Isolation of cytochrome P450 from hepatopancreas microsomes of the spiny lobster, Panulirus argus, and determination of catalytic activity with NADPH cytochrome P450 reductase from vertebrate liver.
One major form of cytochrome P450 has been isolated from the hepatopancreas of untreated spiny lobsters, Panulirus argus. This form, termed here D1, was purified to a specific content of 12.1 +/- 1.8 nmol/mg protein. Two minor forms, termed D2 and D3 were partially purified to 4.6 +/- 1.6 and 2.3 +/- 0.2 nmol P450/mg protein, respectively. No NADPH-cytochrome P450 reductase activity was detected in spiny lobster hepatopancreas microsomes and no purification of spiny lobster reductase was achieved in this study. Very low NADPH-cytochrome c reductase activity was found in hepatopancreas microsomes and also in cytosol. Indirect evidence suggested that proteolysis of spiny lobster P450 reductase during the preparation of hepatopancreas microsomes may in part account for the lack of detectable monooxygenase activity in hepatopancreas microsomes. The catalytic activities of the D1 or D2 forms of spiny lobster P450 were measured by mixing D1 or D2 with NADPH-cytochrome P450 reductase isolated from pig or rat liver microsomes. D2 was very efficient in demethylating benzphetamine, with a turnover number of 122 per minute, and D1 was an efficient catalyst of progesterone 16 alpha-hydroxylation, with a turnover number of 43 per minute. Other good substrates for D1 and D2 forms were aminopyrine, testosterone, benzo(a)pyrene, and 7-ethoxycoumarin. Little activity was found with methyl-, ethyl-, pentyl-, or benzyl-phenoxazone ethers as substrates. The profile of metabolites formed by D1 or D2 with benzo(a)pyrene as substrate were more similar to those formed with uninduced rat liver microsomes than to those formed by liver microsomes from uninduced flatfish species.
James
M O
MO
Department of Medicinal Chemistry, University of Florida, Gainesville 32610.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004064:Digestive System / Q000201:enzymology*; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006882:Hydroxyapatites; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D008121:Nephropidae; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D039181:Steroid 16-alpha-Hydroxylase; D013552:Swine
D000596:Amino Acids; D006882:Hydroxyapatites; D011189:Potassium Chloride; D003577:Cytochrome P-450 Enzyme System; D017886:Durapatite; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase