PLANT PHYSIOLOGY , Vol 109, Issue 2 505-511, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Galactinol Synthase from Kidney Bean Cotyledon and Zucchini Leaf (Purification and N-Terminal Sequences)
J. J. Liu, W. Odegard and B. O. de Lumen
Department of Nutritional Sciences, University of California, Berkeley, California 94720
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 [mu]mol 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 (Cucurbita 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.
