Characterization of the ATP:2-deoxyecdysone 22-phosphotransferase (2-deoxyecdysone 22-kinase) in the follicle cells of Schistocerca gregaria

Abstract

Follicle cells were isolated from their oocytes using 0.15% collagenase and low speed centrifugation. Incubation of the follicle cell pellet with [³H]2-deoxyecdysone yielded its 22-phosphate ester conjugate. Addition of ATP/Mg²⁺ or GTP/Mg²⁺ to follicle cell homogenate incubated with 2-deoxyecdysone increased the phosphotransferase activity by 4-fold for ATP and 2-fold for GTP. In the case of intact cells, only ATP was effective. The enzyme had a cytosolic subcellular localization and its $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for 2-deoxyecdysone was 3 μM. The phosphotransferase activity required the presence of both ATP and Mg²⁺, and had an apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP of 0.83 mM, with maximum activity being obtained in the presence of 10 mM Mg²⁺. The activity was strongly inhibited in the presence of Ca²⁺ with $\text{IC}{}_{50}\text{= 1}$ mM. The reaction rate was linear for 10 min and with increasing protein concentrations up to 1 mg/ml. Optimal pH was about 7.4 and the optimal temperature was 37°C. The phosphotransferase activity survived freezing at −20°C, but was totally abolished by heat at 60°C for 10 min. Investigation of the variation in activity of the phosphotransferase during ovarian development revealed a peak at the end of oogenesis in excellent agreement with the titre of ecdysteroid 22-phosphates found in the oocytes just before chorionation and egg-laying.