdmCl) can also be applied to ETI covalently attached to a solid matrix.
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PMID:Erythrina caffra trypsin inhibitor retains its native structure and function after reducing its disulfide bonds. 819 58

Protein S-thiolation, the formation of mixed disulphides of cysteine residues in proteins with low-molecular-mass thiols, occurs under conditions associated with oxidative stress and can lead to modification of protein function. In the present study, we examined the site of S-thiolation of the enzyme creatine kinase (CK), an important source of ATP in myocytes. Inactivation of this enzyme is thought to play a critical role in cardiac injury during oxidative stress, such as during reperfusion injury. Reaction of rabbit CK M isoenzyme with GSSG, used to model protein S-thiolation, was found to result in enzyme inactivation that could be reversed by GSH or dithiothreitol. Measurement of GSH that is released during the thiolation reaction indicated that the maximum extent of CK thiolation was approx. 1 mol of GSH/mol of protein, suggesting thiolation on one reactive cysteine residue. Accordingly, matrix-assisted laser-desorption ionization MS confirmed that the molecular mass of CK was increased, consistent with addition of one GSH molecule/molecule of CK. Using trypsin digestion, HPLC and MS analysis, the active-site cysteine residue (Cys(283)) was identified as the site of thiolation. Reversal of thiolation was shown to be rapid when GSH is abundant, rendering dethiolation of CK thermodynamically favoured within the cell. We conclude that S-glutathionylation of CK could be one mechanism to explain temporary reversible loss in activity of CK during ischaemic injury. The maintainance of GSH levels represents an important mechanism for regeneration of active CK from S-glutathionylated CK.
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PMID:Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue. 1076 88


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