Solubilization of galactosyltransferase that synthesizes 1,4-beta-galactan side chains in pectic rhamnogalacturonan I.

Abstract

beta-1,4-Galactan galactosyltransferase (GT) activity was solubilized from potato microsomal membranes in the presence of 78 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid. The solubilized GT activity transferred 14[C]galactose from UDP-14[C]galactose onto the acceptor-substrates composed of rhamnogalacturonan (RG) with short galactan chains (RG-A, approximately 1.2 MDa, mol% Gal/Rha = 0.7; RG-B, approximately 21 kDa, mol% Gal/Rha = 1.2). However, shorter RG containing short galactan chains (approximately 2 kDa and 1.2 kDa), RG oligomers without galactosyl-residues, galactan, and galactooligomers did not act as acceptor-substrates. Optimal pH for 14[C] incorporation onto RG-A and RG-B was around 5.6 and 7.5, respectively. The 14[C]-labelled products synthesized upon RG-A and RG-B could be digested with a RG specific lyase into smaller RG fragments. 1,4-beta-Endogalactanase could not digest the former product, whereas the latter product was digested to 14[C]galactobiose and 14[C]galactose. This demonstrates that at least two GT activities were solubilized from potato microsomal membranes. One had optimal pH around 5.6 to transfer galactosyl residues onto RG-A, whereas the other had optimal pH around 7.5 to transfer galactosyl residues onto RG-B. Both synthesized galactan attached to the RG backbone of RG-A and RG-B, and the galactan synthesized onto the RG-B acceptor was 1,4-beta-linked.