Figure 6.

Aldolase Stimulates V-ATPase Hydrolytic Activity by Increasing Affinity for ATP.

(A) V-ATPase hydrolytic activity (bafilomycin-sensitive and azide- and vanadate-insensitive) was estimated by spectrophotometric measurement of inorganic phosphate release as described in Methods. Activity was measured in tonoplast vesicles (15 μg protein) isolated from M. crystallinum over a range of aldolase concentrations. Data represent means ± se of three replicate experiments. Each replicate experiment was performed using independent membrane preparations. The solid lines show the fit of the kinetic data with the Michaelis-Menten equation, and from this the rate constants K_s and V_max were calculated. K_s refers to the concentration of aldolase that gives half the maximal velocity, and V_max refers to the velocity of the enzyme catalyzed reaction at saturating aldolase concentrations. The χ² value indicates the goodness of fit and confirmed that the data fitted the equation at a probability level of at least P < 0.01.

(B) V-ATPase hydrolytic activity was measured over a range of ATP concentrations in the presence of increasing amounts of aldolase. Data represent means ± se of three replicate experiments performed using independent membrane preparations. The solid lines show the fit of the data with the Michaelis-Menten equation. Units for V_max are μmol P_i mg⁻¹ protein min⁻¹. The χ² values, indicating the goodness of fit of the data to the equation, gave probabilities of at least P < 0.05.

[See online article for color version of this figure.]