Purification, kinetic characterization, and molecular cloning of a novel enzyme ecdysteroid-phosphate phosphatase.
From eggs of the silkworm Bombyx mori, we isolated a novel enzyme that is involved in the conversion of physiologically inactive conjugated ecdysteroids, such as ecdysone 22-phosphate and 20-hydroxyecdysone 22-phosphate, to active free ecdysteroids. This enzyme, called ecdysteroid-phosphate phosphatase (EPPase), was located in the cytosol fraction and differed from nonspecific lysosomal acid phosphatases in various enzymic properties. EPPase was purified about 3,000-fold to homogeneity by seven steps of column chromatography. The cDNA clone encoding EPPase was isolated by reverse transcription polymerase chain reaction using degenerate primers on the basis of the partial amino acid sequence obtained from purified EPPase and by subsequent 3'- and 5'-rapid amplification of cDNA ends. The full-length cDNA of EPPase was found to be composed of 1620 bp with an open reading frame encoding a protein of 331 amino acid residues. A data base search showed that there was no functional protein with the amino acid sequence identical to that of EPPase. Northern blot analysis revealed that EPPase mRNA was expressed predominantly during gastrulation and organogenesis in nondiapause eggs but was not detected in diapause eggs whose development was arrested at the late gastrula stage. In nondiapause eggs, the developmental changes in the expression pattern of EPPase mRNA corresponded closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs.
Yamada
Ryouichi
R
Department of Life and Functional Material Science, Graduate School of Natural Sciences, Konan University, Kobe 658-8501, Japan.
Sonobe
Haruyuki
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000196:embryology / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D026461:Ecdysteroids / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010063:Ovum / Q000201:enzymology; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014018:Tissue Distribution
D018076:DNA, Complementary; D026461:Ecdysteroids; C476613:ecdysteroid-phosphate phosphatase; D010744:Phosphoric Monoester Hydrolases