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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cytochrome P-450 (P-450SG1) was purified from a lanosterol 14 alpha-demethylase (P-450(14DM)) defective mutant of Saccharomyces cerevisiae, strain SG1, by a method similar to that used in the purification of the wild type enzyme (Yoshida, Y., and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660). P-450SG1 had the same apparent Mr as and was immunochemically identical to P-450(14DM). Peptide maps of P-450SG1 made by limited proteolysis with Staphylococcus aureus V8 proteinase, chymotrypsin, or papain followed by gel electrophoresis were identical to corresponding peptide maps of P-450(14DM). However, P-450SG1 showed no lanosterol 14 alpha-demethylase activity and its mode of interaction with diniconazole [(E)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-y1)-1- penten-3- o1], a specific inhibitor of P-450(14DM), was fundamentally different from that of P-450(14DM). The absorption spectrum of ferric P-450SG1 was unusual for a native low-spin cytochrome P-450 and was superimposable on that of 1-methylimidazole complex of P-450(14DM), indicating that P-450SG1 has a histidine 6th ligand trans to the thiolate 5th ligand, while the 6th ligand of other ferric low-spin cytochrome P-450s is a water molecule or a hydroxyl group of an oxyamino acid. It is concluded that P-450SG1 is an altered P-450(14DM). Difference in the primary structure between P-450SG1 and P-450(14DM) may be slight and was not detected by peptide mapping. However, the alteration caused significant change in the substrate site and heme environments of the cytochrome. P-450SG1 is the first example of a cytochrome P-450 having a histidine axial ligand trans to thiolate and of a genetically altered cytochrome P-450 isolated in a homogeneous state.
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PMID:Isolation and characterization of an altered cytochrome P-450 from a yeast mutant defective in lanosterol 14 alpha-demethylation. 311 90

A thermolytic hydrolysis of maleinated fragment F1 has been performed, resulted in isolation of 44 peptides; their complete amino acid sequence has been determined. Non-overlapping thermolytic peptides of fragment F1 involve 178 amino acid residues, which comprises about 71% of its amino acid sequence. Also, the cleavage and structural investigation of some tryptophan-containing peptides obtained from the limited trypsinolysis of fragment F1 were carried out; reconstitution of the polypeptide chain of the fragment is completed. The cyanogen bromide cleavage of carboxymethylated cytochrome P-450 was achieved and 17 peptides, comprising almost the whole polypeptide chain of the protein molecule (91%), was isolated. To investigate structure of the cyanogen bromide peptides, we hydrolysed them at tryptophan residues with trypsin, chymotrypsin, proteinase from Staphylococcus aureus, and BNPS-skatole. The data obtained and those published earlier led to the complete primary structure of the cholesterol-hydroxylating cytochrome P-450. The proteins polypeptide chain consists of 481 amino acid residues and has the precise molecular mass 56 407.7.
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PMID:[Primary structure of 20S,22R-cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria. IV. Structure of peptides of thermolytic and limited tryptic hydrolysis of the fragment F1; peptides of cyanogen bromide hydrolysis of cytochrome P-450. Complete amino acid sequence]. 390 59

In the present studies, a novel form of highly purified cytochrome P-450 (cytochrome P-452) isolated from the hepatic microsomes of clofibrate-pretreated rats has been compared to the major isozymes isolated from the hepatic microsomes of rats pretreated with phenobarbital (cytochrome P-450) and 2-naphthoflavone (cytochrome P-447) using a number of biochemical criteria. The results show that these three isozymes exhibit marked structural differences from each other as judged by a complete lack of immunochemical cross-reactivity between the isozymes and the heterologous rabbit serum antibodies using Ouchterlony double diffusion, and non-identity between the limited proteolytic digestion maps of the three isozymes obtained in the presence of chymotrypsin, papain and Staphylococcus aureus V8 proteases. Furthermore, the three isozymes exhibited clear differences in their monomeric molecular weights determined on calibrated sodium dodecyl sulphate/polyacrylamide gel electrophoresis in gels of varying acrylamide concentration. Substantial differences were also observed in the substrate specificities of the isozymes, which were reflected in differences in the turnover rates and positional selectivities of the hemoproteins for some model substrates. In addition, the isozymes differed in their substrate binding affinities and their ability to interact with purified hepatic microsomal cytochrome b5, as judged using difference spectrophotometry. Finally, subtle differences were detected in the ultraviolet visible absorbance spectra of the hemoproteins in the ferric, ferrous, and carbonmonoxyferrous states. Taken collectively, the above data provides compelling evidence that fundamental differences exist between these cytochrome P-450 isozymes, further establishing the uniqueness of the major form of cytochrome P-450 induced by clofibrate pretreatment.
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PMID:Multiple forms of hepatic cytochrome P-450. Purification, characterisation and comparison of a novel clofibrate-induced isozyme with other major forms of cytochrome P-450. 669 12

