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)

1. Human and murine liver cytosolic epoxide hydrolase (CEH) had an apparent Mw of 59,000 by SDS-PAGE. 2. Peptide maps of CNBr, trypsin and Staphylococcus aureus V8 digests, as well as amino acid analysis, showed that human and murine CEH were similar. Uninduced and clofibrate induced murine CEH appeared qualitatively identical. 3. The CEHs shared antigenic determinants as determined by Western blotting. 4. Circular dichroism spectra indicate that human CEH had 39% alpha-helix. Uninduced and clofibrate induced murine CEH had 38 and 35% alpha-helix, respectively.
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PMID:Human and murine cytosolic epoxide hydrolase: physical and structural properties. 234 24

Cytosolic epoxide hydrolases purified from livers of control and clofibrate-induced male C57B1/6 mice were compared. The proteins were reduced, alkylated and cleaved with trypsin and chymotrypsin. The digests were analyzed by HPLC and no qualitative differences were observed in the peptide mapping profiles of the two types of epoxide hydrolase preparation. The amino acid compositions and N-terminal residues of selected tryptic peptides also gave identical results for the control and clofibrate-induced mice. Both intact proteins have alpha-amino-blocked N-termini. The two enzyme forms are concluded to have highly similar, if not identical, primary structures.
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PMID:Cytosolic epoxide hydrolase from liver of control and clofibrate-treated mice. Structural comparison by HPLC peptide mapping. 344 28

Two forms of cytochrome P-450 have been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated with imidazole. Several criteria indicate that the cytochrome of higher electrophoretic mobility is identical with ethanol-inducible isozyme 3a. "Imidazole-3a" and "ethanol-3a" exhibit the same chromatographic characteristics and have identical electrophoretic mobilities upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, the two protein preparations have the same absorbance maxima and absorption coefficients in the oxidized, reduced, and reduced-CO states. A single immunoprecipitin band exhibiting complete identity was observed upon reaction of imidazole-3a and ethanol-3a with the immunoglobulin G fraction from sheep immunized with the latter protein. The amino acid composition and first 10 residues of the amino terminus of the two protein preparations are indistinguishable, as are the high-performance liquid chromatographic maps of the peptides obtained upon cleavage with trypsin, Staphylococcus aureus V8 protease, or Lys C endoproteinase . Furthermore, these preparations have very similar activities in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline. Evidence was obtained that the cytochrome of lower electrophoretic mobility isolated from imidazole-treated rabbits is probably identical with isozyme 6; the spectral characteristics, amino acid composition, and carboxyl-terminal sequence are described. As an inducer, imidazole has the advantage over ethanol of being less variable in its effects and requiring a shorter period of treatment. From the resulting liver microsomes, one can readily isolate, in addition to P-450 isozymes 3a and 6, isozymes 3c and 4 as well as epoxide hydrolase.
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PMID:Purification of liver microsomal cytochrome P-450 isozymes 3a and 6 from imidazole-treated rabbits. Evidence for the identity of isozyme 3a with the form obtained by ethanol treatment. 642 1

The environmental influence of various drugs on the epoxide hydrolase with styrene oxide (EHSO) or benzo(a)pyrene-4,5-oxide (EHBPox) as substrate and the aryl hydrocarbon hydroxylase (AHH) activity was studied in monolayer cultures of human fetal hepatocytes (HFH) obtained at legal abortions. Hepatocytes were isolated by trypsin treatment of liver fragments and primary HFH cultures were maintained in Eagle's minimum essential medium supplemented with 15% newborn calf serum. The HFH were plated on culture dishes and allowed to 'settle' for one day before adding various drugs (in 1 microliter dimethylsulfoxide/ml) or solvent only and assay 1-2 days later. The basal AHH activity [assayed with 3H-benzo(a)pyrene as substrate] varied between 2 and 8.4 pmoles/min/mg protein and the basal EHSO activity was 0.3-4.9 nmoles/min/mg protein (n = 6) after one or two days' culture. The corresponding activity of EHBPox was 0.23-1.48 nmoles/min/mg protein (n = 5). Exposure of cultures to 2 mM phenobarbital (Pb), 2.5-25.0 microM benzanthracene (BA), 0.1 mM trans-stilbene oxide (TSO), or 5 microM beta-naphtoflavone (beta NF) resulted in a 1.2-3.7-fold induction of EHSO. Induction of EHBPox was also observed with Pb, beta NF, BA and TSO as inducers. Pb gave a dose-dependent induction of both EH at 0.1, 1.0 and 2.0 mM. Our results demonstrate that EH and AHH activities in HFH cultures are inducible by classical in vivo inducers. Although difficult to prove, it is plausible that such induction takes place also in intrauterine life.
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PMID:Human fetal liver cultures: basal activities and inducibility of epoxide hydrolases and aryl hydrocarbon hydroxylase. 653 14

