Solubilization of rhamnogalacturonan I galactosyltransfrases from membranes of a flax cell suspension.
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, beta-D-galactosyl residues were specifically transferred onto 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 degrees C. an apparent Km for UDP-Gal and exogenous RG-I substrate of 460 +/- 40 microM and 1.1 +/- 0.1 mg ml(-1) respectively, and a Vmax of 3.0 +/- 0.5 pkat mg(-1) protein.
Peugnet
I
I
University of Rouen, SCUEOR, ESA 6037 CNRS, Mont Saint Aignan, France.
Goubet
F
F
Bruyant-Vannier
M P
MP
Thoiron
B
B
Morvan
C
C
Schols
H A
HA
Voragen
A G
AG
D002462:Cell Membrane; D002473:Cell Wall / Q000737:chemistry; D002478:Cells, Cultured; D003902:Detergents / Q000494:pharmacology; D019597:Flax / Q000201:enzymology*; D005700:Galactosyltransferases / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008861:Microsomes / Q000201:enzymology; D010368:Pectins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000493:pharmacokinetics; D014531:Uridine Diphosphate Galactose / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics
D003902:Detergents; C042491:rhamnogalacturonan I; D014531:Uridine Diphosphate Galactose; D010368:Pectins; D005700:Galactosyltransferases; D006026:Glycoside Hydrolases