Solubilization and partial characterization of homogalacturonan-methyltransferase from microsomal membranes of suspension-cultured tobacco cells

Abstract

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.

Figure 1

Effect of CHAPS concentration on the solubilization of HGA-MT. The data are the average counts per minute (±se) of product recovered from duplicate samples from at least four independent experiments except for the 0.1% and 1% (w/v) CHAPS points, which are from duplicate samples from one experiment.

Figure 2

Effect of solubilization time on the solubilization of HGA-MT. The data are the average counts per minute (±se) incorporated into product in triplicate samples from one experiment for the 0-, 30-, and 60-min points and from three independent experiments for the other points.

Figure 3

Effect of pH on solubilized HGA-MT activity. The data are the average counts per minute (±se) incorporated into product in duplicate samples from five independent experiments.

Figure 4

Time course of the incorporation of ¹⁴C-methyl into precipitable product. The data are the average counts per minute (±se) incorporated into product in duplicate samples from three independent experiments.

Figure 5

Hanes-Woolf plot of the production of methylated product by solubilized HGA-MT. A, Hanes-Woolf plot for SAM. Reactions contained with 1 mg mL⁻¹ of pectin (HGA or pectin [30% DE]). B, Hanes-Woolf plot for pectin (HGA and pectin [30% DE]). Reactions contained 100 μm SAM. The data represent the average initial velocities (±se) in duplicate samples from at least two independent experiments. Initial velocity (V₀) is in picomoles of methyl incorporated per second per milligram of protein.