Co-ordinate variations in methylmalonyl-CoA mutase and methionine synthase, and the cobalamin cofactors in human glioma cells during nitrous oxide exposure and the subsequent recovery phase

We investigated the co-ordinate variations of the two cobalamin (Cbl)-depen dent enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MCM), and measured the levels of their respective cofactors, methylcobalamin (CH( 3)Cbl) and adenosylcobalamin (AdoCbl) in cultured human glioma cells during nitrous oxide exposure and during a subsequent recovery period of culture in a nitrous oxide-free atmosphere (air). In agreement with published data, MS as the primary target of nitrous oxide was inactivated rapidly (initial rate of 0.06 h(-1)), followed by reduction of CH(3)Cbl (to < 20%,). Both e nzyme activity and cofactor levels recovered rapidly when the cells were su bsequently cultured in air, but the recovery was completely blocked by the protein-synthesis inhibitor, cycloheximide. During MS inactivation, there w as a reduction of cellular AdoCbl and holo-MCM activity (measured in the ab sence of exogenous AdoCbl) to about 50%, of pre-treatment levels. When the cells were transferred to air, both AdoCbl and holo-MCM activity recovered, albeit more slowly than the MS system. Notably, the regain of the holo-MCM and AdoCbl was enhanced rather than inhibited by cycloheximide. These find ings confirm irreversible damage of MS by nitrous oxide; hence, synthesis o f the enzyme is required to restore its activity. In contrast, restoration of holo-MCM activity is only dependent on repletion of the AdoCbl cofactor. We also observed a synchronous fluctuation in AdoCbl and the much larger h ydroxycobalamin pool during the inactivation and recovery phase, suggesting that the loss and repletion of AdoCbl reflect changes in intracellular Cbl homoeostasis. Our data demonstrate that the nitrous oxide-induced changes in MS and CH(3)Cbl are associated with reversible changes in both MCM holoa ctivity and the AdoCbl level, suggesting co-ordinate distribution of Cbl co factors during depletion and repletion.