NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

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Abstract
Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD(+)- and NADP(+)-isocitrate dehydrogenases, NAD(+)- and NADP(+)-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP(+)-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP(+)-malate dehydrogenase activity is probably due to unspecificity of the NAD(+)-malate dehydrogenase. NADP(+)-specific isocitrate dehydrogenase had much lower K(m)s for NADP(+) and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD(+)-specific enzyme (101 micromolar for NAD(+) and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP(+)-specific isocitrate dehydrogenase whereas the NAD(+)-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP(+) stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP(+) is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP(+)-reducing activities of malate dehydrogenase and the NADP(+)-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.