Reevaluation of the Role of ATP in Autolysis of Liver Protein 1

Abstract

Evidence for a postulated energy requirement for protein degradation in vivo was investigated by measuring the effects of potential energy sources on the release of ninhydrin-positive material during autolysis of rat liver homogenate at pH 7.5. Addition to the reaction mixture of low levels of ATP (9.1 × 10^-2 mM), an amino acid mixture, or both ATP (9.1 × 10^-2 mM) plus amino acids all failed to stimulate autolysis of liver proteins as measured by release of ninhydrin-positive material. Large nonphysiological concentrations of ATP (4.8 or 8.5 mM) or of 5′-AMP (8.5 mM) markedly stimulated the process, however about 80% of the added ATP disappeared within the first 5 min of incubation, whereas ninhydrin-positive material accumulated with time. Measurement of ammonia release during incubation of homogenate with high concentrations of ATP or AMP indicated that after 90 min the amount of ammonia released was proportional to the amount of ATP added from 0–4.8 mM ATP and that addition of amino acids to the incubation mixture did not further increase ammonia release. As ammonia increased, so did production of inosine and hypoxanthine. The results indicate that ATP does not enhance proteolysis and provide evidence that ATP is a source of substrate for deamination reactions which contribute ninhydrin-positive material during autolysis of liver.

Since the 1950's several publications (1–3) have indicated that an energy source such as ATP may be required for intracellular protein degradation in the liver, however the role of an energy source in this process is not clearly established. Recently Brostrom and Jeffay (4) concluded from a study of protein catabolic activity in several types of rat liver preparations (slice, homogenate, and a soluble sonicated preparation), that liver protein catabolism may require a “structural component” (a recognition site, possibly related to interaction of substrate with catabolic enzymes, that would permit specificity in the degradation of proteins) the integrity of which depends upon a supply of metabolic energy, whereas proteolytic activity probably has no such requirement.