Galactinol synthase from kidney bean cotyledon and zucchini leaf. Purification and N-terminal sequences

• Published in: Plant physiology (Bethesda) 1995-10, Vol.109 (2), p.505-511
• Main Authors: Liu, J.J.J. (University of California, Berkeley, CA.), Odegard, W, De Lumen, B.O
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Biochemistry and Enzymology; Biological and medical sciences; BIOSINTESIS; BIOSYNTHESE; Chromatography; Chromatography, Affinity; Chromatography, DEAE-Cellulose; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; COTILEDONES; COTYLEDON; Cotyledons; CUCURBITA PEPO; DOSAGE BIOLOGIQUE; Electrophoresis, Polyacrylamide Gel; ENSAYO BIOLOGICO; ENZIMAS; ENZYME; Enzymes; Fabaceae - enzymology; FEUILLE; Fundamental and applied biological sciences. Psychology; Galactosyltransferases - chemistry; Galactosyltransferases - isolation & purification; Galactosyltransferases - metabolism; Gels; GLICOSILTRANSFERASAS; GLYCOSYLTRANSFERASE; GRAINE; HOJAS; Kidneys; Kinetics; Leaves; Metabolism; Molecular Sequence Data; OLIGOSACARIDOS; OLIGOSACCHARIDE; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; PHASEOLUS VULGARIS; Plant Leaves; Plant physiology and development; Plants, Medicinal; PROTEINAS; PROTEINE; Proteins; Quaternary ammonium compounds; Seeds; SEMILLA; Sequence Homology, Amino Acid; Soybeans; Sulfates; TECHNIQUE ANALYTIQUE; TECNICAS ANALITICAS; Vegetables - enzymology; VIA BIOQUIMICA DEL METABOLISMO; VOIE BIOCHIMIQUE DU METABOLISME
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.109.2.505

Galactinol synthase (GS) was purified 1591-fold with a 3.9% recovery from the cotyledon of kidney bean (Phaseolus vulgaris) by a novel scheme consisting of ammonium sulfate fractionation followed by diethylaminoethyl, Affi-Gel Blue, and UDP-hexanolamine affinity chromatography. The purified enzyme had a specific activity of 8.75 micromol mg-1 min-1, a pH optimum of 7.0, and requirements for manganese ion and DTT. The enzyme exhibited a Km = 0.4 mM for UDP-galactose and a Km = 4.5 mM for myo-inositol. It was identified as a 38-kD peptide that co-purified with a 41- and a 43-kD peptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purification to homogeneity was achieved by isolating the 38-kD peptide from the SDS-PAGE gel. To clarify conflicting reports in the literature about the relative molecular mass of purified GS from zucchini leaf (Cururbita pepo), a similar scheme with modified eluting conditions was used to purify GS from this source. Zucchini leaf GS was purified to homogeneity and identified as a 36-kD peptide on SDS-PAGE. Partial N-terminal sequences of the 38-kD peptide from kidney bean cotyledon and the 36-kD peptide from zucchini leaf were obtained. To facilitate identification of GS during the purification, an assay utilizing thin-layer chromatography and an isotopic analytic imaging scanner was developed