Five functional isoforms of a novel plant thiol methyl-transferase from the leaves of cabbage (Brassica oleracea L.) were purified to electrophoretic homogeneity. Pooled, partly purified preparations of the enzyme were previously shown to methylate thiol compounds released upon the hydrolysis of glucosinolates. The enzyme could also accept halide ions as substrates. The estimated molecular masses of the purified isoforms ranged between 26 and 31 kDa. The three most abundant isoforms of the enzyme could all catalyze the S-adenosyl-L-methionine-dependent methylation of thiocyanate, a number of organic thiols and iodide. However, the kinetic properties of these forms toward various substrates differed widely. None of the isoforms examined methylated the O- and N-equivalents of the thiol substrates. The three isoforms also had distinct pH optima, covering the range from 5 to 9. Their kinetic analysis indicated that they shared a sequential substrate binding mechanism and an Ordered Bi Bi mechanism for substrate binding and product release. Partial internal amino acid sequence from one isoform showed high similarity to an Arabidopsis EST of unknown function, and to a recently cloned methyl chloride transferase from Batis maritima. The differences in the pH optima and kinetic properties of the isoforms suggest that each may methylate a specific substrate or a narrow group of substrates under cellular conditions. (C) 2000 Academic Press.