On the Occurrence and Structure of Subunits of Endopolygalacturonase Isoforms in Mature-Green and Ripening Tomato Fruits

Abstract

Endopolygalacturonase [poly(1,4-α-galacturonide) glycanohydrolase EC 3.2.1.151 occurs in tomato fruit in three molecular forms- PG1, PG2A, PG2B. Trace amounts of PG1, 1-10 pkat g^-1 are shown to occur in mature-green fruit as compared to 17 nkat in ripe fruit. As polygalacturonase activity increases through ripening, the percentage of the activity due to PG1 decreases progressively from 100 to less than 20. On fully or partly demethylated substrates, PG1 is more active than PG2 when the ionic strength is that expected in the tissue apoplast. A method for purifying PGI from ripe fruit is described. PG1 preparations contain polypeptides of Mr 45, 43 and 38 thousand. The Mr 43 thousand and 45 thousand components correspond in size to PG2A and PG2B and are detected by antisera raised against PG2A. The M, 38 thousand polypeptide is immunologically distinct. From carbohydrate and amino acid analyses, this polypeptide appears to contain 2870 carbohydrate as glucosamine, mannose, xylose and fucose attached to a polypeptide of estimated Mr 28 342 that is rich in tyrosine and glycine. A method for purifying the subunits of PG1 by cation exchange chromatography in 6 M urea is described. PG2A and PG2B were separated by column chromatography and shown to have identical N-terminal sequences, and serine at the C-terminus. PG2A and PG2B are confirmed as two glycoforms of the one polypeptide. The possibility that PGl consists of populations of molecules containing either PG2A or PG2B coupled with the Mr 38 thousand polypeptide is discussed.