A comparison of the effects of gentle heating,acetone, and the sulfhydryl reagent bis (4-fluoro-3-nitrophenyl) sulfone on the ATPase activity and pellet height response of tetrahymena cilia

Incubation of glycerol-extracted, Triton X-100 demembranated Tetrahymena cilia with 2–10 vol % acetone caused an enhancement of ATPase activity by 2- to 3- fold, depending on concentration and time of incubation. Axonemal ATPase activity was also increased upon incubation with bis (4-fluoro-3-nitrophenyl) sulfone (FNS). Acetone and FNS enhanced the activity of solubilized 30S dynein, but slightly inhibited that of 14S dynein. Heating at 38°C, incubation with FNS, and incubation with acetone activated axonemal ATPase to the same extent. Subsequent studies of (1) the effect of time of preincubation with a spin-labeled maleimide (SLM) at 25°C as a function of pH on the ATPase activity, (2) the concentration dependence of the inhibition of ATPase activity by N-ethylmaleimide or SLM, (3) the ratio of ATPase activity assayed at 25°C to that assayed at 0°C, and (4) the ratio of ATPase activity at pH 8.6 to that at pH 6.9 did not reveal any difference in the properties of the axonemal ATPase after near maximal enhancement by the heat, acetone, or FNS treatments. It was concluded that enhancement of ATPase activity by gentle heat treatment, by incubation with acetone (or other organic solvents), or by FNS results from a conformation change of 30S dynein. The effect of acetone and of FNS on the pellet height response (a measure of the increase in height of the pellet of cilia precipitated by brief centrifugation in the presence of ATP as compared to the absence of ATP) was also determined. Enhancement of ATPase by these reagents did not lead to a decrease in pellet height response. This observation, in conjunction with other data, indicates that there are at least 3 states of the cross-bridge cycle of dynein arms in cilia.