Changes in Polygalacturonase Isoenzymes During the 'ripening' of Normal and Mutant Tomato Fruit

1. Polygalacturonase activity is not detectable in mature green tomato fruits but appears as fruits begin to change colour and continues to increase during the ripening period. There is a sequential appearance of two isoenzymes, polygalacturonase 1 and 2, during ripening. These isoenzymes have been purified and their properties compared. Polygalacturonase 1 has a Mr of 100,000, is 50% inactivated at 78 degrees C and has a density of 1.343 g cm-3 in caesium chloride. Polygalacturonase 2 has a Mr of 42,000, is 50% inactivated at 57 degrees C and has a density of 1.300 g cm-3 in caesium chloride. 2. Fruits from isogenic lines homozygous for the 'Neverripe' (Nr) mutation do not ripen normally and contain reduced amounts of polygalacturonase. Only polygalacturonase 1 is produced in Nr fruit. Tomatoes from isogenic lines homozygous for the 'ripening inhibitor' (rin) mutation do not ripen normally and produce very little detectable polygalacturonase. 3. Although polygalacturonases 1 and 2 have different properties they both give rise to a single polypeptide on electrophoresis in polyacrylamide gels in the presence of sodium dodecyl-sulphate (Mr = 46,000). A comparison of the major fragments produced by limited proteolysis of polygalacturonase 1 and 2 with chymotrypsin suggests that the polypeptides from the two isoenzymes are similar. The same conclusion was reached from a comparison of polygalacturonase 1 and 2 by radioimmunoassay, using antibody prepared against polygalacturonase 2 and 125I-labelled polygalacturonase 2. 4. The results from radioimmunoassay of extracts from green and ripening fruits suggest that the increase in polygalacturonase activity during ripening is due to net synthesis of protein.