Solubilization of rhamnogalacturonan I galactosyltransferases from membranes of a flax cell suspension

Abstract.

Galactosyltransferases (GalTs), capable of transferring a galactosyl residue from UDP-galactose (UDP-Gal) to polysaccharide acceptor, were solubilized from flax (Linum usitatissimum L.) membranes using 0.5% CHAPS. The observed requirement for a rhamnogalacturonan I (RG-I) exogenous substrate to stimulate the solubilized GalT activity provided the first evidence for the presence of RG-I GalT activities in flax cells. An assay to measure specifically the products of this RG-I GalT activity was designed, based on size-exclusion chromatography. Labelled products were characterized as an RG-I polymer by using purified RG-I hydrolase or lyase. At pH 8 and in the presence of 5 mM CaCl₂, β-D-galactosyl residues were specifically transferred onto RG-I branches of short β-(1→4)-D-galactan side chains. These side chains were liable to hydrolysis by β-galactosidase and endo-β-(1→4)-D-galactanase. The RG-I GalT had a temperature optimum of 30 °C, an apparent K _m for UDP-Gal and exogenous RG-I substrate of 460±40 µM and 1.1±0.1 mg ml^–1 respectively, and a V _max of 3.0±0.5 pkat mg^–1 protein.