Protonic inhibition of the mitochondrial adenosine 5'-triphosphatase in ischemic cardiac muscle. Reversible binding of the ATPase inhibitor protein to the mitochondrial ATPase during ischemia.

Twenty minutes of ischemia in canine cardiac muscle produced a 50% to 60% inhibition of the mitochondrial ATPase. The inhibition has been shown to be triggered by a drop in cell pH under the non-energizing conditions which prevail in ischemic cells (Rouslin, W J Biol Chem 258, 9657-9661 (1983). In the present study we showed that the ATPase inhibition produced in situ in ischemic cardiac muscle was preserved in submitochondrial particles (SMP) prepared from mitochondria isolated from the ischemic tissue. The ischemic SMP ATPase was 45 +/- 3% as active as that of control particles. Measurements of the amounts of ATPase inhibitor protein of Pullman and Monroy present in extracts of control and ischemic SMP by two independent methods, titration of rat heart SMP ATPase and radioimmunoassay, revealed that control SMP contained 62 +/- 4% as much inhibitor as ischemic SMP as estimated by the titration procedure and 66 +/- 3% as much as estimated by the RIA. The results suggest that about one-third of the inhibitor was displaced from the control SMP. Finally, submitochondrial particles prepared from 20 min ischemic heart muscle showed a 2.5-fold increase in ATPase specific activity and a concomitant release of 35% of their inhibitor as a result of subsequent reenergization in vitro. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented both ATPase reactivation and inhibitor release. These findings support the hypothesis that the observed in situ ATPase inhibition is inhibitor protein mediated. Moreover, they suggest a pathophysiological function for the inhibitor protein in cardiac muscle.

Regulation of the composition and positioning of saturated and monoenoic fatty acids in phosphatidylcholine.

The mechanism by which polyenoic acids control the amount and positioning of monounsaturated fatty acids in choline phosphoglycerides from baby hamster kidney cells was studied. Under normal growth conditions monoenoic acids were derived from the desaturation of saturated fatty acids and comprised over 50% of the fatty acids at position 1 of the glycerol moiety. The monoene content of positions 1 and 2 decreased in response to the addition of di- and polyenoic acids to the culture medium. All the di- and polyenoic acid supplements tested inhibited the desaturation of palmitic and stearic acid and replaced monoenes at position 2. However only linoleic, linolenic, and eicosadienoic acids replaced monoenes at position 1. The results suggest that under appropriate conditions up to 25% of the choline phosphoglyceride fraction consisted of a stable molecular species containing di- or trienoic fatty acids at both the 1 and 2 positions of glycerol moiety. With eicosatrienoic or arachidonic acid supplements, on the other hand, the monoenes at position 1 were replaced with saturated fatty acids. The magnitude of these effects, particularly at position 1, was proportional to the concentration of the fatty acid supplement. The results suggest that polyenes with at least 20 carbon atoms can play a key role in determining the ultimate composition and positioning of fatty acids in baby hamster kidney choline phosphoglycerides and that this control is mediated by their ability to inhibit delta 9 desaturase and by a retailoring system specific for these polyenes.

Amino acid sequence of the protein inhibitor of mitochondrial adenosine triphosphatase.

The complete amino acid sequence of the mitochondrial ATPase inhibitor peptide was determined. The molecule contains 84 residues of which 40 are charged amino acids that occur in clusters along the chain. A section of the chain, located at the COOH-terminal end, contains several duplicated regions, the most prominent of which are pentapeptides. This section of the chain also contains all of the five histidines present in the molecule. Some of the physicochemical properties of the protein and an improved purification procedure are described.