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)

Highly selective chromatography of microsomal enzymes has been carried out on columns of immobilized cytochrome b5 that was obtained by detergent solubilization (d-b5) of the complete amphipathic molecule. Several partially purified isozymes of cytochrome P-450 are resolved on d-b5 columns, and one high-affinity isozyme has been readily purified to homogeneity. Chromatographic selectivity and correlation of elution order of isozymes of cytochrome P-450 with direct spectral measurements of affinity constants suggests affinity chromatography on d-b5 columns. Substantial one-step enrichments of NADH-cytochrome-b5 reductase and an unstable cytochrome b5-dependent oxidase of cholesterol synthesis, 4-methyl sterol oxidase, have been obtained on d-b5 columns which further supports this conclusion. Comparison of chromatographic behavior on columns of immobilized cytochrome b5 that was obtained by trypsin solubilization (t-b5) with d-b5 columns shows marked differences which must be attributed to the absence of the hydrophobic domain of the t-b5 molecule. NADH-cytochrome-b5 reductase and the high affinity isozyme of cytochrome P-450 purified by d-b5 affinity chromatography are poorly retained on t-b5 columns. A different cytochrome P-450 isozyme with lower affinity for cytochrome b5 is only retained on d-b5 columns. Cytochrome-P-450 reductase is not retained on either column. Because affinity chromatography is suggested on d-b5 columns, the procedure may be generally applicable for predicting protein-protein interactions of microsomal electron transport components that either donate electrons to, or receive electrons from, cytochrome b5. In addition, the procedure should have considerable utilitarian application for enzyme enrichment.
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PMID:Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5. 394 90

The complete covalent structure of a cytochrome P-450, form 4, isolated from liver microsomes of beta-naphthoflavone-induced rabbits was determined. The S-carboxyamidomethylated protein was cleaved with cyanogen bromide, endoproteinase Lys-C, and trypsin before and after succinylation. Selected peptides from CNBr digests of alkylated rabbit cytochrome P-450 forms 3a and 3c were also isolated and sequenced. Form 4 exhibited microheterogeneity due to the presence of several truncated forms. The existence of multiple NH2-terminal residues for form 4 was confirmed by the isolation and sequence analysis of the corresponding tryptic peptides. The predominant form contained 514 residues, corresponding to Mr 58,030. A peptide having Gly-232 and Gln-246 replaced by Ser and Asn residues, respectively, was also found in the isozyme preparation investigated here. The amino acid sequences of form 4 and selected peptide sequences from forms 3a and 3c were compared with the primary structures of forms 2 and 3b (previously determined in this laboratory). This comparison identified some 90 invariant residues. A cysteinyl residue at position 456, earlier reported as the heme-binding cysteine 436 (Heineman, F. S., and Ozols, J. (1982) J. Biol. Chem. 257, 14988-14999), was also present in forms 4, 3a, and 3c. Other single invariant residues identified were form 4/forms 2,3b, Trp-132/121, and His 270/252. The tyrosyl residues at positions 71/62 and 365/348 were also invariant. The latter is present in the "conserved segment" of the protein, residues 363/346 to 375/359, and may be involved in the substrate binding of cytochrome P-450. Also a lysyl residue, formerly identified by other laboratories to be involved in the electron transfer between the reductase and cytochrome P-450 form 2, was invariant in all five species. This lysyl residue corresponds to Lys-402 in form 4 or Lys-384 in the other forms.
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PMID:Complete amino acid sequence of a cytochrome P-450 isolated from beta-naphthoflavone-induced rabbit liver microsomes. Comparison with phenobarbital-induced and constitutive isozymes and identification of invariant residues. 394 97

