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.

Reduction of polygalacturonase activity in tomato fruit by antisense RNA

“…Fruit pericarp tissue was homogenized with a Brinkmann Polytron in 3 vol of 1.3 M NaCl/0.05 M sodium phosphate, pH 6.0/40 mM 2-mercaptoethanol. The supernatant was recovered after centrifugation (11,000 x g, 30 min) and assayed for PG activity as described (6).…”

“…PG is involved in cell wall degradation and is the most extensively studied enzyme involved in ripening (6,7). Characterizations of PG cDNAs have indicated that the polypeptide has a molecular weight of S51,000, including a 71-amino acid N-terminal sequence which may be involved in export of PG to the cell wall (3,5).…”

The beta subunit of tomato fruit polygalacturonase isoenzyme 1: isolation, characterization, and identification of unique structural features.

“…Although there is only a single gene for the catalytic PG polypeptide, the total PG activity isolated from ripe tomato fruit is attributable to a mixture of several closely related, post-translationally derived isoenzymes, PG1, PG2A, and PG2B (Pressey and Avants, 1973;Ali and Brady, 1982;Moshrefi and Luh, 1983;DellaPenna and Bennett, 1988). The PG2A and PG2B isoenzymes (herein referred to as the PG2 isoenzymes) accumulate late in the ripening process and are each composed of a single catalytic PG polypeptide differing only in the degree of glycosylation (Ali and Brady, 1982;DellaPenna and Bennett, 1988). The PG1 isoenzyme accumulates first during ripening and is thought to be composed of one or two catalytic PG2 polypeptides tightly associated with an ancillary glycoprotein, the p subunit protein (Moshrefi and To whom correspondences should be addressed.…”

Tomato Fruit Cell Wall

“…Previous reports focused on the physical properties of the enzyme including temperature of inactivation and specific ion interactions (3). These features of PG action have generally been evaluated using substrates, usually citrus pectin, of unspecified composition.…”