Reconstitution of highly purified proton-translocating pyrophosphatase from Rhodospirillum rubrum

Overt carnitine palmitoyl transferase (CPT₁) activity was measured in liver mitochondria from foetal rats (21 days gestation) and from neonatal rats (1 day post-partum). Birth was accompanied by a 6-fold increase in CPT₁ activity, a 14-fold decrease in sensitivity to inhibition by malonyl CoA and an increase in the n_H and the S_0.5 from palmitoyl CoA. The activity of latent enzyme (CPT₂) was unaffected at birth.     

Purification of the membrane-bound proton-translocating inorganic pyrophosphatase from Rhodospirillum rubrum

The proton translocating membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum S1, has been solubilized with good yield from chromatophores using Triton X-100 (9–10 oxyethylene groups) in the presence of high concentrations of MgCl₂ and ethyleneglycol. The enzyme has been purified 80-fold by hydroxylapatite column chromatography, to a state of near homogeneity, according to polyacrylamide-gelelectrophoresis. The enzyme appears to be a very hydrophobic integrally bound membrane protein. Phospholipids or Triton X-100 reconstitutes the enzyme activity after solubilization and purification. The purified enzyme preparation has a specific activity of 24 units. Both the purified and the chromatophore-bound enzyme are inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzo-2-oxo-1,3-diazol (NBF-Cl), sodium fluoride, imidodiphosphate, methylenediphosphonate and the antibiotic Dio-9 (energy-transfer inhibitor). In the solubilized state the purified enzyme is not stimulated by uncouplers or inhibited by dicyclohexylcarbodiimide in contrast to the chromatophore-bound pyrophosphatase. When reconstituted into liposomes the purified enzyme regains the stimulation by uncouplers.     

The native F0F1-inhibitor protein complex from beef heart mitochondria and its reconstitution in liposomes

A functional F₀F₁ ATP synthase that contains the endogenous inhibitor protein (F₀F₁I) was isolated by the use of two combined techniques [Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975).Biochemistry 14, 1727–1735; Dreyfus, G., Celis, H., and Ramirez, J. (1984).Anal. Biochem. 142, 215–220]. The preparation is composed of 18 subunits as judged by SDS-PAGE. A steady-state kinetic analysis of the latent ATP synthase complex at various concentrations of ATP showed aV _max of 1.28mol min^–1 mg^–1, whereas theV _max of the complex without the inhibitor was 8.3mol min^–1 mg^–1. In contrast, theK _m for Mg-ATP of F₀F₁ I was 148M, comparable to theK _m value of 142M of the F₀F₁ complex devoid of IF₁. The hydrolytic activity of the F₀F₁I increased severalfold by incubation at 60C at pH 6.8, reaching a maximal ATPase activity of 9.5mol min^–1 mg^–1; at pH 9.0 a rapid increase in the specific activity of hydrolysis was followed by a sharp drop in activity. The latent ATP synthase was reconstituted into liposomes by means of a column filtration method. The proteoliposomes showed ATP-Pi exchange activity which responded to phosphate concentration and was sensitive to energy transfer inhibitors like oligomycin and the uncouplerp-trifluoromethoxyphenylhydrazone.