Purification and aminopyrine monooxygenase activity of liver microsomal cytochrome P-450 from alloxan-induced diabetic rats.
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.
Shimeno
H
H
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Fukuoka University, Japan.
Toda
A
A
Ogata
S
S
Nagamatsu
A
A
D000595:Amino Acid Sequence; D000632:Aminopyrine / Q000378:metabolism*; D000633:Aminopyrine N-Demethylase / Q000378:metabolism; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D004790:Enzyme Induction; D007328:Insulin / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D007328:Insulin; D000632:Aminopyrine; D003577:Cytochrome P-450 Enzyme System; D000633:Aminopyrine N-Demethylase; D009251:NADPH-Ferrihemoprotein Reductase