Protein Degradation at Neutral pH. Possible Enzymic and Control Mechanisms 1

Abstract

Several investigators (1-3) have shown that in tissue slices the process of protein degradation at neutral pH can be significantly reduced by the uncoupling or inhibition of the oxidative phosphorylation. Complementary studies have indicated that the addition of adenosine-5′-triphosphate (ATP) to preparations of intracellular organelles (4) or to whole tissue homogenates (5, 6) greatly increase the rate of proteolysis, and that this activating effect is mediated by aspartic acid, presumably through the formation of an adenylate derivative (7). This activating effect of ATP was found to be widespread in the organs of the rat (6) and to be able to return to a normal level the reduced proteolytic activity of the liver of rats subjected to an acute stress (8).

All these studies indicate that not only is there a regulatory mechanism for the control of protein degradation at neutral pH, but also that the same mechanism may be involved in the correlation of the rate of protein degradation with the rate of protein synthesis during turnover. This possibility has increased the need to elucidate the nature of the proteolytic enzymes responsible for the neutral proteolytic activity and particularly, the need to identify and characterize the enzyme or enzymes activated by ATP.

This article describes an effort to determine the main types of proteolytic enzymes active in liver homogenates at pH 7.5 and describes their behavior toward ATP during 2 hr of autolysis. These studies have allowed the formulation of an hypothesis, which provides a mechanism for the control of neutral proteolysis and for the interaction of this process with the process of protein synthesis.

Materials and Methods. These studies were carried out with 10% liver homogenates made in 0.22 M phosphate buffer (pH 7.5)-0.2% Triton X-100. The amount of protein in the homogenate was determined according to the method of Lowry et al. (9) using bovine serum albumin as standard.

Studies with synthetic substrates and modified albumin. Qualitative studies were carried out with 50 mM solutions of the following synthetic substrates³: L-leucine amide, L-phenylalanine amide, glycyl-L-phenylalanine, glycyl-L-phenylalanine amide, glycyl-L-phenylalanyl-L-phenylalanine, L-phenylalanyl-L-phenylalanine, L-phenylalanyl-L-tyrosine, L-leucyl-L-tyrosine, L-tyrosyl-L-leucine, N-carbobenzoxy-glycyl-L-tyrosine, N-carbobenzoxy-L-glutamyl-L-tyrosine, and α-N-benzoyl-L-arginine amide. Two hundred microliter of a peptide solution were incubated for 2 hr at 37° with the same volume of a 4% homogenate made by diluting a 10% homogenate with 0.22 M phosphate buffer, pH 7.5. At this time 200 μl of 10% tricholoroacetic acid (TCA) were added, and 50 μl of the supernatant were spotted on a 20 X 10-in. sheet of Whatman No. 1 paper. For identification purposes a similar volume of a 5 mM reference solution of each one of the possible products of hydrolysis of the peptide in question was simultaneously spotted. The constituents in the spots were separated with a solvent mixture containing n-butanol: acetic acid: pyridine: water (15:3:10:12) by descending chromatography after 6 hr of equilibration. The formation of Ninhydrin-positive products other than the products of hydrolysis of the synthetic peptides was monitored by spotting the TCA supernatant of incubation mixtures in which the liver homogenate had been previously heated at 95° or in which the substrate solution had been substituted for phosphate buffer. The products were developed with a Ninhydrin spray and were identified by comparing their rate of migration with the rate of the appropriate reference solutions.