Molecular cloning of NADPH-cytochrome P450 oxidoreductase from silkworm eggs. Its involvement in 20-hydroxyecdysone biosynthesis during embryonic development.
Using RT-PCR, a cDNA fragment of NADPH-cytochrome P450 oxidoreductase from silkworm, Bombyx mori, was cloned from three-day-old nondiapause eggs. RACE was used to isolate the ends of the DNA. The full-length cDNA obtained was composed of 3471 bp with an open reading frame encoding a protein of 687 amino-acid residues with a relative molecular mass of 77 700. The protein, fused with glutathione S-transferase, was expressed in Escherichia coli and purified to homogeneity. The fused protein not only had NADPH-dependent cytochrome c-reducing activity, but also acted as an electron carrier from NADPH to bovine adrenal 21-hydroxylase P450 in the steroid hydroxylation reaction, confirming that the protein is the silkworm NADPH-cytochrome P450 oxidoreductase. Ecdysone 20-hydroxylase activity in the nondiapause egg microsomes increased until the fourth day after oviposition, and then decreased, little being detected on the ninth day. An antibody raised against the P450 reductase inhibited the ecdysone hydroxylation. Immunoblot analyses of the microsomes indicated that the P450 reductase protein appeared distinctly in the three-day-old nondiapause eggs and, in contrast to the developmental pattern of ecdysone hydroxylase activity, continued to increase as the embryos developed. These results suggest that ecdysone hydroxylation in the early stage of embryogenesis is dependent on the presence of both P450 reductase and ecdysone 20-hydroxylase P450, but its gradual reduction in the later stage may be due to the decrease in the level of ecdysone 20-hydroxylase P450.
Horike
N
N
Department of Molecular Physiological Chemistry, Osaka University Medical School, Japan.
Takemori
H
H
Nonaka
Y
Y
Sonobe
H
H
Okamoto
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D012831:Bombyx / Q000201:enzymology*; D002417:Cattle; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004441:Ecdysterone / Q000096:biosynthesis*; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D005982:Glutathione Transferase; D006900:Hydroxylation; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics*; D016366:Open Reading Frames; D010063:Ovum / Q000201:enzymology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013255:Steroid 21-Hydroxylase / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000378:metabolism; D013997:Time Factors
D018076:DNA, Complementary; D007074:Immunoglobulin G; D004441:Ecdysterone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D013255:Steroid 21-Hydroxylase; C020095:ecdysone 20-hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D005982:Glutathione Transferase