Reaction Mechanism of 21 S Dynein ATPase from Sea Urchin Sperm. I. Kinetic Properties in the Steady State

Abstract

21 S Dynein ATPase [EC 3. 6. 1. 3] from axonemes of a Japanese sea urchin, Pseudocentrotus depressus, and its subunit fractions were studied to determine their kinetic properties in the steady state, using [γ-³²P]ATP at various concentrations, 5mM divalent cations, and 20mM imidazole at pH 7.0 and 0°C. The following results were obtained. 1. 21 S Dynein had a latent ATPase activity of about 0.63 pmol P₁/(mg•min) in 1mM ATP, 100mM KCI, 4mM MgSO₄, 0.5mM EDTA, and 30 mM Tris-HC1 at pH 8.0 and 25°C. Its exposure to 0.1% Triton X-100 for 5min at min) 25°C induced an increase in the ATPase activity to about 3.75 μmol P₁/(mg) and treatment at 40°C for 5min also induced a similar activation. 2. The doublereciprocal plot for the ATPase activity of dynein activated by the treatment at 40°C consisted of two straight lines, while that of nonactivated 21 S dynein fitted a single straight line. 3. In low ionic strength solution, the Mg- and Mn-ATPase of 21 S dynein showed substrate inhibition at ATP concentrations above 0.1mM; the inhibition decreased with increasing ionic strength. Ca- and Sr-ATPase showed no substrate inhibition. 4. Both the V_max and Km values of dynein ATPase decreased reversibly upon addition of about 40% (v/v) glycerol. In the presence of glycerol, the dynein ATPase showed an initial burst of P, liberation. The apparent P₁-burst size was 1.0 mol/(10⁶g protein) and the true size was calculated to be 1.6 mol/1, 250 K after correcting for the effect of P₁ liberation in the steady state and the purity of our preparation. 5. One of the subunit fractions of 21 S dynein which was obtained by the method of Tang et al. showed substrate inhibition and an initial burst of P₁ liberation of 1.4 mol/(10⁶g protein) in the presence of 54% (v/v) glycerol.