A GLYCOPROTEIN INHIBITOR OF PECTIN METHYLESTERASE IN KIWI FRUIT - PURIFICATION BY AFFINITY-CHROMATOGRAPHY AND EVIDENCE OF A RIPENING-RELATED PRECURSOR

The pectin methylesterase inhibitor from kiwi fruit (Actinidia chinens is) was purified by a single-step procedure based on affinity chromato graphy. Partially purified tomato pectin methylesterase was covalently bound to Sepharose. The affinity resin strongly and selectively binds the inhibitor, which could be eluted in high yield as a single, homog eneous and sharp peak by high salt concentration at pH 9.5 without los s of inhibitory activity. The purified protein possesses a molecular m ass of 18 kDa, as estimated by SDS/PAGE, whereas by gel filtration und er native conditions, its molecular mass appears to be 25 kDa. The inh ibitor interacts with pectin methylesterase, forming a 1:1 complex, as demonstrated by gel-filtration experiments. The inhibitor was glycosy lated. Its glycidic portion can be removed by digestion with N-glycosi dase F after protein denaturation and, to a minor extent, by digestion with N-glycosidase H. No glycidic residue could be removed by digesti ng the native protein with those N-glycosidases. Antibodies against pe ctin methylesterase inhibitor were raised in rabbits and used to evide nce protein expression during fruit ripening. The results showed that the inhibitor is present in the unripe fruit as an inactive precursor with a higher molecular mass (30 kDa) and is transformed into the acti ve protein, most likely by proteinase action, during the course of the ripening process.