The possible interaction of two haloalkanes - bromotrichloromethane and 1,2-dibromo-1,2-dichlorethane - with stearate desaturase was assessed in hepatic microsomes from rats fed a high carbohydrate diet which elevates the levels of stearate desaturase. Both compounds shifted the redox steady state of NADPH reduced hepatic microsomal cytochrome b-5 towards ferricytochrome b-5 and enhanced the re-oxidation of NADH reduced hepatic microsomal cytochrome b-5. The equilibrium constants for the enhancement of microsomal electron transfer by the haloalkanes in these preparations were 2.2 +/- 0.3 mM and 0.46 +/- 0.1 mM for bromotrichloromethane and 1,2-dibromo-1,2-dichlorethane, respectively. The haloalkane mediated enhancement of the oxidation of cytochrome b-5 in hepatic microsomes from rats fed a high carbohydrate diet was diminished by KCN and the inhibitors of cytochrome P-450, CO and/or metyrapone, as well as by fasting of the experimental animals. The I50 values for KCN inhibition of the effects of the haloalkanes on the re-oxidation of cytochrome b-5 (01 mM) were identical to the I50 for KCN inhibition of stearate desaturase (Oshino et al., 1966). The haloalkanes did not affect the activity of hepatic microsomal NADH- or NADPH-cytochrome c reductase, the autoxidation of purified trypsin-cleaved ferrocytochrome b-5 or the conversion of stearoyl CoA to oleate. It is concluded that bromotrichloromethane and 1,2-dibromo-1,2-dichloroethane stimulate hepatic microsomal electron transfer from NADH via cytochrome b-5 by interacting with cytochrome P-450 and with stearate desaturase.
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PMID:Influence of two haloalkanes on the redox behavior of hepatic microsomal cytochrome b-5 and its possible relationship to stearate desaturase. 611 52

Electron transfer to rat liver microsomal cytochrome P-450 of 14 alpha-methyl group demethylation of 24,25-dihydrolanosterol (C30-sterol) has been studied with a new radio-high-performance liquid chromatography assay. The monooxygenase is dependent upon NADPH plus oxygen, insensitive to CN-, and sensitive to CO. Microsomal oxidation is also sensitive to trypsin digestion, and reactivation is dependent upon the addition of purified, detergent-solubilized cytochrome P-450 reductase. Electron transport of C-32 sterol demethylation can be fully supported by very low concentrations of NADPH (approximately 10 microM) only in the presence of saturating concentrations of NADH (approximately 200 microM) suggesting involvement of cytochrome b5-dependent electron transfer in addition to the NADPH-supported pathway. The cytochrome P-450 of 14 alpha-demethylation has been solubilized with detergents, resolved chromatographically from cytochrome P-450 reductase and cytochrome b5, and fully active C-32 demethylase reconstituted. Incubation of intact microsomes with NADH and very low concentrations of NADPH described above leads to interruption of demethylation without 14 alpha-methyl group elimination. Under these conditions, C-32 oxidation products of the C30-sterol substrate accumulate at the expense of formation of demethylated, C29-sterol products. This enzymic interruption of C-32 demethylation, accumulation of oxygenated C30-sterols, along with subsequent demethylation of the isolated C30-oxysterols under similar oxidative conditions supports the suggestion that 14 alpha-hydroxymethyl and aldehydic sterols are metabolic intermediates of sterol 14 alpha-demethylation. Only very modest inductions of the constitutive cytochrome P-450 isozyme of 14 alpha-methyl sterol oxidase can be obtained with just 2 out of 12 known, potent inducers of mammalian hepatic cytochrome P-450s. Alternatively, administration of complete adjuvant in mineral oil drastically reduces amounts of total microsomal cytochrome P-450 while activity of 14 alpha-methyl sterol oxidase is not affected dramatically. Thus, as much as 2.5-fold enhancement of C-32 oxidase specific activity is obtained when expressed per unit of cytochrome P-450.
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PMID:Cytochrome P-450-dependent oxidation of lanosterol in cholesterol biosynthesis. Microsomal electron transport and C-32 demethylation. 620 95

