Inhibition par la N-éthylmaleimide des transports du glutamate et du phosphate dans les mitochondries

Abstract

Summary We have studied the inhibitory effect of NEM at the level of phosphate and glutamate transport in rat liver and pig heart mitochondria. Two kinds of methods have been utilised: a kinetic method (swelling of mitochondria), a metabolic method where glutamate oxidation can be considered as a resultant between transport and substrate metabolism. Following results have been obtained : 1.— Phosphate and glutamate transport, estimated by swelling methods, are inhibited by NEM in rat liver mitochondria : 0.1 mM NEM, 15 seconds, results in a 50 p. cent inhibition of phosphate transport, whereas under the same conditions glutamate transport is affected by 15 p. cent. These results are consistent with a different localisation of the two transport systems in the internal mitochondrial membrane. 2.— Phosphate transport inhibition by NEM (rat liver and pig heart mitochondria) is protected by substrate. That is to say, phosphate decreases the inhibition by NEM when added to the preincubation medium 30 seconds before NEM. This « protectionis more extensive in pig heart mitochondria : 2 mM phosphate decreases NEM inhibition by 75 p. cent (NEM 0.2 mM, 45 seconds). In rat liver mitochondria the following values are obtained 10 mM phosphate decreases NEM inhibition by 50 p. cent (NEM 0.1 mM, 30 seconds). In the case of glutamate transport, in rat liver mitochondria, there is no protection by substrate. 3.— Glutamate oxidation, in rat liver mitochondria, is inhibited by 0.1 mM NEM. The same results are observed for coupled and uncoupled mitochondria by ClCCP. In the coupled state glutamate and phosphate transport are concerned, in the uncoupled state only glutamate transport is concerned. Succinate oxidation is less inhibited by NEM. All the data are consistent with a simultaneous effect of NEM at different sites : the phosphate and glutamate carriers but also the electron transfer enzymes. Pig heart mitochondria do not swell in 100 mM ammonium glutamate.