Effects of temperature on the degradation of proteins in rabbit reticulocyte lysates and after injection into HeLa cells.
Bovine serum albumin, pyruvate kinase, hemoglobin, and the Fc fragment of IgG were labeled and introduced into HeLa cells by erythrocyte-mediated microinjection. Degradation of the injected proteins was then measured in cells cultured at temperatures between 6 degrees C and 37 degrees C. Arrhenius plots revealed a constant Ea of 27 +/- 5 kcal/mol over this temperature interval. Similarly, the apparent Ea for the degradation of long-term endogenously labeled HeLa proteins was 22-26 kcal/mol. Both local protein unfolding and proteolysis by defined enzymes, such as trypsin or papain, proceed with EaS between 5 and 15 kcal/mol. The 2-fold higher values obtained in this study indicate that protein unfolding or simple proteolysis is not rate limiting in the degradation of injected or long-lived endogenous HeLa proteins. Moreover, the relatively uniform EaS suggest that a similar biochemical event is rate limiting in the degradation of a specific protein independent of its half-life. This event may involve a reaction in the ATP-dependent proteolytic pathway from rabbit reticulocyte lysates because we observed that EaS for ATP-dependent proteolysis in this system were also 27 +/- 5 kcal/mol.
D000818:Animals; D006367:HeLa Cells / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments; D007700:Kinetics; D051379:Mice; D008845:Microinjections; D009113:Muramidase / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011506:Proteins / Q000008:administration & dosage / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
D006454:Hemoglobins; D007141:Immunoglobulin Fc Fragments; D011506:Proteins; D012710:Serum Albumin, Bovine; D011770:Pyruvate Kinase; D009113:Muramidase; D010447:Peptide Hydrolases