SOLUBILIZATION AND CHARACTERIZATION OF A GALACTURONOSYLTRANSFERASE THAT SYNTHESIZES THE PECTIC POLYSACCHARIDE HOMOGALACTURONAN

Polygalacturonate 4-alpha-galacturonosyltransferase (PGA-GalAT), the g lycosyltransferase that synthesizes the plant cell wall pectic polysac charide homogalacturonan, has previously been identified and partially characterized in tobacco membranes. Membrane bound PGA-GalAT catalyze s the transfer of galacturonic acid from UDP-galacturonic acid (UDP-Ga lA) onto an endogenous acceptor to produce polymeric homogalacturonan (Doong et al., (1995) Plant Physiol. 109, 141-152). It is shown here t hat a galacturonosyltransferase is solubilized from tobacco membranes with a HEPES buffer, pH 6.8, containing 40 mM CHAPS and 2 mM EDTA. The solubilized galacturonosyltransferase was identified as putative PGA- GalAT because it transfered [C-14]GalA from UDP-[C-14]GalA onto exogen ous homogalacturonan accepters with degrees of polymerization (DP) of greater than or equal to 10. Maximal solubilized PGA-GalAT activity in the presence of 0.9 mu M UDP-[C-14]GalA required approximately 125 mu M exogenous homogalacturonan acceptor [e.g. oligogalacturonide (OGA) of DP 15]. Solubilized PGA-GalAT was active over a broad pH range of 6 .3-7.8, and had an apparent K-m for UDP-GalA of 37 mu M and a V-max of 290 pmol min(-1) mg(-1) protein. Approximately 44% of the PGA-GalAT a ctivity in detergent-dispersed membranes, corresponding to 21% of the PGA-GalAT activity in intact membranes, was solubilized. PGA-GalAT sol ubilized with 40 mM CHAPS was shown, by exopolygalacturonase treatment in combination with size exclusion and high performance anion exchang e chromatographies, to add a single alpha-1,4-linked galacturonic acid residue onto an OGA exogenous acceptor of DP 15 to yield an OGA produ ct of DP 16. The significance of the apparent lack of processivity of the solubilized PGA-GalAT is discussed.