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 (M_r) 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 M_r 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, M_r 43 000, reacted also with the larger enzymes. After denaturation in sodium dodecyl sulfate, the largest (M_r 115 000) and smallest (M_r 43 000) enzymes yielded polypeptides of identical size while the third enzyme (M_r 46 000) was slightly larger. All three enzymes were glycoproteins. From fully ripe fruits, enzyme preparations with specific activities of 1.7 µkat mg^-1 were obtained. This specific activity exceeds those described previously for plant polygalacturonases.