Development of a filter assay for measuring homogalacturonan: α-(1,4)-Galacturonosyltransferase activity

Abstract

α-(1,4)-Galacturonosyltransferases (GalATs) catalyze the addition of (1,4)-linked α- d-galacturonosyl residues onto the nonreducing end of homogalacturonan chains. The nucleotide-sugar donor for the enzymatic reaction is uridine diphospho- d-galactopyranosyluronic acid (UDP- d-Gal pA). Many GalAT activity assays are based on the incorporation of d-[ 14C]Gal pA from UDP- d-[ 14C]Gal pA onto exogenously added homogalacturonan acceptors. Reactions based on this method can be time-consuming because multiple labor-intensive centrifugations and washes with organic solvents are required to remove the unincorporated UDP- d-[ 14C]Gal pA from the 14C-labeled products. Here we report the development of an alternative GalAT filter assay based on the ability of homogalacturonan to bind to cetylpyridinium chloride (CPC). GalAT assay reaction products made using radish ( Raphanus sativus) microsomal membranes or solubilized proteins from tobacco ( Nicotiana tabacum L. cv. Samsun) and Arabidopsis thaliana (cv. Columbia) were spotted onto Whatman 3MM paper treated with 2.5% (w/v) CPC. Unincorporated UDP- d-[ 14C]Gal pA was selectively removed from the filters by washing with 150–250 mM NaCl. The versatility of this assay is demonstrated by using it to identify GalAT activity in fractions obtained during the partial purification of tobacco GalAT by SP Sepharose cation exchange chromatography and by detecting the GalAT-catalyzed incorporation of d-[ 14C]Gal pA onto endogenous acceptors from Arabidopsis membranes.