Three N-aralkylated derivatives of 1-aminobenzotriazole as potent and isozyme-selective, mechanism-based inhibitors of guinea pig pulmonary cytochrome P-450 in vitro.

The potency and cytochrome P-450 (P-450) isozyme selectivity of 1-aminobenzotriazole (ABT) and three of its N-aralkylated analogues, N-benzyl-1-aminobenzotriazole (BBT), N-alpha-methylbenzyl-1-aminobenzotriazole (alpha MB), and the newly synthesized N-alpha-ethylbenzyl-1-aminobenzotriazole (alpha EB), as mechanism-based inhibitors were compared in pulmonary microsomes of untreated and beta-naphthoflavone (beta-NF)-induced guinea pigs. All four compounds were suicide substrates for pulmonary P-450, resulting in the loss of spectrally assayed hemoprotein (up to 50%). Monooxygenase activities were measured with isozyme-selective/specific substrates; the O-dealkylation of 7-pentoxyresorufin (PRF) for the guinea pig ortholog of rabbit P-450IIB4, the O-deethylation of 7-ethoxyresorufin for P-450IA1, and the N-hydroxylation of the aromatic amine 4-aminobiphenyl for P-450IVB1, BBT, alpha MB, and alpha EB were selective for the suicidal inhibition of P-450IIB4; for example, 1 microM alpha MB inactivated 95% of P-450IIB4-, and approximately 10% of P-450IA1- and IVB1-catalyzed, activity in microsomes from beta-NF-induced lungs. Isozyme selectivity was approximately the same for alpha EB and slightly lower for BBT, which inactivated relatively more P-450IA1. At low concentrations, 1 and 10 microM, respectively, ABT preferentially inactivated P-450IVB1, consistent with the efficient N-hydroxylation of aromatic amines by this form of P-450. alpha EB also was shown to efficiently inactivate P-450IIB4-catalyzed PRF activity in microsomes prepared from liver of phenobarbital-induced guinea pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on lysine:N6-hydroxylation by cell-free systems of Aerobacter aerogenes 62-1.

Electron microscopic examination has revealed the vesicular nature of the membrane component, of the cell-free system of Aerobacter aerogenes 62-1, which catalyses lysine: N6-hydroxylation. Regardless of the orientation of the vesicles, N-hydroxylation process is still stimulated by pyruvate. Both pyruvate oxidation and lysine: N6-hydroxylation were inhibited by protonophores and Gramicidin S.

Direct evidence for the participation of pyruvate in N-hydroxylation of lysine.

The contribution of pyruvate to the formation of N6-acetyl-N6-hydroxylysine by a cell-free system of Aerobacter aerogenes 62-1 involved in the production of the dihydroxamate siderophore, aerobactin, has been assessed by a study of the influence of its analogs as well as of inhibitors of thiamine pyrophosphate-dependent decarboxylation reactions. These studies have provided unequivocal evidence for pyruvate functioning not only as a source of reducing equivalents in the initial step of N-hydroxylation of lysine but also as a precursor of the acetyl moiety in the subsequent conversion of the N-hydroxy amino to its N6-acetyl derivative.