1. As previously reported (Yang and Davis 1992), N-methylcarbazole (NMC) is converted to N-hydroxymethylcarbazole (NHMC), and 3-hydroxy-N-hydroxymethylcarbazole (3-OH-NHMC), two relatively stable carbinolamine metabolites by the fungus Cunninghamella echinulata (ATCC 9244). Decomposition of these two carbinolamines yields the corresponding dealkylated metabolites, carbazole and 3-hydroxycarbazole. In the present study, the possible involvement of cytochrome P450 in the requisite N-alkyl hydroxylation reaction was examined. 2. Carbon monoxide, a classical P450 inhibitor, markedly inhibited the formation of NHMC, as did potassium cyanide. 1-Benzylimidazole, piperonyl butoxide and SKF-525A inhibited the formation of both NHMC and 3-OH-NHMC, while beta-naphthoflavone (5,6-benzoflavone) induced their formation. 3. The source of the oxygen atom in the metabolite NHMC was examined by GC/MS analysis of NHMC formed during incubation of NMC in H218O-enriched medium which resulted in no incorporation of labelled oxygen into the metabolite. 4. An intermolecular isotope effect was not observed for the formation of NHMC suggesting that C-H bond cleavage is not a rate limiting step in the formation of this metabolite under the conditions examined. 5. It was concluded that P450 enzymes may be involved in the N-demethylation of NMC catalyzed by this fungal model of mammalian metabolism, and provides further support for biochemical and mechanistic parallels between mammalian metabolism and microbial systems catalyzing phase-1 biotransformations.

The presence of membrane-associated proteins which stereospecifically bind cyclosporin A and react with anti-cyclophilin antibodies has been documented in rat tissues. Extraction of membranes with 6 M urea or 0.5% Chaps releases cyclosporin-binding activity that is 5 -12% of that found in cytosol. Cyclosporin-A-binding proteins are present in most subcellular organelles of liver, but microsomes contain the greatest activity. These proteins can be purified by adsorption onto a cyclosporin-A affinity column and elution with cyclosporin A. Two major fractions are resolved on SDS/PAGE: an 18-kDa fraction is comprised of two isoforms that are similar if not identical to the two major cytosolic isoforms of cyclophilin. In addition, in microsomes an approximately equal quantity of a 22-kDa glycoprotein was detected. Based on partial sequencing (five peptides, 89 amino acids) this protein is similar but not identical to human cyclophilin B. This 22-kDa isoform is poorly recognized by affinity-purified anti-cyclophilin antibodies and comprises several predominant isoforms (pl z 9.3 -9.6). Selective binding of membrane 22-kDa cyclophilin to peanut lectin suggests the oligosaccharides contain a terminal galactosyl-N-galactosamine residue.The major cytosolic binding protein for cyclosporin in mammalian tissues is known as cyclophilin [I, 21. This 17.8-kDa protein exhibits remarkable stereospecificity for binding cyclosporin A, exists in several isoforms, catalyzes the cis-trans isomerization of peptidylproline bonds in oligopeptides and accelerates the rate-limiting step in the refolding of selected proteins [3,4]. A recent report indicates that cyclophilin, when complexed with cyclosporin, can associate with calcineurin, a protein phosphatase, and can inhibit this enzyme activity even though neither cyclophilin nor cyclosporin alone has this property [5].Similar cyclosporin-A-binding proteins are found throughout the animal kingdom and also in plants and yeasts [6 -101. The wide distribution of peptidylproline cis-transisomerase (PPIase) activity implies a fundamental biological role of the cyclophilins beyond cyclosporin-A-mediated immunosuppression [6]. Although a gene has been characterized for the predominant cyclophilin species in humans, multiple cyclophilin-related DNA sequences are found in mammalian DNA [l 11. In parallel studies, the immunosuppressive drugs FK506 and rapamycin have been shown to bind to a small protein, the FK506-binding protein, which also possesses PPI activity, [12, 131.

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