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 large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional protein consisting of a unique N-terminal protein kinase domain and a ribonucleotide reductase domain. Previous studies showed that the two functional domains are linked by a protease sensitive site. Here we provide evidence for two subdomains, of 30K and 53K, within the reductase domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0.4 micrograms/ml) of trypsin or V8 protease, cleavage between the subdomains was prevented by the presence of the N-terminal protein kinase domain. At higher protease concentrations (1 micrograms/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two domain structure, and that the interdomain linking region is protected by the unique N terminus.
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PMID:Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase. 799 27

Ferricyanide reductase activity of plasma membranes isolated from Ehrlich ascites tumour cells was very sensitive to trypsin treatment. The decreases of activity observed after treatment with different glycosidases suggests that ferricyanide reductase is a glycoprotein. The opposite effects of phospholipase A2 and phospholipase C on the redox activity indicate that the phospholipidic environment plays an important role in the function of ferricyanide reductase. Sodium ions at millimolar concentrations, and some divalent cations at micromolar concentrations (Ca2+, Mg2+, Sr2+, and Mn2+) behaved as stimulators of ferricyanide reductase activity.
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PMID:Characterization of plasma membrane redox activity from Ehrlich cells. 804 92

Spheroplasts prepared from a molybdenum cofactor-deficient mutant of Rhodobacter sphaeroides f. sp. denitrificans secreted dimethyl sulfoxide (DMSO) reductase which had no molybdenum cofactor and therefore no activity, whereas those from wild-type cells secreted the active reductase. The inactive DMSO reductase proteins were separated by nondenaturing electrophoresis into two forms: form I, with the same mobility as the native enzyme, and form II, with slower mobility. Both forms had the same mobility on denaturing gel. Form I and active DMSO reductase had the same profile on gel filtration chromatography. Form II was eluted a little faster than the native enzyme, suggesting that DMSO reductase form II was not an aggregated form but a compactly folded form very similar to the native enzyme. Form II was digested by trypsin and denatured with urea, whereas form I was unaffected, like native DMSO reductase. These results suggested that form II was a partially unfolded but compactly folded apoprotein of DMSO reductase.
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PMID:Secretion of both partially unfolded and folded apoproteins of dimethyl sulfoxide reductase by spheroplasts from a molybdenum cofactor-deficient mutant of Rhodobacter sphaeroides f. sp. denitrificans. 813 56

Ocular tissues appear to require high levels of ascorbic acid and the elucidation of the mechanisms by which those tissues maintain the vitamin in its reduced state remains an important objective. The regeneration of ascorbate from its oxidative by-product, dehydroascorbic acid (DHAA), was studied in bovine iris-ciliary body. Iris-ciliary body was removed by scalpel, weighed, minced, and homogenized in 20 mM MOPS, 62 mM sucrose, and 0.1 mM EDTA at pH 7.0. The homogenate was centrifuged and precipitated with ammonium sulfate such that maximal DHAA reducing activity was enhanced in a 50-75% ammonium sulfate fraction. This fraction was employed for subsequent characterization of DHAA reduction by iris-ciliary body. Results indicate that the iris-ciliary body enzymatically reduces DHAA to ascorbate at a rate significantly greater than can be accounted for by a nonenzymatic glutathione-dependent mechanism. In addition, saturation kinetics are observed, and the enzymatic activity is dependent on protein concentration, DHAA concentration, and reduced glutathione (GSH) concentration. The activity is sensitive to pH, to high temperature, and to digestion by trypsin and is greatest in the presence of both GSH and NADPH. The reducing activity is therefore attributed to one or more proteins that are distinct from the known ascorbate regenerating enzyme, GSH-dependent DHAA reductase (EC 1.8.5.1).
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PMID:Ascorbic acid regeneration by bovine iris-ciliary body. 822 19

