Molecular Pharmacology, Vol 17, 415-420, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Quantitation of Two Forms of Pulmonary Cytochrome P-450 in Microsomes, Using Substrate Specificities

¹ Laboratory of Pharmacology, National Institute of Environmental Health Sciences, NIH, P. 0. Box 12233, Research Triangle Park, North Carolina 27709

The metabolism of N-hydroxyamphetamine by the rabbit pulmonary microsomal fraction results in the formation of a metabolic intermediate complex with almost all of the cytochrome P-450. However, only half of the cytochrome P-450 forms a complex during the metabolism of norbenzphetamine, benzphetamine, or piperonyl butoxide. Immunological studies in which antibodies against each of the two known forms of pulmonary cytochrome P-450 were added to microsomes and monooxygenase systems containing purified enzymes demonstrate that one form of cytochrome P-450 complexes with both norbenzphetamine and N-hydroxyamphetamine, whereas a second form complexes only with N-hydroxyamphetamine. The cytochrome P-450-metabolic intermediate complexes can be solubilized from the microsomal membrane and separated from each other by chromatography on DEAE-cellulose. The complexes isolated in this manner are the same as those formed in purified systems. As determined by metabolic intermediate complex formation, pulmonary microsomal fractions contain equal amounts of the two forms of cytochrome P-450.

Note:
ACKNOWLEDGMENTS The authors would like to thank Zadock McCoy for the preparation of rabbit lung microsomes. Norbenzphetamine (N-benzyl--methylphenethylamine) was a gift from the Upjohn Co. (Kalamazoo, Mich.), and N-hydroxyamphetamine was a gift from Smith Kline and French Laboratories (Philadelphia, Pa.).