The alpha-oxidation of [1-14C]phytanic acid of high specific activity was studied in postnuclear and various subcellular fractions from rat liver. alpha-Oxidation in the postnuclear fraction required ATP, Mg2+, nicotinamide, and molecular oxygen for activity. alpha-Oxidation was inhibited by iron-specific chelating agents and respiratory chain inhibitors. Partial inhibition by carbon monoxide indicated a possible involvement of cytochrome P-450. However, phenobarbital-treated rat liver postnuclear fraction did not stimulate phytanic acid alpha-oxidation above that of control. Subcellular fractionation indicated that in addition to the mitochondrial fraction, cytosol was required for activity. The cytosolic factor appeared to be dialyzable; it was inactivated by heat treatment, but not affected by trypsin digestion. NAD, CoA, ascorbic acid, and catalase did not replace cytosolic activity nor did the recently characterized heat-stable factors in brain hydroxylation, namely, adenosine nucleotides and glutamate.
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PMID:Phytanic acid alpha-oxidation in rat liver. Requirement of cytosolic factor. 623 33

1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH-cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH-cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH-cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH-cytochrome P-450 reductase and cytosolically orientated membrane factor(s).
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PMID:Asymmetric distribution of cytochrome P-450 and NADPH--cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver. 625 76

Rabbit lung and liver microsomes were subjected to three procedures which decreased NADPH cytochrome c reductase activity; flavoprotein antibody, trypsin and subtilisin digestion. The effects on benzphetamine and p-nitroanisole demethylation and amine metabolic-intermediate complex formation were investigated. In general, the proteolytic digestion had a greater inhibitory effect on oxidation reactions for a given loss of NADPH cytochrome c reductase activity than did flavoprotein antibody; and of the two proteases, subtilisin, which also diminishes the cytochrome b5 reduction pathway, had a greater inhibitory effect than trypsin. Subtilisin digestion had similar effects in both liver and lung microsomes; a loss of flavoprotein without a loss of cytochrome P-450; but whereas all three oxidative reactions decreased in unison as the flavoprotein was lost in the liver, benzphetamine demethylation was less susceptible to flavoprotein depletion than the other two reactions in lung microsomes. With trypsin digestion flavoprotein was removed without loss of cytochrome P-450 only in lung microsomes; in liver microsomes the cytochrome P-450 was susceptible to tryptic degradation. In lung microsomes, benzphetamine and p-nitroanisole demethylations were less susceptible to flavoprotein loss than metabolic-intermediate complex formation.
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PMID:The influence of in vitro procedures which diminish NADPH cytochrome c reductase activity on oxidative drug metabolism in lung and liver microsomes. 641 23

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

Highly specific antibodies against hemeprotein were obtained by immunizing rabbits with a highly purified cholesterol-hydroxylating cytochrome P-450scc from adrenocortical mitochondria. The antibodies do not specifically interact with other components of the adrenocortical electron transport chain, e. g., adrenodoxin reductase and adrenodoxin. Using double immunodiffusion technique (Ouchterlony method), it was shown that the antibodies did not precipitate the microsomal cytochromes P-450 LM2 and LM4, cytochrome b5 and 11 beta-hydroxylating cytochrome P-450 from adrenocortical mitochondria. Antibodies against cytochrome P-450scc inhibited the cholesterol side chain cleavage activity of cytochrome P-450scc in a reconstituted system. Limited proteolysis with trypsin and immunoelectrophoresis in the presence of specific antibodies revealed that antigenic determinants are present of the heme-containing catalytic domain of cytochrome P-450scc (F1) as well as on the domain responsible for the interaction with the phospholipid membrane (F2).
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PMID:[Immunochemical characteristics of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria]. 644 2

The degradation of cytochrome P-450 as a result of proteolytic action of trypsin is a biphasic process. Lipid peroxidation (LPO) increases the rate of the fast phase of cytochrome P-450 degradation and its accessibility to protease. The efficiency of this process depends on the mode of LPO induction and decreases in the following order: NADPH----NADH----ascorbate-dependent LPO. The induction of the monooxygenase system increases the efficiency of proteolysis. LPO and proteolysis seem to be mutually enhancing processes which provide for a high efficiency of cytochrome P-450 degradation. LPO can be regarded as a triggering mechanism which makes various forms of cytochrome P-450 accessible to endogenous proteases.
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PMID:Mechanisms of disassembly of a mixed function oxygenase system in liver endoplasmic reticulum: II. The interrelation between lipid peroxidation and proteolytic degradation of cytochrome P-450. 648 26


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