Inside-out Submitochondrial Particles Research Articles

4-Azido-2-nitrophenyl phosphate, a new photoaffinity derivative of inorganic phosphate. Study of its interaction with the inorganic phosphate binding site of beef heart mitochondrial adenosine triphosphatase.

4-Azido-2-nitrophenyl phosphate (ANPP) was synthesized and characterized. ANPP, unlabeled or labeled by 32P, was used as a photoreactive analogue of Pi to study the Pi binding site(s) in isolated F1-ATPase and inside-out particles from beef heart mitochondria. In the dark, the phosphate bond of ANPP was cleaved by alkaline phosphatase but not by mitochondrial F1-ATPase. ANPP bound reversibly to the phosphate site of F1-ATPase as shown by competitive inhibition of binding of Pi to F1-ATPase by ANPP in the dark; the Ki value was 60 microM. Upon photoirradiation with visible light, [32P]ANPP bound covalently to F1-ATPase and inactivated the enzyme. Part of the added ANPP was, however, photolyzed with release of Pi. By extrapolation, it could be calculated that complete inactivatin of F1-ATPase was accompanied by incorporation of 32P radioactivity corresponding to 1 mol of [32P]ANPP per mol of F1-ATPase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [32P]-ANPP-labeled F1-ATPase revealed only one radioactive peptide with a Mr of 50000. This peptide was characterized as the beta subunit of F1-ATPase by specific labeling with [14C]dicyclohexylcarbodiimide [Pougeois, R., Satre, M., & Vignais, P. V. (1979) Biochemistry 18, 1408-1413]. Photoirradiation of inside-out submitochondrial particles with [32P]ANPP resulted in the labeling of two peptides with a Mr of 50000 and 30000-32000; both labelings were significantly decreased by incubation of the particles with Pi prior to photoirradiation. The Mr 50000 peptide is most probably the beta subunit of F1-ATPase; the other peptide might be the Pi carrier protein.

The electrogenic nature of ADP/ATP transport in inside-out submitochondrial particles

1. 1. Beef heart submitochondrial particles prepared by sonication loaded with ADP and MgCl 2 are competent in ADP/ATP transport. The Δψ and ΔpH developed by these particles have been determined, and the electrogenic nature of the ADP/ATP transport that they catalyze has been assessed. 2. 2. MgCl 2 particles, prepared by sonication, oxidizing succinate in the presence of oligomycin develop a Δψ of 95 mV (positive inside) and a ΔpH of 1.5 pH units (acidic inside), as measured by the distribution of S 14CN − (Δψ) and [ 14C]methylamine (ΔpH) using the flow dialysis technique. The Δψ values determined with the thiocyanate electrode are somewhat higher (110–130 mV). Δψ and ΔpH are partly interchangeable. 3. 3. The fluorescent dye, dipropylthiodicarbocyanine, and the absorbing dye, neutral red, are suitable probes for continuous, qualitative monitoring of Δψ and ΔpH respectively. The Δψ and ΔpH responses to respiratory activity in particles prepared by sonication are opposite to those found with intact mitochondria, in agreement with the reverse polarity of the membrane in particles prepared by sonication. 4. 4. ATP added to oligomycin-pretreated particles prepared by sonication is taken up in exchange for internal ADP. Under these conditions, the fluorescence intensity of dipropylthiodicarbocyanine is transitorily quenched, which corresponds to the net influx of an excess of negative charges inside the particles. A subsequent addition of ADP results in a transient rise in fluorescence. The ATP-induced fluorescence quenching of dipropylthiocarbocyanine is abolished by addition of SCN −, a strong permeant anion which collapses the Δψ. It is inhibited by ADP in a competitive manner. This fluorescence quenching is specific for ATP among a number of nucleotides tested, is inhibited by bongkrekic acid and is highly dependent on temperature. All these features are typical properties of the ADP/ATP carrier. 5. 5. Assays with neutral red as a pH indicator show that the internal phase of particles prepared by sonication treated by oligomycin becomes more acidic upon addition of ATP. However the pH changes are markedly diminished at high concentration of K + and totally abolished by K + plus valinomycin and, on the contrary, enhanced by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. This suggests that the ATP-dependent changes of pH are not characteristic of the mechanism of the ADP/ATP carrier, but correspond to a passive transport of H +. 6. 6. In respiring particles prepared by sonication, addition of SCN − at a concentration of 5 mM, sufficient to collapse the Δψ, strongly modifies the kinetics of ATP 4− ex/ADP 3− in exchange, but not that of ADP 3− ex/ADP 3− in. It increases the K m value for ATP ex and decreases the maximal velocity of transport. Nigericin has no effect. 7. 7. In non-respiring particles prepared by sonication, addition of a sufficiently high concentration of SCN − (30 mM) induces a transient diffusion potential which results in the inhibition of ATP 4− ex/ADP 3− in exchange. When the excess of negative charges carried inside the particles by SCN − are neutralized by the simultaneous entry of K +, the ATP 4− ex/ADP 3− in exchange remains unaltered. 8. 8. The above data indicate that ADP/ATP transport across the mitochondrial membrane is primarily electrogenic.