ON THE RELATIONSHIP BETWEEN THE METABOLIC AND THERMODYNAMIC STABILITIES OF T4 LYSOZYMES - MEASUREMENTS IN EUKARYOTIC CELLS

We have measured the metabolic stabilities of wildtype and 17 temperat ure-sensitive mutants of T4 lysozyme in HeLa cells, in Xenopus egg ext ract, and in reticulocyte lysate. [S-35]Methionine-labeled T4 lysozyme s were expressed in Escherichia coil, purified, injected into HeLa cel ls, and their degradation rates were determined. Wild-type T4 lysozyme has a half-life of 4 h; the half-lives of 16 lysozyme variants ranged from 2 to 10 h. Surprisingly, the most temperature-sensitive enzyme i n the set, R96H, was significantly more stable (half-life = 10 h). Dif ferent T4 lysozyme variants yield conflicting answers to the proposed relationship between thermal and metabolic stabilities. For mutations at Thr(157) there is no correlation between melting temperature and ha lf life. By contrast, T4 lysozymes mutated at various positions show a definite correlation between the two parameters. Treatment of injecte d HeLa cells with the lysosomotropic agents chloroquine or ammonium ch loride did not alter the stability of T4 lysozyme. However, the enzyme 's half-life increased 10-fold in HeLa cells depleted of ATP. Although T4 lysozyme is degraded rapidly within HeLa cells, the molecule is st able in reticulocyte lysate and Xenopus egg extract. Presumably, there is a specific proteolytic event(s) in HeLa cells which is not manifes t in the in vitro extracts.