Solubilization and characterization of a galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan

被引:62
作者
Doong, RL
Mohnen, D
机构
[1] Univ Georgia, Complex Carbohydrate Res Ctr, Athens, GA 30602 USA
[2] Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
关键词
D O I
10.1046/j.1365-313X.1998.00042.x
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
Polygalacturonate 4-alpha-galacturonosyltransferase (PGA-GalAT), the glycosyltransferase that synthesizes the plant cell wall pectic polysaccharide homogalacturonan, has previously been identified and partially characterized in tobacco membranes. Membrane bound PGA-GalAT catalyzes the transfer of galacturonic acid from UDP-galacturonic acid (UDP-GalA) onto an endogenous acceptor to produce polymeric homogalacturonan (Doong et al., (1995) Plant Physiol. 109, 141-152). It is shown here that 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 [(14)C]GalA from UDP-[(14)C]GalA onto exogenous 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-[(14)C]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 activity in detergent-dispersed membranes, corresponding to 21% of the PGA-GalAT activity in intact membranes, was solubilized. PGA-GalAT solubilized with 40 mM CHAPS was shown, by exopolygalacturonase treatment in combination with size exclusion and high performance anion exchange chromatographies, to add a single alpha-1,4-linked galacturonic acid residue onto an OGA exogenous acceptor of DP 15 to yield an OGA product of DP 16. The significance of the apparent lack of processivity of the solubilized PGA-GalAT is discussed.
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页码:363 / 374
页数:12
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