Isolation,characterization, and properties of factor F1 from mitochondrial preparations of housefly (Musca domestica L.) thorax

A water-soluble Mg²⁺-dependent ATPase (coupling factor F₁) was isolated from the mitochondria of housefly thorax. It comprised about 14% of the proteins from a crude preparation. The F₁ preparation was nearly homogeneous as assessed by gel electrophoresis, isoelectric focusing, and electron microscopy. It was composed of five subunits with the following apparent molecular weights: α, 68,000; β, 61,000; γ, 38,000; δ, 27,000; and ?, 17,500. The isoelectric pH (pI) of this protein was 7.3. F₁ had a pH optimum of 8.2 and a temperature optimum between 37 and 45°C. The enzyme was fairly stable at 25°C. Nearly complete loss of activity was noticed at 0°C, while at 0 or 25°C, glycerol (20%) partially stabilized the enzyme activity against such inactivation. The K_m value of the enzyme with respect to ATP was 0.4 mM. The activity was stimulated by low concentrations of 2,4-dinitrophenol. The enzyme was inhibited by azide, p-hydroxymercuribenzoate, and guanidine hydrochloride. Oligomycin and the pesticides pyrethrin, cyhexatin, and DDT have no effect on the enzyme activity. However, all of these chemicals inhibited intact Mg²⁺- ATPase. The results are discussed in the light of differential responses of soluble and intact ATPase to these pesticides.