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Synthesis and inactivation of bacterial luciferase determined by immunochemical techniques. Comparison with total protein synthesis and turnover.

1982; Elsevier BV; Volume: 257; Issue: 2 Linguagem: Inglês

10.1016/s0021-9258(19)68304-3

1083-351X

Carole Reeve, Thomas Baldwin,

Advanced Chemical Sensor Technologies

The luciferase of the luminous marine bacterium Vibrio harveyi is rapidly inactivated during late log phase and stationary phase.A n analysis of the rates of synthesis and degradation of total protein and of luciferase throughout growth of the culture shows that the rate of total protein synthesis per cell was maximal in midlog phase and declined steadily to a very low level in stationary phase.The synthesis of luciferase was maximal in early log phase and declined throughout log phase growth to near zero at about the time that luciferase inactivation began.Luciferase was stable throughout exponential growth and accumulated in the cells, but during stationary phase, luciferase cross-reacting material was lost from the 100,000 X g supernatant of cell extracts at the same rate as luciferase activity.Neutralization of luciferase activity in uncentrifuged lysates with antiluciferase antibody, however, suggested an accumulation of inactive cross-reacting material during stationary phase.The antibody was shown to complex and precipitate proteolytic fragments of luciferase produced in vitro.Furthermore, the antibody was shown to recognize chymotrypsintreated luciferase as well as native luciferase in the luciferase activity neutralization assay.Chloramphenicol blocked protein synthesis quite effectively while by comparison, cyanide acted very slowly and ineffectively to block protein synthesis.Cyanide blocked total protein turnover immediately while the effect of chloramphenicol upon protein turnover was not apparent until 1-2 h after addition.Even though cyanide blocked total protein turnover immediately, luciferase inactivation continued for about 1 h, and, as in the experiment without cyanide, the luciferase protein was lost from centrifuged lysates at the same rate as luciferase activity.Inactivation of bacterial luciferase appears to be associated with a decrease in solubility, such that the inactive protein is separated from the active luciferase in lysates by centrifugation.