Cell-Free Synthesis of Pectin (Identification and Partial Characterization of Polygalacturonate 4-[alpha]-Galacturonosyltransferase and Its Products from Membrane Preparations of Tobacco Cell-Suspension Cultures)

Abstract

Polygalacturonate 4-[alpha]-galacturonosyltransferase (EC 2.4.1.43) activity has been identified in microsomal membranes isolated from tobacco (Nicotiana tabacum L. cv Samsun) cell-suspension cultures. Incubation of UDP-[14C]galacturonic acid with tobacco membranes results in a time-dependent incorporation of [14C]galacturonic acid into a chloroform-methanol-precipitable and 65% ethanol-insoluble product. The optimal synthesis of product occurs at a pH of 7.8, 25 to 30[deg]C, an apparent Km for UDP-D-galacturonic acid of approximately 8.9 [mu]M, and a Vmax of approximately 150 pmol min-1 mg-1 protein. The product was characterized by scintillation counting, thin-layer chromatography, high-performance anion-exchange chromatography, and gel-filtration chromatography in combination with enzymatic and chemical treatments. The intact product has a molecular mass of approximately 105,000 D based on dextran molecular standards. The product was treated with base to hydrolyze ester linkages (e.g. methyl esters), digested with a homogeneous endopolygalacturonase (EPGase), or base and EPGase treated. Base and EPGase treatment results in cleavage of 34 to 89% of 14C-labeled product into components that co-chromatograph with mono-, di-, and trigalacturonic acid, indicating that a large portion of product contains contiguous 1,4-linked [alpha]-D-galactosyluronic acid residues. Optimal EPGase fragmentation of the product requires base treatment prior to enzymatic digestion, suggesting that 45 to 67% of the galacturonic acid residues in the synthesized homogalacturonan are esterified. At least 40% of the base-sensitive linkages were shown to be methyl esters by comparing the sensitivity of base-treated and pectin methylesterase-treated products to fragmentation by EPGase.