Molecular Pharmacology, Vol 13, 911-923, Copyright © 1977 by the American Society for Pharmacology and Experimental Therapeutics

Preparation and Properties of Highly Purified Cytochrome P-450 and NADPH-Cytochrome P-450 Reductase from Pulmonary Microsomes of Untreated Rabbits

¹ Department of Biochemistry and Center in Environmental Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Rabbit lung microsomal cytochrome P-450 has been purified to a specific content of 14.9 nmoles/mg of protein by n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE-cellulose chromatography. NADPH-cytochrome P-450 reductase was purified from the same microsomal preparation to a specific activity of 47,700 nmoles of cytochrome c reduced per minute per milligram of protein, using n-octylamino-Sepharose 4B and 2',5'-ADP-agarose affinity chromatography. Both proteins were apparently homogeneous as judged from polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the respective yields of the two proteins were greater than 20% and 40%. A less highly purified and apparently distinct cytochrome P-450 fraction was also separated in lower yield in the course of purification, suggesting the presence of multiple forms of cytochrome P-450 in the lung. The major lung cytochrome P-450 fraction, when combined with lung NADPH-cytochrome P-450 reductase and phospholipid, was active in demethylating benzphetamine; activity was also demonstrated toward the substrates benzo[a]pyrene, 7-ethoxycoumarin, cyclohexane, dimethylnitrosamine, N-methyl-4-aminoazobenzene, N,N-dimethyl-4-aminoazobenzene, 4-ipomeanol, 2-(N-ethylcarbamoylhydroxymethyl)furan, and diethyl p-nitrophenylphosphorothionate (parathion). The NADPH-cytochrome P-450 reductases prepared from lung and liver microsomes appeared nearly identical as judged by their apparent subunit molecular weights, isoelectric focusing patterns, activities toward cytochrome c, and abilities to support benzphetamine demethylation with lung and liver cytochromes P-450. The major lung microsomal cytochrome P-450 has the same apparent subunit molecular weight (49,000) as the major form of liver microsomal cytochrome P-450 (P-450_LM-2) induced by phenobarbital (and differs from the other cytochromes P-450 of the liver in this regard) and reacts strongly with antibody prepared to this hepatic enzyme [but not antibody prepared to -naphthoflavone-induced liver microsomal cytochrome P-450 (P-450_LM-4)] in Ouchterlony double-diffusion analyses. These hepatic and pulmonary cytochromes are, however, distinguished by their apparent isoelectric points and specificities toward certain substrates.

Note:
ACKNOWLEDGMENTS I am grateful to Mr. J. S. French and Dr. M. J. Coon of the University of Michigan for carrying out the immunological experiments with antibodies prepared in their laboratory.

This article has been cited by other articles:

				C. Serabjit-Singh, C. Wolf, R. Philpot, and C. Plopper Cytochrome p-450: localization in rabbit lung Science, March 28, 1980; 207(4438): 1469 - 1470. [Abstract] [PDF]