Solubilization and partial characterization of homogalacturonan-methyltransferase from microsomal membranes of suspension-cultured tobacco cells.
The transfer of a methyl group from S-adenosyl-L-methionine onto the carboxyl group of alpha-1,4-linked-galactosyluronic acid residues in the pectic polysaccharide homogalacturonan (HGA) is catalyzed by an enzyme commonly referred to as pectin methyltransferase. A pectin methyltransferase from microsomal membranes of tobacco (Nicotiana tabacum) was previously characterized (F. Goubet, L.N. Council, D. Mohnen [1998] Plant Physiol 116: 337-347) and named HGA methyltransferase (HGA-MT). We report the solubilization of HGA-MT from tobacco membranes. Approximately 22% of the HGA-MT activity in total membranes was solubilized by 0.65% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid containing 1 mM dithioerythritol. The addition of phosphatidylcholine and the methyl acceptors HGA or pectin (30% degree of esterification) to solubilized enzyme increased HGA-MT activity to 35% of total membrane-bound HGA-MT activity. Solubilized HGA-MT has a pH optimum of 7.8, an apparent K(m) for S-adenosyl-L-methionine of 18 microM, and an apparent V(max) of 0. 121 pkat mg(-1) of protein. The apparent K(m) for HGA and for pectin is 0.1 to 0.2 mg mL(-1). Methylated product was solubilized with boiling water and ammonium oxalate, two conditions used to solubilize pectin from the cell wall. The release of 75% to 90% of the radioactivity from the product pellet by mild base treatment showed that the methyl group was incorporated as a methyl ester rather than a methyl ether. The fragmentation of at least 55% to 70% of the radiolabeled product by endopolygalacturonase, and the loss of radioactivity from the product by treatment with pectin methylesterase, demonstrated that the bulk of the methylated product produced by the solubilized enzyme was pectin.
Goubet
F
F
Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, 220 Riverbend Road, Athens, Georgia 30602-4712, USA.
Mohnen
D
D
D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002478:Cells, Cultured; D002793:Cholic Acids / Q000378:metabolism; D004226:Dithioerythritol; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology*; D010070:Oxalates / Q000378:metabolism; D010368:Pectins / Q000378:metabolism*; D010947:Plants, Toxic; D011096:Polygalacturonase / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology*; D014867:Water / Q000378:metabolism
D002413:Cations, Divalent; D002793:Cholic Acids; D010070:Oxalates; D014867:Water; D004226:Dithioerythritol; D010368:Pectins; D008780:Methyltransferases; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase; D011096:Polygalacturonase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; C003181:polygalacturonic acid