Polarographic Study of Dicarboxylic-Acid-dependent Export of Reducing Equivalents from Illuminated Chloroplasts

Abstract

Isolated pea chloroplasts, prepared by differential centrifugation, catalyzed 02 evolution in the light in the presence of 0.03 to 3 millimolar malate, 0.12 to 1.2 millimolar NAD, 4 milimolar pyruvate and exogenous NAD-malate dehydrogenase and lactate dehydrogenase. The reaction, which did not occur in the absence of any one of these factors, was accompanied by the consumption of pyruvate; the ratio of 02 evolved to pyruvate consumed was <0.5. As discussed in recent reviews (5, 9, 17) there is evidence that reducing equivalents generated by the light reactions of chloroplasts can be transferred to the cytoplasm. Since the chloroplast envelope is impermeable to NADP(H) and NAD(H) (5,7, 17), transfer of the reducing equivalents must be mediated indirectly via a carrier.The light-dependent reduction of OAA' to malate, which is mediated via NADP-MDH in the chloroplast (2), has been implicated as a key reaction in the proposed OAA/malate shuttle (5, 9, 17) (see Fig. 4 Massey Gem) were raised in a growth cabinet as before (8) and harvested 13 or 14 days after imbibition. Method 1 chloroplasts were prepared as in reference 1 and method 2 chloroplasts as in reference 2. In summary, method 1 entails extracting pea shoots (100 g) in an icy slush of medium (250 ml) containing 0.33 M glucose, 50 mm Na2HPO4, 50 mm KH2PO4, 5 mM MgCl2, 0.1%