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Purification and characterization of the raffinose oligosaccharide chain elongation enzyme, galactan : galactan galactosyltransferase (GGT), from Ajuga reptans leaves
Author(s) -
Haab Canan Inan,
Keller Felix
Publication year - 2002
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1034/j.1399-3054.2002.1140305.x
Subject(s) - galactan , raffinose , galactosyltransferase , stachyose , biochemistry , chemistry , oligosaccharide , enzyme , melibiose , molecular mass , chromatography , sucrose
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 (K m = 42 m M ) and stachyose (K m = 58 m M ). 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 m M .