Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 reductase.
The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059 bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 +/- 0.5 and 2.6 +/- 0.4 nmol/min/mg protein (mean +/- SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 +/- 6.01 microM, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6 kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species.
Kong
Sandra
S
Sansom Institute, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA, Australia.
Ngo
Suong N T
SN
McKinnon
Ross A
RA
Stupans
Ieva
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D007614:Macropodidae; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis* / Q000235:genetics*; D048368:Phascolarctidae / Q000235:genetics*; D051381:Rats; D017208:Rats, Wistar
D009251:NADPH-Ferrihemoprotein Reductase