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.
Cells of Bacillus megaterium GW1 and Escherichia coli W7-M5 were specifically radiolabeled with 2,2'-diamino[G-3H]pimelic acid ([3H]DAP) as models of gram-positive and gram-negative bacteria, respectively. Two experiments were conducted to study the in vivo metabolism of 2,2'-diaminopimelic acid (DAP) in sheep. In experiment 1, cells of [3H]DAP-labeled B. megaterium GW1 were infused into the rumen of one sheep and the radiolabel was traced within microbial samples, digesta, and the whole animal. Bacterially bound [3H]DAP was extensively metabolized, primarily (up to 70% after 8 h) via decarboxylation to [3H]lysine by both ruminal protozoa and ruminal bacteria. Recovery of infused radiolabel in urine and feces was low (42% after 96 h) and perhaps indicative of further metabolism by the host animal. In experiment 2, [3H]DAP-labeled B. megaterium GW1 was infused into the rumens of three sheep and [3H]DAP-labeled E. coli W7-M5 was infused into the rumen of another sheep. The radioactivity contents of these mutant bacteria were insufficient to use as tracers, but the metabolism of DAP was monitored in the total, free, and peptidyl forms. Free DAP, as a proportion of total DAP in duodenal digesta, varied from 0 to 9.5%, whereas peptidyl DAP accounted for 8.3 to 99.2%. These data reflect the extensive metabolism of bacterially bound DAP within the gastrointestinal tracts of ruminant animals and serve as a serious caution to the uncritical use of DAP as a marker of bacterial biomass in the digesta of these animals.
Protozoa and bacteria were isolated from the rumen of a sheep given a concentrate and hay diet and were incubated separately with either free 2,2'‐diaminopimelic acid (A 2 pm) as [G‐ 3 H]A 2 pm or bacterially‐bound A 2 pm in the form of [G‐ 3 H]A 2 pm‐labelled Bacillus megaterium GW1. Lysine was the only radiolabelled metabolite produced in pellet and supernatant fluid when ciliates were incubated with 0·1 mmol/l free [G‐ 3 H]A 2 pm; pipecolate was an additional product when 1·0 and 8·0 mmol/l A 2 pm were used. As well as incorporating A 2 pm, rumen bacteria decarboxylated it and produced a further three unidentified metabolites in the supernatant fluids. Protozoa rapidly engulfed and digested [G‐ 3 H]A 2 pm‐labelled B. megaterium GW1. Radioactive A 2 pm and lysine were present in the protozoal pellets. A 2 pm, lysine and three other radiolabelled compounds were excreted into the supernatant fluids; the major product, designated G, appeared to be a series of A 2 pm‐containing peptides and derivatives. When rumen bacterial cells were incubated with [G‐ 3 H]A 2 pm‐labelled B. megaterium GW1 they accumulated A 2 pm and lysine. Only A 2 pm and G were detected in bacterial supernatant fluids. The significance of these results to the use of A 2 pm as a marker of bacterial outflow from the rumen is assessed.
Abstract1. Intra-gastric administration of brotizolam (0.1–200 mg/kg) daily for three days to rats resulted in no significant changes in the hepatic and intestinal cytochrome P-450-dependent or P-448-dependent mixed-function oxidases, or in the hepatic flavoprotein dimethylaniline N-oxidase.2. Liver microsomes from mouse, rat and man metabolized brotizolam by hydroxylation of the diazepine ring and of the methyl group at rates which were greater for mouse > rat > man. Brotizolam and its metabolites generated by rat-liver microsomes in vitro were not mutagenic in the Ames' test.3. Brotizolam, at 200 mg/kg per day for two to six weeks, depleted liver glutathione concentration and markedly increased liver γ-glutamyl transpeptidase, glutathione reductase and glutathione transferase activities. Similar changes were not seen at the lower dose of 0.3 mg/kg.4. The observed increases in glutathione metabolism and the decreased tissue concentration of glutathione are indicative of high levels of glutathione conjugation, and provide a possible explanation for the equivocal increase in tumorigenicity seen in rats receiving brotizolam at high dosage.
The role of cytochromes P-450 and P-447 in the activation of 4-aminobiphenyl to mutagens in the Ames test was studied using S9 preparations and highly purified isozymes. S9 preparations from β-naphthoflavone-pretreated rats were more efficient in converting 4-aminobiphenyl to mutagens than the corresponding preparations from phenobarbitone-pretreated animals. Similarly, reconstituted systems comprising purified cytochrome P-447 were twice as efficient as cytochrome P-450 in activating the carcinogen. Of all the known Phase I metabolites of 4-aminobiphenyl, only the N-hydroxy-derivative was mutagenic in the Ames test. These findings indicate that arylamine N-hydroxylase is a cytochrome P-450 dependent enzyme, and the nature of the isozyme of the cytochrome is an important determinant of its mutagenicity.
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