Identification of elongating beta-1,4-galactosyltransferase activity in mung bean (Vigna radiata) hypocotyls using 2-aminobenzaminated 1,4-linked beta- D-galactooligosaccharides as acceptor substrates.
Galactosyltransferase (GalT) activity that results in the transfer of galactose (Gal) from UDP-Gal to exogenous (1-->4)-beta-galactooligosaccharides labeled with 2-aminobenzamide (2AB) at their reducing ends was identified in a particulate preparation obtained from 2-day-old mung bean (Vigna radiata L. Wilezek) hypocotyls. The enzymes responsible were shown, by high-performance anion-exchange chromatography and normal-phase liquid chromatography-electrospray ionization mass spectrometry, to transfer up to eight Gals to the non-reducing end of 2AB-labeled galactooligosaccharide. Using 1H nuclear magnetic resonance spectroscopy, and beta-galactosidase and endo-beta-(1-->4)-galactanase treatments of the enzymatically formed 2AB-labeled galactooligosaccharides, the newly incorporated Gal residues were shown to be beta-(1-->4) linked. Time-course studies indicated that at least two different types of GalT isoform are involved in the elongation of the acceptor substrates. 2AB-labeled galactoheptaose was the most effective acceptor substrate analyzed, although galactooligosaccharides with a degree of polymerization between 4 and 6 were also acceptor substrates. 2AB-labeled penta- and heptasaccharides (RG5 and RG7) generated from rhamnogalacturonan I (RG-I) were not acceptor substrates, suggesting that the GalTs were not capable of adding Gal residues directly to the RG-I backbone. Maximum GalT activity was obtained at pH 6.5 and 20 degrees C in the presence of 25 mM Mn2+ and 0.75% (w/v) Triton X-100. The enzyme had an apparent Km of 20 microM for 2AB-labeled galactoheptaose and 32 microM for UDP-Gal. The characteristics of the enzyme in mung bean microsomal membranes and the usefulness of fluorogenic 2AB-labeled galactooligosaccharides for the assay of GalT are discussed.
Ishii
Tadashi
T
Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, 305-8687 Ibaraki, Japan. tishii@ffpri.affrc.go.jp
Ohnishi-Kameyama
Mayumi
M
Ono
Hiroshi
H
D002853:Chromatography, Liquid; D007887:Fabaceae / Q000201:enzymology* / Q000378:metabolism; D005685:Galactans / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D018546:Hypocotyl / Q000201:enzymology* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008861:Microsomes / Q000201:enzymology; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D062367:ortho-Aminobenzoates / Q000737:chemistry*
D005685:Galactans; D009844:Oligosaccharides; D062367:ortho-Aminobenzoates; D014531:Uridine Diphosphate Galactose; D016695:Glycosyltransferases; C000219:anthranilamide