IDENTIFICATION AND PARTIAL CHARACTERIZATION OF THE PECTIN METHYLTRANSFERASE HOMOGALACTURONAN-METHYLTRANSFERASE FROM MEMBRANES OF TOBACCO CELL-SUSPENSIONS

A membrane preparation from tobacco (Nicotiana tabacum L.) cells conta ins at least one enzyme that is capable of transferring the methyl gro up from S-adenosyl-methionine (SAM) to the C6 carboxyl of homogalactur onan present in the membranes. This enzyme is named homogalacturonan-m ethyltransferase (HGA-MT) to distinguish it from methyltransferases th at catalyze methyletherification of the pectic polysaccharides rhamnog alacturonan I or rhamnogalacturonan II. A trichloroacetic acid precipi tation assay was used to measure HGA-MT activity, because published pr ocedures to recover pectic polysaccharides via ethanol or chloroform:m ethanol precipitation lead to high and variable background radioactivi ty in the product pellet. Attempts to reduce the incorporation of the C-14-methyl group from SAM into pectin by the addition of the alternat ive methyl donor 5-methyltetrahydrofolate were unsuccessful, supportin g the role of SAM as the authentic methyl donor for HCA-MT. The pH opt imum for HCA-MT in membranes was 7.8, the apparent Michaelis constant for SAM was 38 mu M, and the maximum initial velocity was 0.81 pkat mg (-1) protein. At least 59% of the radiolabeled product was judged to b e methylesterified homogalacturonan, based on the release of radioacti vity from the product after a mild base treatment and via enzymatic hy drolysis by a purified pectin methylesterase. The released radioactivi ty eluted with a retention time identical to that of methanol upon fra ctionation over an organic acid column. Cleavage of the radiolabeled p roduct by endopolygalacturonase into fragments that migrated as small oligomers of HCA during thin-layer chromatography, and the fact that H GA-MT activity in the membranes is stimulated by uridine 5'-diphosphat e galacturonic acid, a substrate for HCA synthesis, confirms that the bulk of the product recovered from tobacco membranes incubated with SA M is methylesterified HGA.