Reduction of Nitrate via a Dicarboxylate Shuttle in a Reconstituted System of Supernatant and Mitochondria from Spinach Leaves

Abstract

Substantial rates of nitrate reduction could be achieved with a reconstituted system from spinach leaves containing supernatant, mitochondria, NAD+, oxaloacetate (OAA), and an oxidizable substrate. Appropriate substrates were glycine, pyruvate, citrate, isocitrate, fumarate or glutamate. The reduction of NO3- with any of the substrates could be inhibited by n-butyl malonate, showing that the transfer of reducing power from the mitochondria to the supernatant involved the malate exchange carrier. The addition of ADP to the reconstituted system decreased NO3- reduction and this decrease could be reversed by the addition of rotenone or antimycin A. The operation of the OAA/malate shuttle was achieved most quickly in the system when low concentrations (.ltoreq. 0.1 mM) of OAA were added. A corresponding increase in the lag time for the operation of the OAA/malate shuttle was observed when the OAA concentration was increased. Concentrations for half-maximal activity of OAA, glycine, NAD+ and NO3- in the reconstituted system were 42 .mu.M, 0.5 mM, 0.25 mM and 26 .mu.M, respectively. The transfer of reducing power from the mitochondria to the soluble phase via the OAA/malate shuttle can not only provide NADH for cytoplasmic reduction but can also sustain oxidation of tricarboxylic cycle acids and the generation of .alpha.-ketoglutarate independently of the respiratory electron transport chain.