CHARACTERIZATION OF ATP - ECDYSONE 3-ACETATE 2-PHOSPHOTRANSFERASE (ECDYSONE 3-ACETATE 2-KINASE) IN SCHISTOCERCA-GREGARIA LARVAE

Soluble ATP:ecdysone 3-acetate 2-phosphotransferase from fat body of d ay 7 last instar larvae of Schistocerca gregaria was characterized. Th e phosphotransferase activity was present in all tissues examined, but the highest activity was obtained in fat body cytosol. A single peak of ATP:ecdysone 3-acetate 2-phosphotransferase activity was obtained b y DEAE-cellulose chromatography. The enzyme has a molecular weight of ca 45kDa, determined by gel filtration on Sephadex G-150. Ecdysone 3-a cetate was the preferred substrate for the enzyme, whereas ecdysone 2- acetate and ecdysone were not significantly phosphorylated. The phosph otransferase activity survived freezing at -20-degrees-C but was total ly abolished by heat at 60-degrees-C for 10 min. The reaction rate was linear for 60 min and with increasing protein concentration up to 2 m g/ml. Optimal pH was about 7.5. The phosphotransferase activity requir ed the presence of ATP, the apparent K(m) for ATP being 91.1 muM. The enzyme exhibited an apparent K(m) for ecdysone 3-acetate of 10.5 muM a nd a maximal specific activity of 91.1 pmol/min/mg protein. Ecdysone a nd 20-hydroxyecdysone did not inhibit the phosphotransferase activity. Maximal enzyme activity was obtained in the presence of 5-10 mM Mg2while Ca2+ was inhibitory with an IC50 value of 1 mM. Investigation of the variation in activity of the phosphotransferase during developmen t of the last instar larvae showed that the phosphotransferase activit y increased significantly during the second half of the instar when th e titre of endogenous ecdysteroids is at its highest level.