ldentification and Partia1 Characterization of Polygalacturonate 4-a- Galacturonosyltransferase and Its Products from Membrane Preparations of Tobacco Cell-Suspension Cultures

Polygalacturonate 4-a-galacturonosyltransferase (EC 2.4.1.43) activity has been identified in microsomal membranes isolated from tobacco (Nicotiana tabacum L. cv Samsun) cell-suspension cultures. lncubation of UDP-('4C)galacturonic acid with tobacco membranes results in a time-dependent incorporation of ('4C)galacturonic acid into a chloroform-methanol-precipitable and 65% ethanol-insolu- ble product. The optimal synthesis of product occurs at a pH of 7.8, 25 to 3OoC, an apparent K, for UDP-D-galacturonic acid of approx- imately 8.9 p~, and a V,,, of approximately 150 pmol min-' mg-' protein. lhe 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 hydro- lyze ester linkages (e.g. methyl esters), digested with a homoge- neous endopolygalacturonase (EPCase), or base and EPCase treated. Base and EPCase 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 a-D-galactosylu- ronic acid residues. Optimal EPCase 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 sensi- tivity of base-treated and pectin methylesterase-treated products to fragmentation by EPCase.