EVIDENCE FOR A CYTOSOLIC NADP-MALIC ENZYME IN TOMATO

The similarity and cellular location of NADP(+)-malic enzyme (NADP-ME, EC 1.1.1.40) in developing fruit and other parts of the tomato (Lycop ersicon esculentum Mill.) plant were investigated in order to clarify the role of the enzyme in metabolism. There appeared to be a single en zyme (native MW 260 kD, subunit MW 66 kD) in mature green tomato fruit ; this was purified to a specific activity of 42.5 mu mol mg(-1) min(- 1) and apparent homogeneity on SDS-PAGE. Polyclonal antibodies raised against this protein achieved 90% precipitation of enzyme activity, bu t required purification in order to detect specifically the 66 kD prot ein on Western blots. The purified antibodies recognized a similar pro tein in tomato leaves, roots and stems. The specific activity of NADP- ME was at least nine times higher in supernatant than in chloroplast f ractions in tomato fruit and leaf, consistent with the distribution of a cytoplasmic marker (alcohol dehydrogenase), but in contrast to a ch loroplast marker (rubisco). The purified NADP-ME antibodies did not de tect a 66 kD protein in chloroplast preparations. A partial NADP-ME cD NA isolated from a fruit library specifically hybridized with a 2.1 kb transcript in RNA preparations from tomato leaves and fruits at diffe rent stages of development. The existence of a minor, possibly chlorop lastidic NADP-ME in tomato cannot be excluded; the major NADP-ME is a cytosolic protein which is present in all plant organs analysed.