Purification of NADPH-P450 reductase (NPR) from Xenopus laevis and the developmental change in NPR expression

Abstract

NADPH-P450 reductase (NPR) was purified from hepatic microsomes of Xenopus laevis. The electron transfer activity of purified NPR was 23.8 units/min/mg with horse cytochrome c. The aminopyrine demethylation activity of rat CYP2B1 with Xenopus NPR was 58.1 nmol/min/nmol. The corresponding cDNA was isolated from Xenopus liver. The homology in amino acid sequence deduced from NPR cDNA isolated from Xenopus liver was 80%, 78%, and 81% with human, rat, and rabbit NPR, respectively. Antibody against Xenopus NPR was prepared. The expression of NPR was investigated in various tissues and in early development by Western blotting. NPR was most abundantly expressed in the kidney, followed by the liver, lung, and heart. The brain had very low levels of NPR. The level of NPR protein was almost the same at all stages, 2-cell stage (st. 2), blastula (st. 8), gastrula (st. 12), tail bud (st. 26) and larva (st.35), examined in this study. We further investigated the distribution of NPR using whole-mount in situ hybridization. NPR mRNA was expressed in cement gland, lens placode, ear vesicle, mesencephalon, rhombencephalon, lymphatic vessel, and heart anlage in the embryo at stage 29. Xenopus NPR has similar properties to mouse and rat NPRs. Localization of NPR in Xenopus embryo was consistent with the abnormal region caused by NPR deficiency in mice.