Rats pretreated with xylene or phenobarbital, and then exposed to n-hexane, exhibited a markedly increased peak serum concentration of the neurotoxic metabolite 2,5-hexanedione. In order to elucidate the mechanism underlying this synergistic effect, the major liver microsomal cytochrome P-450 isozymes induced by xylene and phenobarbital, respectively, were purified. In a reconstituted system both isozymes showed a high enzymatic activity with n-hexane as the substrate. Turnover numbers for the formation of 2-hexanol were 24 and 27 for the xylene- and phenobarbital-induced isozyme, respectively. The turnover numbers for 7-ethoxycoumarin, benzo[a]pyrene, and 1,1,2,2-tetrachloroethane were also in the same range for the two cytochrome P-450 preparations. The isozyme induced by xylene had an amino acid composition very similar to that of the phenobarbital-induced isozyme, and the purified proteins had identical electrophoretic mobilities on polyacrylamide gels in the presence of sodium dodecyl sulfate. Furthermore, similar peptide maps were obtained following digestion with alpha-chymotrypsin and papain, and each isozyme yielded a single immunoprecipitin band upon reaction with the immunoglobulin G fraction from rabbits immunized with the phenobarbital-induced enzyme. We conclude that xylene induces a rat liver microsomal cytochrome P-450 isozyme very similar to the major isozyme induced by phenobarbital and that this induction is the probable explanation for the enhanced formation of 2,5-hexanedione from n-hexane in vivo.
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PMID:Xylene induces a cytochrome P-450 isozyme in rat liver similar to the major isozyme induced by phenobarbital. 686 1

A new isozyme of cytochrome P-450 has been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated chronically with ethanol. Several criteria indicate that the ethanol-inducible cytochrome, which has a minimal molecular weight of 51,000 and is designated form 3a on the basis of its relative electrophoretic mobility, is distinct from the known isozymes of P-450. As judged spectrally, the new isozyme is high spin in the oxidized state, as is form 4, but differs in that the spin state is unperturbed by nonionic detergents. The absolute spectrum of the ferrous carbonyl complex of form 3a is red shifted as compared to that of forms 2, 3b, 3c, 4, and 6 and exhibits a maximum at 452 nm. The amino acid composition of form 3a is different from that of the other isozymes, and both the NH2- and COOH-terminal sequences are distinct; form 3a has an NH2-terminal alanine and a carboxyl-terminal leucine residue. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following treatment with papain, chymotrypsin, or Staphylococcus aureus V8 protease and by high performance liquid chromatography following trypsinolysis indicates that form 3a is a unique gene product. This cytochrome displays the highest activity of all of the rabbit isozymes in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline when reconstituted with NADPH-cytochrome P-450 reductase and phospholipid in the presence of NADPH and oxygen.
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PMID:Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits. 708 77

The effects of trypsin, phospholipase A, and chymotrypsin on NADPH-cytochrome c reductase and cytochrome P-450 of microsomes from cryptorchid mouse testes and liver were compared. Trypsin released both enzymes almost completely from testis microsomes, while it readily released only NADPH-cytochrome c reductase from liver microsomes. Chymotrypsin alone, even under conditions where 30-40% of the microsomal protein was hydrolyzed, had little effect on localization or activity of either enzyme in either tissue. Phospholipase A destroyed cytochrome P-450 in testicular microsomes but had little effect on this enzyme in hepatic microsomes or on NADPH-cytochrome c reductase in either preparation. When, however, the microsomes were incubated with chymotrypsin in the presence of a detergent, the effects were similar to those of trypsin alone; testicular cytochrome P-450 was destroyed, while hepatic cytochrome P-450 was only slightly solubilized, and NADPH-cytochrome c reductase from both types of microsomes was both solubilized and activated. From these results we conclude that arginyl and/or lysyl bonds may play a significant role in the junction between the hydrophobic region of the membrane and the anchor region of the reductase molecule and that cytochrome P-450 of testicular microsomes is more superficially located in the lipid bilayer than is hepatic microsomal cytochrome P-450.
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PMID:The environment of cytochrome P-450 in testicular microsomes. 720 24

Two forms of rabbit pulmonary cytochrome P-450, P-450I and P-450II, are distinguished by unique peptides observed upon electrophoresis of the products of limited proteolysis (with papain or chymotrypsin) in the absence or presence of sodium dodecyl sulfate. In contrast, the peptides from the proteolytic digestions of pulmonary cytochrome P-450I are indistinguishable from those of the major form of hepatic cytochrome P-450 induced by phenobarbital (P-450PB). Electrophoresis of the fragments of P-450, and P-450II produced by treatment with cyanogen bromide also shows different peptides, whereas the peptides produced from P-450I and P-450PB are the same. The amino acid composition and P-450II is different from that of P-450I or P-450PB. P-450II is distinguished further from the other two cytochromes on the basis of amino acid composition and subunit molecular weight (either as calculated from the amino acid composition or as estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis). The sequence of the first five residues at the NH2-terminal segment of P-450I and P-450PB are identical, while that of P-450II is different.
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PMID:The rabbit pulmonary monooxygenase system. partial structural characterization of the cytochrome P-450 components and comparison to the hepatic cytochrome P-450. 746 52

