Purification and aminopyrine monooxygenase activity of liver microsomal cytochrome P-450 from alloxan-induced diabetic rats

H Shimeno, A Toda, S Ogata and A Nagamatsu

Department of Biochemistry, Faculty of Pharmaceutical Sciences, Fukuoka University, Japan.

We purified two diabetes-inducible and insulin-sensitive forms of cytochrome P-450, named P-450AL-1 and AL-2, from the liver microsomes of alloxan-diabetic male rats, using sodium cholate solubilization, octylamino-Sepharose 4B chromatography, and HPLC with diethylaminoethyl- 5PW and hydroxyapatite columns. The purified forms gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 50,000 for P-450AL-1 or 48,500 for P-450AL- 2. The CO-reduced spectral maximum of these forms was at 452 nm for P- 450AL-1 and 451 nm for P-450AL-2. The two purified forms had the low- spin state of heme in the oxidized form. Both P-450AL-1 and AL-2 were active in the metabolism of aniline, benzphetamine, and 7- ethoxycoumarin. However, the catalytic activity of P-450AL-2 for these substrates was obviously higher than that of AL-1. The NH2-terminal sequences of P-450AL-1 and AL-2 differed from each other, and did not agree with those of the other P-450 forms purified from diabetic rats previously. Furthermore, we examined the metabolism of aminopyrine in a reconstituted system with the purified cytochromes P-450. The diabetes- inducible forms of P-450 had high aminopyrine 3-hydroxylation and low N- demethylation activities. These findings provide clear evidence supporting our previous results, which have shown an increase in 3- hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazolin-5-on e and a decrease in 4-monomethylaminoantipyrine in intact diabetic rats.

Volume 19, Issue 2, pp. 291-297, 03/01/1991
Copyright © 1991 by American Society for Pharmacology and Experimental Therapeutics