Purification and Characterization of the Polygalacturonases of Tomato Fruits

Abstract

Endopolygalacturonase [poly(1,4-α-D-galacturonide) glycanohydrolase, EC 3.2.1.15] was purified from ripening tomato fruits. The dominant enzyme in fruits at an early stage of ripening had a native molecular weight (Mr) of about 115 000, an isoelectric point (pI) of 8.6, and retained 50% of activity after 10 min at 70°C. Divalent metal ions enhanced the activity of this enzyme and ethylenediaminetetraacetate (EDTA) inhibited activity. In fully ripe fruits, two enzymes of Mr about 43 000 and 46 000 were most prominent. These enzymes each had a pI of 9.4; when a mixture of these two enzymes was heated for 10 min at 50°C, 50% of activity was lost. Antibodies raised against the smallest enzyme, Mr 43 000, reacted also with the larger enzymes. After denaturation in sodium dodecyl sulfate, the largest (Mr 115 000) and smallest (Mr 43 000) enzymes yielded polypeptides of identical size while the third enzyme (Mr 46 000) was slightly larger. All three enzymes were glycoproteins. From fully ripe fruits, enzyme preparations with specific activities of 1.7 �kat mg-1 were obtained. This specific activity exceeds those described previously for plant polygalacturonases.