The 3,6-dideoxyhexoses, usually confined to the cell wall lipopolysaccharide of gram-negative bacteria, are essential to serological specificity and are formed via a complex biosynthetic pathway beginning with CDP-D-hexoses. In particular, the biosynthesis of CDP-ascarylose, one of the naturally occurring 3,6-dideoxyhexoses, consists of five enzymatic steps, with CDP-6-deoxy-delta 3,4-glucoseen reductase (E3) participating as the key enzyme in this catalysis. This enzyme has been previously purified from Yersinia pseudotuberculosis by an unusual procedure (protocol I) including a trypsin digestion step (O. Han, V.P. Miller, and H.-W. Liu, J. Biol. Chem. 265:8033-8041, 1990). However, the cloned gene showed disparity with the expected gene characteristics, and upon expression, the resulting gene product exhibited no E3 activity. These findings strongly suggested that the protein isolated by protocol I may have been misidentified as E3. A reinvestigation of the purification protocol produced a new and improved procedure (protocol II) consisting of DEAE-Sephacel, phenyl-Sepharose, Cibacron blue A, and Sephadex G-100 chromatography, which efficiently yielded a new homogeneous enzyme composed of a single polypeptide with a molecular weight of 39,000. This highly purified protein had a specific activity nearly 8,000-fold higher than that of cell lysates, and more importantly, the corresponding gene (ascD) was found to be part of the ascarylose biosynthetic cluster. Presented are the identification and confirmation of the E3 gene through cloning and overexpression and the culminating purification and unambiguous assignment of homogeneous E3. The nucleotide and translated amino acid sequences of the genuine E3 are also presented.
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PMID:CDP-6-deoxy-delta 3,4-glucoseen reductase from Yersinia pseudotuberculosis: enzyme purification and characterization of the cloned gene. 828 41

The brain has a high level of ascorbic acid which is thought to act as a reducing agent, e.g. in protecting tissues against oxidative stress. The mechanism by which ascorbate is maintained in the useful, reduced state in the CNS is evaluated herein. Cerebrum from rat or calf was minced and homogenized in buffer. The endogenous levels of ascorbic acid, dehydro-L-ascorbic acid (DHAA) and reduced glutathione (GSH) were determined by HPLC with coulometric electrochemical detection. We also quantitated tissue capacity to regenerate ascorbic acid from DHAA, which is a product of electron transfer reactions of ascorbic acid. The homogenate was fractionated by centrifugation in steps up to 110,000 x g and dialyzed free of low molecular weight components. The activity for reducing DHAA was approximately equal in the various supernatants; resuspended pellets had little activity. The active component has several properties of a protein, including being precipitated by solid ammonium sulfate addition to the tissue extract; most activity appeared in the 40-80% saturated fraction. The activity was stable up to a temperature of 80 degrees C, but was lost at 95 degrees C. The protein was digested by trypsin. The results suggest that a cytosolic component of cerebrum regenerates ascorbic acid in a step that preferentially uses GSH and NADPH as reducing cofactors. At least one form of DHAA reductase exists in brain.
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PMID:Cerebral metabolism of oxidized ascorbate. 831 69

We report the initial identification of a 37.5-kDa putative aldoketo reductase in human colon carcinoma cells. An aminoterminal trypsin fragment was sequenced and found to be identical to bovine prostaglandin F synthase in 19 of 21 amino acids. Levels of this cytosolic human aldo-keto reductase, assessed by immunoblots using polyclonal antibodies raised against this protein, increased 30-fold in cells resistant to the Michael reaction acceptor ethacrynic acid and increased with time and ethacrynic acid concentration after treatment of wild-type cells. Induction of the reductase appeared to be cell type and drug specific. It was induced by the Michael reaction acceptors dimethyl maleate, t-butylhydroquinone, and hydroquinone but not by the nitrogen mustard chlorambucil. Ethacrynic acid and dimethyl maleate induced the reductase in a second human colon cell line but not in human prostate cells. NADPH-dependent metabolism of aldoketo reductase substrates by cytosol from colon but not prostate cells was enhanced 2-3-fold when cells were grown in the presence of either ethacrynic acid or dimethyl maleate. The discrepancy between induced reductase activity and protein levels may be due to the multiplicity of constitutively expressed NADPH-dependent reductases that compete for substrate. Ethacrynic acid-resistant cells exhibited low levels of cross-resistance to Adriamycin, mitomycin C, and the bovine prostaglandin F synthase substrates phenylglyoxal and prostaglandin D2. Thus, significant overexpression of a human aldo-keto reductase structurally related to bovine prostaglandin F synthase may result from exposure of cells to Michael reaction acceptors and may give rise to an enhanced capacity to metabolize exogenous and endogenous substrates, thereby contributing to the drug-resistant phenotype.
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PMID:Overproduction of a 37.5-kDa cytosolic protein structurally related to prostaglandin F synthase in ethacrynic acid-resistant human colon cells. 831 17

