Immunopurification and Immunocharacterization of the Glucosinolate Biosynthetic Enzyme Thiohydroximate S-Glucosyltransferase

Abstract

Preparing homogeneous UDP-glucose:thiohydroximate S-glucosyltransferase (S-GT), the penultimate biosynthetic enzyme of glucosinolates, by standard chromatographic methods has yielded too little protein for adequate purity evaluation, identity verification, and structural analysis. The low yields were apparently due to low abundance in source tissues, aggravated by enzyme instability. Here we describe an immunological method for purification of workable quantities from florets of Brassica oleracea ssp. botrytis (cauliflower). Florets that had undergone browning due to exposure to sunlight contained higher S-GT activities than are normally found in Brassica tissues. S-GT was adsorbed from crude tissue extracts onto an agarose-monoclonal antibody complex. Elution from the complex required harsh alkaline conditions (pH 11.5), giving extremely variable activity recoveries (maximum 20%). The eluate contained two proteins that could be separated readily by preparative polyacrylamide gel electrophoresis or anion-exchange chromatography. The overall S-GT protein recovery was estimated at less than 200 [mu]g/kg of cauliflower tissue. Molecular weight determinations with homogeneous cauliflower S-GT gave relative molecular weight (Mr) values of 55,500 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 57,600 by gel chromatography; isoenzymes with isoelectric point values of 4.80 and 4.95 were identified. A polyclonal antibody raised against denatured enzyme showed broad cross-reactivity in immunoblots with S-GT from a number of Brassica species and other crucifers. The monoclonal antibody that was used in the immunopurification was much more specific; it exclusively precipitated S-GT isoenzymes that had their genomic origin in the primary diploids B. oleracea and Brassica campestris. Thus, all of the S-GT was precipitated from the amphidiploid Brassica napus, which is a hybrid of B. orleracea and B. campestris. About half of the S-GT was precipitated from the amphidiploids Brassica carinata and Brassica juncea, which have B. oleracea and B. campestris as one of their parents, respectively. It was shown that the S-GT isoenzymes of B. juncea with Mr 55,500 and about 57,000 originate from the parents B. campestris and B. nigra, respectively.