Evidence for a cytosolic NADP-malic enzyme in tomato

Abstract

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 ( Lycopersicon esculentum Mill.) plant were investigated in order to clarify the role of the enzyme in metabolism. There appeared to be a single enzyme (native NW 260 kD, subunit MW 66 kD) in mature green tomato fruit; this was purified to a specific activity of 42.5 μmol mg −1 min −1 and apparent homogeneity on SDS-PAGE. Polyclonal antibodies raised against this protein achieved 90% precipitation of enzyme activity, but required purification in order to detect specifically the 66 kD protein on Western blots. The purified antibodies recognized a similar protein in tomato leaves, roots and stems. The specific activity of NADP-ME was at least nine times higher in supernatant than in chloroplast fractions in tomato fruit and leaf, consistent with the distribution of a cytoplasmic marker (alcohol dehydrogenase), but in contrast to a chloroplast marker (rubisco). The purified NADP-ME antibodies did not detect a 66 kD protein in chloroplast preparations. A partial NADP-ME cDNA isolated from a fruit library specifically hybridized with a 2.1 kb transcript in RNA preparations from tomato leaves and fruits at different stages of development. The existence of a minor, possibly chloroplastidic NADP-ME in tomato cannot be excluded; the major NADP-ME is a cytosolic protein which is present in all plant organs analysed.