Characterization of the Heat - stable Polypeptide of the ATP - dependent Proteolytic System

The energy dependence of intracellular protein breakdown has been recognized for a long time (1,2), but its biochemical mechanisms have begun to be elucidated only recently. This has been initiated by the establishment of several cell-free systems in which protein breakdown is stimulated by ATP (37 ) . We have found previously that the ATP-dependent proteolytic system from reticulocytes is composed of several components (8,9). The first component to be isolated, APF-1,’ is a relatively small, heat-stable polypeptide that has no protease activity by itself but stimulates protein breakdown in the presence of the other components and ATP (8). Subsequently, we have found that ‘251-labeled APF-1 forms covalent conjugates with endogenous reticulocyte proteins or with exogenous protease substrates in an ATP-requiring reaction (10, 11). In this reaction, 1 or more molecules of APF-1 are bound to 1 molecule of the substrate protein (11). Upon the removal of ATP, the breakdown of the conjugates has been observed, with the regeneration of free APF-1. It has been proposed that the conjugation of APF-1 is followed by the proteolytic breakdown of the substrate (11). In the present paper, we describe the purification and characterization of apparently homogeneous APF-1 from rab-