Effects of temperature on the degradation of proteins in rabbit reticulocyte lysates and after injection into HeLa cells.

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Abstract
Bovine serum albumin, pyruvate kinase, Hb and the Fc fragment of IgG were labeled and introduced into [human cervical carcinoma] HeLa cells by erythrocyte-mediated microinjection. Degradation of the injected proteins was then measured in cells cultured at temperatures between 6.degree. C and 37.degree. C. Arrhenius plots revealed a constant Ea [activation energy] 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 Eas for ATP-dependent proteolysis in this system were also 27 .+-. 5 kcal/mol.