The roles of amino acid residues at positions 43 and 45 in microsomal contents and enzymatic functions of rat CYP2D1 and CYP2D2.
The effects of the substitution of amino acid residues at positions 43 and 45 of rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions were examined. The substitution of Val-45 of CYP2D1 by glycine decreased the microsomal content, whereas the substitution of Gly-45 of CYP2D2 by valine increased. The substitution of Leu-43 of CYP2D2 by tryptophan also increased the microsomal protein content. In reduced CO-difference spectra, CYP2D2 showed a P420 peak as well as a P450 peak, whereas CYP2D1 gave only a P450 peak. The substitution of Leu-43 and Gly-45 of CYP2D2 by valine and tryptophan, respectively, markedly decreased the P420 peak in parallel with an increase in P450 content. These substitutions did not cause remarkable changes in drug oxidation capacities (bufuralol 1''-hydroxylation and debrisoquine 4-hydroxylation) of the recombinant enzymes in terms of nmol/min/nmol CYP. The results indicate that amino acid residues at positions 43 and 45 are important for anchoring of the rat CYP2D proteins and their stabilities in the endoplasmic reticulum membrane.
Laboratory of Health Chemistry, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan. shizuo@pharm.okayama-u.ac.jp
Shizuo
S
Narimatsu
Kazuo
K
Imoto
Takashi
T
Isobe
Kimio
K
Kiryu
Shinsaku
S
Naito
Hiroyuki
H
Hichiya
Yoshihiko
Y
Funae
Nobumitsu
N
Hanioka
Shigeo
S
Yamamoto
D000429:Alcohol Oxidoreductases; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis*; D015153:Blotting, Western; D002248:Carbon Monoxide / Q000737:chemistry; D000072467:Cytochrome P450 Family 2; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D003647:Debrisoquin / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004983:Ethanolamines / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008099:Liver / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D014633:Valine / Q000737:chemistry
D000596:Amino Acids; D017931:DNA Primers; D018076:DNA, Complementary; D004983:Ethanolamines; D011994:Recombinant Proteins; D002248:Carbon Monoxide; C010831:bufuralol; D011392:Proline; D000429:Alcohol Oxidoreductases; D001189:Aryl Hydrocarbon Hydroxylases; C504568:Cyp2d1 protein, rat; C119985:Cyp2d2 protein, rat; D000072467:Cytochrome P450 Family 2; D007930:Leucine; D014633:Valine; D010100:Oxygen; D005998:Glycine; D003647:Debrisoquin