Abstract
The Departinent of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka A remarkable decrease in the ratio of the rate of Pi-liberation (v) to the amount of phosphorylated intermediate ([EP]) was observed during the initial phase of the Ca2+-dependent ATPase [ATP phosphohydrolase: EC 3.6.1.3] reaction of SR. The effects of ATP, pH, divalent cations, Triton X-100 and NEM on the transition in v/[EP] were investigated, and the following results were obtained.
1. A typical transition in v/[EP] during the initial phase of the reaction was observed at alkaline pH as well as at neutral pH. At alkaline pH the amount of Pi liberated from ATP during the transition phase was far less than that of the phosphorylation site of SR. The value of v/[EP] in the steady state was unchanged by the presence of oxalate (0 to 5 mm). The extent of the decrease in v/[EP] was not affected by changing the concentration of Mg2+.
2. The extent of decrease in v/[EP] after starting the reaction by adding Ca2+ was less than that after starting the reaction by adding ATP.
3. When SR was pretreated with Triton X-100 (20 μl/mg SR protein), the value of v/[EP] in the steady state was greatly increased and its transition disappeared almost completely. The transition also disappeared on treatment of SR with NEM.
4. The transition in the value of v/[EP] occurred completely even in the pres-ence of ATP, at a much lower concentration than that of the phosphorylation site. The transition in v/[EP] after adding 0.05 μ M ATP was rather slow, and 45 sec-1 and 0.45 sec-1 were obtained as the initial and steady state values, respectively, of v/[EP] at 15°C.
From these results, we concluded that the transition in v/[EP] results from a cooperative conformational change in the vesicular structure of SR induced by ATP.