In order to investigate the involvement of amino acids in the catalytic mechanism of the soluble epoxide hydrolase, different mutants of the murine enzyme were produced using the baculovirus expression system. Our results are consistent with the involvement of Asp-333 and His-523 in a catalytic mechanism similar to that of other alpha/beta hydrolase fold enzymes. Mutation of His-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme. When His-332 was replaced by asparagine, 96.7% of the specific activity was lost and mutation of the conserved His-523 to glutamine led to a more dramatic loss of 99.9% of the specific activity. No activity was detectable after the replacement of Asp-333 by serine. However, more than 20% of the wild-type activity was retained in an Asp-333-->Asn mutant produced in Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and the Asp-333-->Asn mutant enzymes produced in Trichoplusia ni cells. We labeled these enzymes by incubating them with the epoxide containing radiolabeled substrate juvenile hormone III (JH III). The purified Asp-333-->Asn mutant bound 6% of the substrate compared to the wild-type soluble epoxide hydrolase. The mutant also showed 8% of the specific activity of the wild-type. Preincubation of the purified Asp-333-->Asn mutant at 37 degrees C (pH 8), however, led to a complete recovery of activity and to a change of isoelectric point (pI), both of which are consistent with hydrolysis of Asn-333 to aspartic acid. This intramolecular hydrolysis of asparagine to aspartic acid may explain the activity observed in this mutant. Wild-type enzyme that had been radiolabeled with the substrate was digested with trypsin. Using reverse phase-high pressure liquid chromatography, we isolated four radiolabeled peptides of similar polarity. These peptides were not radiolabeled if the enzyme was preincubated with a selective competitive inhibitor of soluble epoxide hydrolase 4-fluorochalcone oxide. This strongly suggested that these peptides contained a catalytic amino acid. Each peptide was characterized with N-terminal amino acid sequencing and electrospray mass spectrometry. All four radiolabeled peptides contained overlapping sequences. The only aspartic acid present in all four peptides and conserved in all epoxide hydrolases was Asp-333. These peptides resulted from cleavage at different trypsin sites and the mass of each was consistent with the covalent linkage of Asp-333 to the substrate.
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PMID:Molecular and biochemical evidence for the involvement of the Asp-333-His-523 pair in the catalytic mechanism of soluble epoxide hydrolase. 771 95

Purification of a novel enantioselective epoxide hydrolase from Aspergillus niger M200 has been achieved using ammonium sulphate precipitation, ionic exchange, hydrophobic interaction, and size-exclusion chromatography, in conjunction with two additional chromatographic steps employing hydroxylapatite, and Mimetic Green. The enzyme was purified 186-fold with a yield of 15%. The apparent molecular mass of the enzyme was determined to be 77 kDa under native conditions and 40 kDa under denaturing conditions, implying a dimeric structure of the native enzyme. The isoelectric point of the enzyme was estimated to be 4.0 by isoelectric focusing electrophoresis. The enzyme has a broad substrate specificity with highest specificities towards tert-butyl glycidyl ether, para-nitrostyrene oxide, benzyl glycidyl ether, and styrene oxide. Enantiomeric ratios of 30 to more than 100 were determined for the hydrolysis reactions of 4 epoxidic substrates using the purified enzyme at a reaction temperature of 10 degrees C. Product inhibition studies suggest that the enzyme is able to differentiate to a high degree between the (R)-diol and (S)-diol product of the hydrolysis reaction with tert-butyl glycidyl ether as the substrate. The highest activity of the enzyme was at 42 degrees C and a pH of 6.8. Six peptide sequences, which were obtained by cleavage of the purified enzyme with trypsin and mass spectrometry analysis of the tryptic peptides, show high similarity with corresponding sequences originated from the epoxide hydrolase from Aspergillus niger LCP 521.
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PMID:Purification and characterisation of a novel enantioselective epoxide hydrolase from Aspergillus niger M200. 1634 76