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

Galactosyltransferases (GalTs), capable of transferring a galactosyl residu e from UDP-galactose (UDP-Gal) to polysaccharide acceptor, were solubilized from flax (Linum usitatissimum L.) membranes using 0.5% CHAPS. The observe d requirement for a rhamnogalacturonan I (RG-I) exogenous substrate to stim ulate the solubilized GalT activity provided the first evidence for the pre sence of RG-I GalT activities in flax cells. An assay to measure specifical ly the products of this RG-I GalT activity was designed, based on size-excl usion chromatography. Labelled products were characterized as an RG-I polym er by using purified RG-I hydrolase or lyase. At pH 8 and in the presence o f 5 mM CaCl2. beta -D-galactosyl residues were specifically transferred ont o RG-I branches of short beta-(1 --> 4)-D-galactan side chains. These side chains were liable to hydrolysis by beta -galactosidase and endo-beta-(1 -- > 4)-D-galactanase. The RG-I GalT had a temperature optimum of 30 degreesC, an apparent K-m for UDP-Gal and exogenous RG-I substrate of 460 +/- 40 muM and 1.1 +/- 0.1 mg ml(-1) respectively, and a V-max of 3.0 +/- 0.5 pkat mg (-1) protein.