Reconstitution of oxidative phosphorylation in submitochondrial particles by a soluble protein phosphoryl transferase.

A protein designated phosphor!// Iransferasr has bee11 isolated from beef heart mitochondria and has been found to increase the ability of suhnlitocllorldrial particles, oxidizing either NASH or snecinatc, to synthesize ,4TP from ADI and inorganic orthophosphate. Increases in the value of P:O ratios between 0.45 and 1 have been observed wit,h srlccinate as snbstrat e. Phosphoryl trnnsferase was released from beef heart mitochondria by sonic disrrlption in the presence of EDTA and was then prlrified by fractionation with ammonillm stllfate, chromatographyon L)EAE-cellrdosc, and recycling molccrk~r sieve chromatography on polyacrylamide gel (BioGel P-200). The isolated phosphoryl trnrrsferase displayed a single peak it1 the analytical Illtracentrifuge and a single band in strip electrophoresis. Its moleclrlnr weight was estimated to be 124,000; its isoelectric poiltt, pH 5.7. The ahsorption spectrum of the protein showed R rnnxim~un at 278-280 rnp, a minimum at 250 II+, and a shoulder at 290 rnF. At pH 13 two distinct maxima appeared at 282 InF and 288.5 ml*. Increases in the P:O ratios of sltbmitocholldrial particles. induced by plnified phosphoryl Iransferase, were observed only at the site of erlergy conservation between reduced coenzyme (2 and cytcjchrome c. The protein has also been isolated from phosphorylating submitochondrial particles, During recycling gel filtration, a protein was separated from the phosphoryl tritusferase which inhibited the ATYase activity of submit ochondrial Darticles :tlld cc~~mterac~tcd the effect of t.he phosphoryl transferase in increasing the P:O ratio.