Chemical modification of cytochrome P450 was used to study the involvement of lysine and arginine residues in the interaction between cytochrome P450 and NADPH-cytochrome P450 reductase. Acetylation of 2.2 and 8.5 mol of lysine/mole of P450 by acetic anhydride led to 38.7 and 95% reductions, respectively, in benzphetamine demethylation activity by NADPH-dependent reconstituted P450/reductase complex, while modification of up to 8.5 mol of lysine/mol of P450 did not inhibit cumene hydroperoxide-supported P450-dependent benzphetamine demethylation. Acetylation of lysine residues by acetic anhydride does not grossly disturb the P450 protein conformation as revealed by absolute, CO-difference and fluorescence spectral studies. Modification of P4502B1 by acetic anhydride did not affect its substrate binding ability either. Lysine residues of P4502B1 putatively involved in the interaction with reductase have been identified by radiolabeling of lysine residues with [14C]acetic anhydride followed by trypsin digestion, HPLC separation, and amino acid microsequencing. Radiolabeled lysines occur at positions 251, 384, 422, 433, and 473. Modification of arginine residues in P4502B1 with phenylglyoxal and 2,3-butanedione seemed to have no significant effect on the benzphetamine demethylation activity of P4502B1 either reconstituted with reductase and NADPH or supported by cumene hydroperoxide. Studies of incorporation of [14C]phenylglyoxal showed no concentration- or time-dependent incorporation of phenylglyoxal into the P4502B1. These results support the hypothesis of a predominant role of lysine residues of P450 in the electrostatic interaction with NADPH-cytochrome P450 reductase.
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PMID:Role of lysine and arginine residues of cytochrome P450 in the interaction between cytochrome P4502B1 and NADPH-cytochrome P450 reductase. 832 89

Aquacobalamin reductase (NADPH), which catalyzes the reduction of aquacobalamin to cob(II)alamin in the synthesis of cobalamin coenzymes, has already been purified from mitochondria of Euglena gracilis and partly characterized. Here, the enzyme was further characterized to clarify its enzymatic properties. The enzyme reduced 2 mol of aquacobalamin per mole of NADPH and had NADPH diaphorase-like activity. The 16 amino acid residues at the NH2-terminal of the enzyme were identical with those of the NADPH diaphorase domain of pyruvate: NADP+ oxidoreductase, which is involved in Euglena wax ester fermentation. Peptide mapping of the aquacobalamin reductase showed that elution during C-18 reversed-phase high-performance liquid chromatography was identical to that of the NADPH diaphorase domain. Immunoblotting indicated that the Euglena aquacobalamin reductase had a higher molecular weight (166,000) in the intact mitochondria than the purified enzyme (65,000), and that the molecular weights of the native and purified enzyme were identical with those of the subunit and the NADPH diaphorase domain, respectively. These results showed that the aquacobalamin reductase isolated earlier was the NADPH diaphorase domain, cleaved by trypsin during preparation of the mitochondrial homogenate from the native enzyme. Purified pyruvate:NADP+ oxidoreductase also had the activity of aquacobalamin reductase, which suggests that the enzyme in Euglena mitochondria has more than one function in the synthesis of cobalamin co-enzymes.
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PMID:Characterization of aquacobalamin reductase (NADPH) from Euglena gracilis. 837 79

Acetaldehyde, the first product of alcohol metabolism, is highly reactive. Several proteins have been shown to be covalently modified by acetaldehyde in vivo. We have previously reported the detection of a cytosolic 37-kd protein-acetaldehyde adduct (-AA) in the liver of alcohol-fed rats. The liver extract from an alcohol-fed rat was subjected to 2-dimensional (2D) sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride (PVDF) membrane, and the 37-kd protein-AA spot was digested with trypsin and sequenced for amino acids. Degenerate oligonucleotides corresponding to a peptide sequence of the protein-AA were used as the probe to screen a lambda gt11 rat liver complementary DNA (cDNA) library. A clone that extended to a potential ATG start codon was identified. The open reading frame was 978 nucleotides long, encoding 326 amino acid residues. The sequence matched that of rat liver delta 4-3-ketosteroid 5 beta-reductase. The cloned cDNA was expressed in Escherichia coli using pGEX-KG as the vector. The expressed protein was found to be of correct molecular weight. It reacted with an antibody that recognized the unmodified liver 37-kd protein by Western blotting. Peptide profiles of tryptic-digested recombinant protein and the purified rat liver 37-kd protein were similar and yielded the same peptide sequence. delta 4-3-ketosteroid 5 beta-reductase catalyzes the reduction of key intermediates during bile acid biosynthesis. Whether modification of the 5 beta-reductase by acetaldehyde affects the enzyme activity and bile acid synthesis remains to be studied.
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PMID:Identification of the 37-kd rat liver protein that forms an acetaldehyde adduct in vivo as delta 4-3-ketosteroid 5 beta-reductase. 855 30


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