On the relationship between the metabolic and thermodynamic stabilities of T4 lysozymes. Measurements in eukaryotic cells.
We have measured the metabolic stabilities of wild-type and 17 temperature-sensitive mutants of T4 lysozyme in HeLa cells, in Xenopus egg extract, and in reticulocyte lysate. [35S]Methionine-labeled T4 lysozymes were expressed in Escherichia coli, purified, injected into HeLa cells, 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 in the set, R96H, was significantly more stable (half-life = 10 h). Different T4 lysozyme variants yield conflicting answers to the proposed relationship between thermal and metabolic stabilities. For mutations at Thr157 there is no correlation between melting temperature and half-life. By contrast, T4 lysozymes mutated at various positions show a definite correlation between the two parameters. Treatment of injected HeLa cells with the lysosomotropic agents chloroquine or ammonium chloride 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 stable in reticulocyte lysate and Xenopus egg extract. Presumably, there is a specific proteolytic event(s) in HeLa cells which is not manifest in the in vitro extracts.
Inoue
I
I
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Rechsteiner
M
M
D000818:Animals; D017122:Bacteriophage T4 / Q000201:enzymology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D006367:HeLa Cells; D006801:Humans; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D013816:Thermodynamics; D014981:Xenopus
D009113:Muramidase