Effect of triiodothyronine on the synthesis and degradation of the small subunit of renal cortical (Na+ + K+)-adenosine triphosphatase.

Abstract

We have previously demonstrated that triiodothyronine (T3) enhanced the de nouo synthesis of the large subunit of renal cortical (Na+ + K+)-dependent adenosine triphosphatase (NaK-ATPase).The present study centers on the effect of TS on the synthesis and degradation of the small subunit of the enzyme.Thyroidectomized rats were injected with single doses of Ts, reverse T3 (50 pg/lOO g of body weight), or the diluent.Twenty hours later the rats were infused via tail vein with [3H]-or [35S]methionine.Eight and 22 h after the infusion the rats were killed, the kidneys (one from each rat) pooled, and the partially purified NaK-ATPase electrophoresed in sodium dodecyl sulfate-polyacrylamide gel.The large subunit (estimated molecular weight = 100,000) of NaK-ATPase was identified by (Na' + Mg2+)-dependent and K+-sensitive incorporation of 12P from [Y-~'P]ATP.The small subunit with apparent molecular weight of 52,000 was identified by incorporation of IH from NaB3H4.Analysis of the amounts of [IHImethionine and [35S]methionine in the protein bands in the gels indicated that reverse TI did not enhance methionine incorporation into the two subunits of NaK-ATPase.Conversely, T3 specifically augmented the incorporation of methionine into the large and small subunits by 35% and 32%, respectively, in the 8-h chase experiments, and by 49% and 538, respectively, in the 22-h chase experiments.Neither reverse TI nor T3 altered the incorporation of methionine into a third protein distinct from NaK-ATPase.Previous studies indicated that TI increased the cellular content of renal cortical NaK-ATPase by enhancing the synthesis rather than retarding the degradation of the large subunit of NaK-ATPase.The effect of T3 on the rate constant of degradation (kd) of the small subunit was tested.Pairs of thyroidectomized rats were injected with either TI or the diluent at 48-h intervals for 4 to 6 days.NaK-ATPase was labeled with [36S]methionine 48 h after the first injection (day zero).The kidneys were resected 48 h after either the second or third injections (Le.day 4 or day 6).NaK-ATPase was isolated from kidney cortices and the partially purified enzyme was labeled separately with either [yS2P]ATP or NaB3H4.The labeled enzyme was then subjected to electrophoresis in sodium dodecyl sulfate-polyacrylamide gel.Radioactivities contained in the two bands representing the two subunits of NaK-ATPase and in a third band representing an unidentified protein were