Studies on in vivo inactivation of gramicidin S synthetase and its retardation

Abstract

The oxygen-dependent in vivo inactivation of gramicidin S synthetase was investigated in Bacillus brevis ATCC 9999. Inhibitors of energy metabolism and of protein synthesis added to aerated cell suspensions did not provide any protection against inactivation, thus indicating that the process does not depend on energy-yielding metabolism nor on de novo protein synthesis. Organic thiols added to anaerobic long-term incubations retarded synthetase inactivation for several hours, whereas in short-term incubations of previously air-exposed cells they resulted in partial restoration of activity. The in vivo inactivation of the enzyme was found to be accompanied by a parallel drop in intracellular thiols. These results implicate enzyme SH oxidation as a mechanism of in vivo inactivation. Retardation of inactivation was achieved upon addition of utilizable carbon sources (glycerol, fructose, inositol) to aerated cell suspensions in buffer, the degree of stabilization being proportional to the ease of uptake and to the concentration of C source. This effect involves actual consumption of the exogenous C source and is accompanied by lower dissolved oxygen levels in the cell suspension. Pulsed additions of C source could retard inactivation but could not restore partly or fully lost activity. The C-source effect was blocked by the uncoupler dinitrophenol, while dissolved oxygen levels in the suspension remained low. C-source-supplemented cell suspensions incubated under air had a decreased intracellular redox state, as revealed by intracellular SH concentration.