Microtubule Assembly and Function in Normal and Glutathione Synthetase-Deficient Polymorphonuclear Leukocytes

Abstract Depression of glutathione (GSH) by specific and non-specific oxidants has been shown before to cause microtubule disassembly and to prevent Concanavalin A (Con A)-induced assembly in human polymorphonuclear leukocytes. These data indicate either that a critical level of GSH is required for assembly or that products of GSH oxidation such as glutathione disulfide (GSSG) cause disassembly. Neutrophils from a patient with GSH synthetase deficiency contain only 20% normal GSH (far below the depressed level associated with microtubule inhibition in normal cells) with no corresponding elevation of GSSG. These cells provide a test system to analyze the biochemical pathway connecting GSH to microtubule integrity. Con A induces a rapid and equal increase in centriole-associated microtubules in both normal and GSH-deficient cells. Thus, levels of GSH above 20% of normal are not essential to promote microtubule assembly. On the other hand, our data emphasize an important role for normal (high) GSH in protecting tubulin against direct attack by oxidants such as H2O2: in normal neutrophils phagocytosis induces microtubule assembly despite concomitant production of H2O2; in GSH-deficient cells microtubule assembly is prevented. Tertiary butylhydroperoxide (BHP) is confirmed to be a highly specific GSH oxidant, inhibiting Con A-induced microtubule assembly in normal but not GSH-deficient cells. In both cases the GSH:GSSG ratio is lowered from approximately 100:1 to below 10:1. However, the absolute level of GSSG achieved after BHP in GSH-deficient cells is only one-fifth that obtained in normals. It is suggested that a critical level of GSSG must be attained to inhibit microtubule assembly and/or cause microtubule disassembly after the specific oxidation of GSH.