Cloning and functional expression of a novel marmoset cytochrome P450 2D enzyme, CYP2D30: comparison with the known marmoset CYP2D19.
Using a primer set designed on the cDNA encoding the known marmoset cytochrome P450 2D19 (CYP2D19), a cDNA encoding a novel CYP2D enzyme (CYP2D30) was cloned from the liver of a female marmoset bred at Kyoto University (KYU). In addition, a cDNA encoding CYP2D19 was cloned from the liver of a female marmoset bred at Kagoshima University (KAU). CYP2D30 and CYP2D19 showed homologies of 93.6 and 93.4% in their nucleotide and amino acid sequences, respectively. Reverse transcription polymerase chain reaction (RT-PCR) and digestion with NdeI demonstrated that the KYU-marmoset liver contained mainly mRNA for CYP2D30, while the KAU-marmoset liver contained mainly mRNA for CYP2D19. Marmoset CYP2D30, like human CYP2D6, exhibited high debrisoquine (DB) 4-hydroxylase activity and relatively low DB 5-, 6-, 7- and 8-hydroxylase activities, whereas CYP2D19 lacked DB 4-hydroxylase but exhibited marked 5-, 6-, 7- and 8-hydroxylase activities. The two marmoset recombinant enzymes showed enantioselective bufuralol (BF) 1"-hydroxylase activities, similar to CYP2D6. BF 1"-hydroxylation by CYP2D30 exhibited product-enantioselectivity of (1"R-OH-BF << 1"S-OH-BF), similar to that observed with human CYP2D6, whereas CYP2D19 showed a reversed selectivity of (1"R-OH-BF > or = 1"S-OH-BF). BF 1"-hydroxylation in marmoset liver microsomes from both sources was inhibited by antibodies raised against rat CYP2D1 in a concentration-dependent manner. A known inhibitor of CYP2D6, quinidine, effectively inhibited the BF 1"-hydroxylation activities in liver microsomal fractions prepared from KYU- and KAU-marmosets. These results suggest that CYP2D19 and CYP2D30 proteins can be expressed as functional enzymes in marmoset livers, although it is unresolved whether both enzymes coexist in the same marmoset liver.
Hichiya
Hiroyuki
H
Laboratory of Health Chemistry, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan.
Kuramoto
Shino
S
Yamamoto
Shigeo
S
Shinoda
Sumio
S
Hanioka
Nobumitsu
N
Narimatsu
Shizuo
S
Asaoka
Kazuo
K
Miyata
Atsuro
A
Iwata
Shinichi
S
Nomoto
Masahiro
M
Satoh
Tetsuo
T
Kiryu
Kimio
K
Ueda
Nobuhiko
N
Naito
Shinsaku
S
Tucker
Geoffrey T
GT
Ellis
S Wynne
SW
D000429:Alcohol Oxidoreductases; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002144:Callithrix; D003001:Cloning, Molecular; D000072467:Cytochrome P450 Family 2; D018076:DNA, Complementary / Q000032:analysis; D005260:Female; D015870:Gene Expression; D006801:Humans; D006900:Hydroxylation; D008099:Liver / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D011802:Quinidine / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D001189:Aryl Hydrocarbon Hydroxylases; C504568:Cyp2d1 protein, rat; D000072467:Cytochrome P450 Family 2; C105144:cytochrome P-450 CYP2D19 (marmoset); C487732:cytochrome P-450 CYP2D30 (marmoset); D011802:Quinidine