Noninbred male guinea-pigs (b. mass 250-300 g) were kept for 35 days on diets in which 50% of standard proteins were replaced for cow milk protein (CMP) 20/80 and its enzymatic hydrolyzate (EH) 20/80 obtained by ultrafiltration. CMP 20/80 and EH 20/80 have been examined for the effect on proteolytic activity of gastric mucosa, small intestinal activity of trypsin and chymotrypsin, hepatic cytochrome P-450 and metabolism of excreted 17-OCS. The animals given EH 20/80 exhibited enhanced total proteolytic activity of trypsin and chymotrypsin, higher total levels of cytochromes P-450 and P-450B in the liver, increased urine excretion of 17-OCS, reduced amount of polar forms in all the glucocorticoid fractions. Potential mechanisms securing the action of enzymatic hydrolyzates on the systems responsible for nonspecific resistance to food allergens are suggested for discussion.
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PMID:[Effect of milk proteins and their enzymatic hydrolyzates on non-specific resistance of some systems to food allergens]. 797 5

Young adult Sprague-Dawley rats were partially hepatectomized (two-thirds organ removal) and administered a basal diet supplemented with various animal- and plant-derived enzymes (trypsin, alpha-chymotrypsin, pepsin, lipase, alpha-amylase, malt diastase, ficin and bromelain) over a post-operative period of up to 10 days. Porcine or bovine dialyzed and lyophilized crystalline trypsin products containing 2400-3200 NF u/mg in addition to enteric-coated tablets with trypsin to chymotrypsin in a ratio of 6:1, were tested at supplementary levels of up to 4980 u/g ration. With the weight of tissue regenerated or the liver increment as indicator, trypsin in excess of 1000-1200 u/g ration proved inhibitory. This effect did not extend to alpha-chymotrypsin (levels of up to 4000 u/g diet) and the remaining 6 enzyme products specified above, nor to the s.c. injection of trypsin daily at 12,860 u/rat for the 1st 7 days. The last route promoted little change in increment with soy bean trypsin inhibitor (8.0 mg/rat daily for days 1 to 9). When a portion of the group fed a trypsin supplement of 2000 u/g was injected with phenobarbital i.p. at 80 mg/kg daily on each of the last 3 days, the resulting liver increment rose to the control range. As with lysine and arginine, acids of pertinence in tryptic proteolysis, no significant change was elicited by feeding a diet supplemented with peptone from tryptic digestion of casein. The enzyme-containing diets fed to sham-operated rats over a similar interval, did not affect the wet- or dry-liver weight per 100 g body weight. Microsomal parameters as total protein, cytochrome P-450 and the enzymes, aminopyrine demethylase and benzo[a]pyrene hydroxylase of livers from the partially hepatectomized or sham-operated rats fed trypsin and the other enzyme diets, presented no significant changes in the respective levels. The possible action of dietary trypsin in conjunction with inhibitors and growth factors controlling liver regeneration is discussed.
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PMID:Liver regeneration in trypsin-fed partially hepatectomized rats. 843 34

In rat kidney, beta-naphthoflavone induced 53 kDa and 55 kDa proteins, which were both recognized by the antibodies against rat liver cytochrome P-450 1A1 (55kDa). The major inducible 53 kDa protein was purified from the beta naphthoflavone-treated rat kidney and shown to be a new cytochrome P-450 having a high aryl hydrocarbon hydroxylase activity. Purified cytochrome P-450, named P-450KAh, was homogeneous on SDS-polyacrylamide gel electrophoresis, and the apparent molecular weight was estimated to be 53 kDa. The absorption spectra of the oxidized form of P-450KAh showed a Soret peak at 416 nm, a characteristic of low-spin hemoprotein, and the Soret peak of the reduced cytochrome P-450-CO complex was at 446 nm. In the reconstituted system, purified P-450KAh showed high catalytic activity for benzo[a]pyrene hydroxylation and 7-ethoxycoumarin O-deethylation. P-450KAh could activate genotoxicities of not only B[a]P, but also 2-acetylaminofluorene and aflatoxin B1 on the umu test. These catalytic properties of P-450KAh were almost the same as those of P-4501A1, a major P-450 form having arylhydrocarbon hydroxylase in liver microsomes of 3-methylcholanthrene-treated rats, and P-450KAh could not be distinguished from P-4501A1 even by immunochemical analysis. However, the electrophoretic peptide patterns after alpha-chymotrypsin or trypsin treatment of P-450KAh were different from those of P-4501A1, and the NH2-terminal 11 amino acid sequence of the P-450 was also different from that of P-4501A1 and any other P-450s of rat.
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PMID:Purification and properties of a new beta-naphthoflavone inducible cytochrome P-450, aryl hydrocarbon hydroxylase from rat kidney. 860 21


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