RECONSTITUTION OF OXIDATIVE PHOSPHORYLATION IN SUBMITOCHONDRIAL PARTICLES BY A SOLUBLE PROTEIN PHOSPHORYL TRANSFERASE

A protein designated phosphoryl transferase has been isolated from beef heart mitochondria and has been found to increase the ability of Submitochondrial particles, oxidizing either NADH or succinate, to synthesize ATP from ADP and inorganic orthophosphate. Increases in the value of P:O ratios between 0.45 and 1 have been observed with succinate as substrate. Phosphoryl transferase was released from beef heart mitochondria by sonic disruption in the presence of EDTA and was then purified by fractionation with ammonium sulfate, chromatography on DEAE-cellulose, and recycling molecular sieve chromatography on polyacrylamide gel (BioGel P-200). The isolated phosphoryl transferase displayed a single peak in the analytical ultra-centrifuge and a single band in strip electrophoresis. Its molecular weight was estimated to be 124,000; its isoelectric point, pH 5.7. The absorption spectrum of the protein showed a maximum at 278-280 mμ, a minimum at 250 mμ, and a shoulder at 290 mμ. At pH 13 two distinct maxima appeared at 282 mμ and 288.5 mμ. Increases in the P:O ratios of Submitochondrial particles, induced by purified phosphoryl transferase, were observed only at the site of energy conservation between reduced coenzyme Q and cytochrome c. The protein has also been isolated from phosphorylating Submitochondrial particles. During recycling gel filtration, a protein was separated from the phosphoryl transferase which inhibited the ATPase activity of Submitochondrial particles and counteracted the effect of the phosphoryl transferase in increasing the P:O ratio. © 1968.