Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.

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Abstract
The heat-stable polypeptide ATP-dependent proteolysis factor 1 (APF-1) of the [rabbit] reticulocyte proteolytic system forms covalent compounds with proteins in an ATP-requiring reaction. APF-1 and [hen egg white] lysozyme, a good substrate for ATP-dependent proteolysis, form multiple conjugates, as was shown by comigration of label from each upon gel electrophoresis. Multiple bands were seen with other substrates of the ATP-dependent proteolytic systems, such as globin or .alpha.-lactalbumin. Analysis of the ratio of APF-1 to lysozyme radioactivities and of the MW of the bands indicated that they consist of increasing numbers of the APF-1 polypeptide bound to 1 molecule of lysozyme. The covalent linkage is probably of an isopeptide nature, because it is stable to hydroxylamine and alkali, and polylysine is able to give conjugates of APF-1. Removal of ATP after formation of the 125I-labeled APF-1 conjugates with endogenous proteins caused the regeneration of APF-1, indicating presence of an amidase. This reaction is thought to compete with proteases that may act on APF-1-protein conjugates, especially those containing several APF-1 ligands. A sequence of reactions in which linkage of APF-1 to the substrate is followed by proteolytic breakdown of the substrate is proposed to explain the role of ATP.