Protein expression, characterization, and regulation of CYP4F4 and CYP4F5 cloned from rat brain.
We previously reported the cDNA cloning of three new forms of P450, CYP4F4, CYP4F5, and CYP4F6, from a rat brain cDNA library. In the present study, we expressed CYP4F4 and CYP4F5 in Escherichia coli using the pCWOri expression vector with a modification of their N-terminal amino acid sequences and the incorporation of a C-terminal [His]4 tag to aid in purification. CYP4F5 recombinant protein was purified to a specific content of 7.7 nmol/mg protein from the membrane fraction of E. coli and showed omega-hydroxylation activity toward leukotriene B4 (LTB4), a chemical mediator of inflammation. On the other hand, the solubilized membrane fraction of CYP4F4-expressed recombinant protein catalyzed the omega-hydroxylation of prostaglandin A1, prostaglandin E1, and 6-trans-LTB4 as well as LTB4. The effects of the peroxisome proliferator, clofibrate, on mRNA expression of CYP4F4, 4F5, and 4F6 were studied by Northern blot analysis. The expression levels of the mRNA of these CYP4Fs were shown to be reduced by clofibrate in liver.
Kawashima
H
H
Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas 77225, USA.
Kusunose
E
E
Thompson
C M
CM
Strobel
H W
HW
D000527:Alprostadil / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D002994:Clofibrate / Q000494:pharmacology; D003001:Cloning, Molecular; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D000960:Hypolipidemic Agents / Q000494:pharmacology; D007975:Leukotriene B4 / Q000378:metabolism; D008297:Male; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011454:Prostaglandins A / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry
D000960:Hypolipidemic Agents; D011454:Prostaglandins A; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007975:Leukotriene B4; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A; D000527:Alprostadil; D002994:Clofibrate; C100573:prostaglandin A1