Characterization of a cytochrome P450 from di(2-ethylhexyl) phthalate-treated rats which hydroxylates fatty acids.
A cytochrome P450 was purified from liver microsomes of rats treated with di(2-ethylhexyl) phthalate (DEHP). DEHP is a member of a group of structurally diverse compounds which have been classified as peroxisome proliferators and are inducers of cytochromes P450 which hydroxylate lauric acid and other fatty acids. The P450 isolated from DEHP-treated rats (P450DEHP) was observed to have a Mr value of 51 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a maximum absorbance of 452 nm in its reduced carbon monoxide bound state. The amino terminal residue for P450DEHP was alanine and an 18-amino acid segment at the N-terminal region was identified. The N-terminal amino acid for the P450 4A1 from clofibrate-treated rats is methionine and alignment of the N-terminal segment of the P450DEHP with P450 4A1 indicated that the first four amino acids were absent. There were two amino acid differences between the two P450s in this 18-amino acid segment; in P450DEHP an alanine and a phenylalanine were substituted for serines in P450 4A1. The P450DEHP was found to catalyze the hydroxylation of several saturated fatty acids, having the highest turnover activity with laurate (82.1 nmol 12-OH-laurate formed/min/nmol P450). Myristate, palmitate, and stearate were also metabolized but at decreasing rates. Cytochrome b5 stimulated laurate 12-hydroxylation 10-fold in a reconstituted system. Laurate was not metabolized at its 11-carbon atom; however, the longer chain length fatty acids were metabolized at the (omega-1)-carbon atom in addition to the omega-carbon atom. A polyclonal antibody to the P450DEHP recognized three protein bands in liver microsomes from control and DEHP-treated rats on Western blot analysis, but only two protein bands from phenobarbital-treated rats.
Okita
R T
RT
Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman 99164.
Okita
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002458:Cell Fractionation; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D004051:Diethylhexyl Phthalate / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005230:Fatty Acids, Nonesterified / Q000378:metabolism*; D006900:Hydroxylation; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007850:Lauric Acids / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D005230:Fatty Acids, Nonesterified; D007527:Isoenzymes; D007850:Lauric Acids; C030358:lauric acid; D003577:Cytochrome P-450 Enzyme System; D004051:Diethylhexyl Phthalate