ATP stimulates proteolysis in reticulocyte extracts by repressing an endogenous protease inhibitor.
An endogenous inhibitor of the reticulocyte ATP-dependent proteolytic system has been purified partially by ammonium sulfate precipitation from rabbit reticulocyte and erythrocyte extracts. Inhibitor-free protease rapidly degrades 21-40% of the substrate [14C]methyl-alpha-casein per hour, resembling ATP-dependent activity in reticulocyte extracts. This proteolytic activity is not stimulated by ATP and does not respond to ubiquitin. Adding back the inhibitory fraction to reticulocyte inhibitor-free protease results in a significant decrease (65-75%) in proteolysis, both in the presence and absence of ATP. In contrast, inhibition is repressed when both ATP and the ubiquitin-containing fraction are present, resulting in an 80-350% stimulation of proteolysis by these components. These results suggest that ATP, in the presence of ubiquitin, may act in releasing the protease(s) from its endogenous inhibitor. Erythrocyte extracts, unlike reticulocyte extracts, exhibit low levels of ATP-dependent proteolytic activity. However, ion-exchange chromatography reveals that erythrocytes contain levels of proteolytic activity that are comparable to the reticulocyte's inhibitor-free protease. Addition of ubiquitin and inhibitor to erythrocyte protease results in a highly ATP-dependent activity that resembles levels of ATP-dependence (3- to 4-fold) seen in reticulocyte extracts. Thus, the proteolytic and inhibitory components of the ATP-dependent proteolytic system appear to be retained with reticulocyte maturation. However, some other component(s) of the system are lost or modified with maturation so that the protease remains inactive.
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002250:Carbon Radioisotopes; D002364:Caseins; D002868:Chromosomal Proteins, Non-Histone; D004912:Erythrocytes / Q000201:enzymology; D007700:Kinetics; D009698:Nucleoproteins; D010447:Peptide Hydrolases / Q000097:blood*; D011480:Protease Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D014452:Ubiquitins
D002250:Carbon Radioisotopes; D002364:Caseins; D002868:Chromosomal Proteins, Non-Histone; D009698:Nucleoproteins; D011480:Protease Inhibitors; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases