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 carbox yl group of ol-l,4-linked-galactosyluronic acid residues in the pectic poly saccharide homogalacturonan (HGA) is catalyzed by an enzyme commonly referr ed to as pectin methyltransferase. A pectin methyltransferase from microsom al membranes of tobacco (Nicotiana tabacum) was previously characterized (F . Goubet, L.N. Council, D. Mohnen [1998] Plant Physiol 116: 337-347) and na med HCA methyltransferase (HGA-MT). We report the solubilization of HCA-MT from tobacco membranes. Approximately 22% of the HCA-MT activity in total m embranes was solubilized by 0.65% (w/v) 3-[(3-cholamidopropyl)dimethylammon io]1-propanesulfonic acid containing 1 mM dithioerythritol. The addition of phosphatidylcholine and the methyl acceptors HCA or pectin (30% degree of esterification) to solubilized enzyme increased HCA-MT activity to 35% of t otal 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 mu M, and an appare nt V-max of 0.121 pkat mg(-1) of protein. The apparent K-m for HCA and for pectin is 0.1 to 0.2 mg mL(-1). Methylated product was solubilized with boi ling water and ammonium oxalate, two conditions used to solubilize pectin f rom 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 inco rporated 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 met hylesterase, demonstrated that the bulk of the methylated product produced by the solubilized enzyme was pectin.