ISSN:
1399-3054
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Biology
Notes:
Galactan: galactan galactosyltransferase (GGT), an enzyme involved in the biosynthesis of the long-chain raffinose family of oligosaccharides (RFOs) in Ajuga reptans, catalyses the transfer of an α-galactosyl residue from one molecule of RFO to another one resulting in the next higher RFO oligomer. This novel galactinol (α-galactosyl-myo-inositol)-independent α-galactosyltransferase is responsible for the accumulation of long-chain RFOs in vivo. Warm treatment (20°C) of excised leaves resulted in a 34-fold increase of RFO concentration and a 200-fold increase of GGT activity after 28 days. Cold treatment (10°C/3°C day/night) resulted in a 26- and 130-fold increase, respectively. These data support the role of GGT as a key enzyme in the synthesis and accumulation of long-chain RFOs. GGT was purified from leaves in a 4-step procedure which involved fractionated precipitation with ammonium sulphate as well as lectin affinity, anion exchange, and size-exclusion chromatography and resulted in a 200-fold purification. Purified GGT had an isoelectric point of 4.7, a pH optimum around 5, and its transferase reaction displayed saturable concentration dependence for both raffinose (Km = 42 mM) and stachyose (Km = 58 mM). GGT is a glycoprotein with a 10% glycan portion. The native molecular mass was 212 kDa as determined by size-exclusion chromatography. Purified GGT showed one single active band after native PAGE or IEF separation, respectively, which separated into three bands on SDS-PAGE at 48 kDa, 66 kDa, and 60 kDa. The amino acid sequence of four tryptic peptides obtained from the major 48-kDa band showed a high homology to plant α-galactosidase (EC 3.2.1.22) sequences. GGT differed, however, in its substrate specificity from α-galactosidases; it neither hydrolysed nor transferred α-galactosyl-groups from melibiose, galactinol, UDP-galactose, manninotriose, and manninotetrose. Galactinol, sucrose, and galactose inhibited the GGT reaction considerably at 10–50 mM.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1034/j.1399-3054.2002.1140305.x
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