Oxidative phosphorylation coupled with the oxidation of α-ketoglutarate by heart-muscle sarcosomes. 1. Kinetics of the oxidative phosphorylation reaction and adenine nucleotide specificity

Abstract

Both oxidation and oxidative phosphorylation proceed at a uniform rate without any detectable time lag from the moment when heart-muscle sarcosomes are added to a suitable reaction mixture containing alpha-ketoglutarate. Unless a very high concn. of hexokinase is used, there is a slight lag in synthesis of hexose monophosphate, due to accumulation of adenosine triphosphate (ATP). The max. yield of oxidative phosphorylation (P:O) was obtained with rat-heart sarcosomes isolated in saline when 35 units or more of hexokinase/mg. sarcosomal protein were used. Adenosine monophosphate (AMP) was phosphorylated at less than 1% of the rate of adenosine diphosphate (ADP). The alpha -ketoglutaric oxidase system and the accompanying phosphorylation have a high affinity for ADP (Michaelis constant about 7 x 10-7, [image]). Freshly prepared sarcomsomes contain 0.02[mu]-mole energy-rich phosphate/mg. sarcosomal protein (0.04[mu] mole total adenosine). This is probably ADP which is present in amts. sufficient to give nearly opt. rates of oxidation in the absence of added adenine nucleotide when a relatively high concn. of sarcosomes (1.25 mg. sarcosomal protein/ml.) is used. Expts. at greater dilutions gave evidence of dissociation of ADP from the alpha-ketoglutaric oxidase system. The affinity of this endogenous ADP for alpha-ketoglutaric oxidase system is of the same order of magnitude as that of added ADP. From the proportionality of the rate of oxidation of succinate to concn. of heart-muscle prepn. in the absence of added cytochrome c it was calculated that the Km of bound cytochrome cannot exceed 4 x 10-11[image].