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The inactivation of pectin depolymerase associated with isolated tomato fruit cell wall: implications for the analysis of pectin solubility and molecular weight
Author(s) -
Huber Donald J.
Publication year - 1992
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1992.tb01307.x
Subject(s) - pectin , pectinase , cell wall , chemistry , lycopersicon , solubility , depolymerization , enzyme , hydrolysis , biochemistry , pectinesterase , chromatography , food science , organic chemistry , botany , biology
An approach commonly employed to assess the potential role of the enzyme polygalacturonase (PG, EC 3.2.1.15) in tomato fruit cell‐wall pectin metabolism includes correlating levels of extractable PG with changes in specific characteristics of cell wall pectins, most notably solubility and molecular weight. Since information on these features of pectins is generally derived from analyses of subfractions of isolated cell wall, assurance of inactivation of the various isoforms of wall‐associated PG is imperative. In the present study, cell wall prepared from ripe tomato ( Lycopersicon esculentum Mill. cv. Rutgers) fruit was examined for the presence of active PG and for the ability of phenolic solvents to inactivate the enzyme. Using pectin solubility and M r (relative molecular mass) changes as criteria for the presence of wall‐associated PG activity, pectins from phenol‐treated and nonphenol‐treated (enzymically active) cell wall from ripe fruit incubated in 50 m M Na‐acetate, 50 m M cyclohexanetrans‐1,2‐diamine tetraacetic acid (CDTA), pH 6.5 (outside the catalytic range of PG), were of similar M r and exhibited no change in size with incubation time. Wall prepared without exposure to the phenolic protein‐denaturants exhibited extensive pectin solubilization and depolymerization when incubated in 50 m M Na‐acetate, 50 m M CDTA at pH 4.5, indicating the presence of active PG. Based on the changes in the M r of pectins solubilized in 50 m M Na‐acetate, 50 m M CDTA, pH 4.5, active PG was also detected in wall exposed during isolation to phenolacetic acid‐water (PAW, 2:1:1, w/v/v), a solvent commonly employed as an enzyme denaturant. Although the depolymerization of pectins in PAW‐treated wall was extensive, oligouronides constituted minor reaction products. Interestingly, PAW‐treated wall did not exhibit PG‐mediated pectin release when incubated under conditions (30 m M Na‐acetate, 150 m M NaCl, pH 4.5) in which nonphenol‐treated cell wall exhibited high autolytic activity. In an alternative protocol designed to inactivate PG, cell wall was exposed to Tris‐buffered phenol (BP). In contrast to pectins released from PAW‐treated wall, pectins solubilized from BP‐treated wall at pH 4.5 were indistinguishable in M r from those recovered from BP‐treated wall at pH 6.5 Even when incubated at pH 4.5 at 34°C, conditions under which pectins from PAW‐treated wall underwent more rapid and extensive depolymerization, pectins from BP‐treated wall exhibited no change in M r , providing evidence that active PG was not present in these wall preparations. The implications of this study in interpreting the solubility and M r of pectin in cell wall from ripening fruit are discussed.