|Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,100,000 (Direct Cost: ¥2,100,000)
In eggs of many insect species, ecdysteroid-phosphates, which are of maternal origin, have widely been accepted as storage forms for supplying active free ecdysteroids during embryonic development (Makka et al., 2002). However, there is little information on the enzyme which specifically catalyzes the phosphorylation of free ecdysteroids. The purpose of this study is to purify and characterize the ecdysteroid kinase (ATP : ecdysteroid phosphotransferase).
Ecdysteroid kinase was assayed by the phosphorylation reaction of [^3H] ecdysone. The enzyme, which was located in the cytosol fraction in mature ovaries of the silkworm Bombyx mori, was purified by about 600-fold to homogeneity by seven steps of column chromatography : Q Sepharose, Chelating Sepharose, Hydroxylapatite, Hitrap Q, Mono Q, 5'AMP Sepharose 4B and Superdex 200pg. The purified enzyme was most active at pH 7.6. The enzyme required the presence of both ATP and Mg^<2+> for expressing the activity. The apparent kinetic parameters for ecdysone kinase was Km=3.30 μM (20-hydroxyecdysone), 3.66 μM (ecdysone), 8.22 μM (3-epiecdysone). It is not clear at present whether ATP : 2-deoxyecdysone 22-phosphotransferase found in Schistocerca gregaria (Kabbouh and Rees 1991) and our ecdysone 22-phosphotransferase (ecdysone kinase) in Bombyx mori are homologous.