Thymidine kinase from ovaries of the cockroach Leucophaea maderae: Regulation by juvenile hormone

Abstract

Thymidine kinase (TK) (ATP: thymidine-5′-phosphotransferase, EC 2.7.1.75) was characterized in ovarian extracts from the cockroach Leucophaea maderae. TK activity was localized in the follicular epithelium surrounding the terminal oocyte, and subcellular fractionation studies indicated that TK was a cytosolic enzyme. Optimal assay conditions for TK were: (a) 5–10 mM ATP, (b) 4–6 mM MgCl 2, (c) pH 7.6–8.5 and (d) an incubation temperature of 45°C. Kinetic studies indicated an apparent Michaelis constant ( K m ) of 0.22 ± 0.07 μM thymidine (TdR) for TK extracted from ovaries of virgin females and 0.17 ± 0.05 μM TdR for that extracted from developing ovaries. Thymidine-5′-triphosphate (TTP) was an effective inhibitor of enzyme activity with an I 50 of approx. 0.062 mM TTP. Thymidine kinase activity was monitored in ovaries during terminal follicle maturation of the first reproductive cycle. Low levels of TK activity (0.5–1.0 pmol/min per ovary) were detected in ovaries of virgin females, peaked in ovaries 5–5.5 mm in length (90 pmol/min per ovary), and then declined to basal levels (1–10 pmol/min per ovary) prior to choriogenesis. Moreover, the profiles of TK activity correlated closely with the profile of DNA synthesis. Decapitation of females arrested TK activity, while injection of 25 μg JH I or III restored activity to normal levels. JH stimulation of the activity was dose-dependent, with maximal activation after injection of 25–50 μg. Peak TK activity was observed approx. 96 hr after injection, and high enzyme activity persisted for 192 hr. Injection of 2 or 4 μg actinomycin D along with the JH inhibited TK activity, suggesting a role for JH in regulation of de novo synthesis of the enzyme.