Protein expression, characterization, and regulation of CYP4F4 and CYP4F5 cloned from rat brain

被引:67
作者
Kawashima, H [1 ]
Kusunose, E [1 ]
Thompson, CM [1 ]
Strobel, HW [1 ]
机构
[1] OSAKA UNIV,SCH MED,TONEYAMA INST TB RES,TOYONAKA,OSAKA 560,JAPAN
关键词
P450; 4F4; 4F5; protein expression; enzyme purification; leukotriene B-4; prostaglandins; clofibrate;
D O I
10.1006/abbi.1997.0342
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 B-4 (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 A(1), prostaglandin E-1, 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. (C) 1997 Academic Press.
引用
收藏
页码:148 / 154
页数:7
相关论文
共 33 条
[1]   EXPRESSION AND ENZYMATIC-ACTIVITY OF RECOMBINANT CYTOCHROME-P450 17-ALPHA-HYDROXYLASE IN ESCHERICHIA-COLI [J].
BARNES, HJ ;
ARLOTTO, MP ;
WATERMAN, MR .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (13) :5597-5601
[2]   IDENTIFICATION OF A NEW P450 SUBFAMILY, CYP4F1, EXPRESSED IN RAT HEPATIC-TUMORS [J].
CHEN, LP ;
HARDWICK, JP .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1993, 300 (01) :18-23
[3]   EXPRESSION OF CYTOCHROME-P450 2D6 IN ESCHERICHIA-COLI, PURIFICATION, AND SPECTRAL AND CATALYTIC CHARACTERIZATION [J].
GILLAM, EMJ ;
GUO, ZY ;
MARTIN, MV ;
JENKINS, CM ;
GUENGERICH, FP .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1995, 319 (02) :540-550
[4]  
IMAI T, 1993, J BIOL CHEM, V268, P19681
[5]  
ISSEMANN I, 1990, NATURE, V347, P645, DOI 10.1038/347645a0
[6]   ESCHERICHIA-COLI EXPRESSION AND CHARACTERIZATION OF CYTOCHROMES P450 2B11, 2B1, AND 2B5 [J].
JOHN, GH ;
HASLER, JA ;
HE, YA ;
HALPERT, JR .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1994, 314 (02) :367-375
[7]   cDNA cloning of three new forms of rat brain cytochrome P450 belonging to the CYP4F subfamily [J].
Kawashima, H ;
Strobel, HW .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1995, 217 (03) :1137-1144
[8]   PURIFICATION AND NH2-TERMINAL AMINO-ACID-SEQUENCES OF HUMAN AND RAT-KIDNEY FATTY-ACID OMEGA-HYDROXYLASES [J].
KAWASHIMA, H ;
KUSUNOSE, E ;
KUBOTA, I ;
MAEKAWA, M ;
KUSUNOSE, M .
BIOCHIMICA ET BIOPHYSICA ACTA, 1992, 1123 (02) :156-162
[9]   Genomic cloning and protein expression of a novel rat brain cytochrome P-450 CYP2D18 catalyzing imipramine N-demethylation [J].
Kawashima, H ;
Sequeira, DJ ;
Nelson, DR ;
Strobel, HW .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (45) :28176-28180
[10]   TRUNCATED HUMAN P450 2D6 - EXPRESSION IN ESCHERICHIA-COLI, NI2+-CHELATE AFFINITY PURIFICATION, AND CHARACTERIZATION OF SOLUBILITY AND AGGREGATION [J].
KEMPF, AC ;
ZANGER, UM ;
MEYER, UA .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1995, 321 (02) :277-288