Partial amino acid sequence of potato solanidine UDP-glucose glucosyltransferase purified by new anion-exchange and size exclusion media.
Solanidine UDP-glucose glucosyltransferase (SGT) is involved in the biosynthesis of steroidal glycoalkaloids in potatoes. This enzyme is present at an extremely low level, is inherently unstable, and copurifies with the major storage protein patatin during isolation. We describe an improved method for isolating SGT from greening potato peel using two new chromatographic supports, Macro-Prep 50 Q anion-exchange and Superdex 75HR size exclusion media, under medium-pressure conditions at room temperature. The enzyme preparation was further resolved by SDS-PAGE and the proteins transferred to PVDF membrane (Immobilon-P). Two protein bands corresponding to active forms of SGT (36 and 37 kDa) were excised and cleaved with cyanogen bromide in trifluoroacetic acid. The resultant peptide mixtures were then separated by Tricine-SDS-PAGE and transferred to a PVDF membrane (Pro-Blott). The two major peptide bands observed in both digests (17 and 19 kDa) were sequenced. Identical N-terminal sequences were obtained from the 19-kDa peptides from both digests.
Stapleton
A
A
Western Regional Research Center-Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710.
Allen
P V
PV
Tao
H P
HP
Belknap
W R
WR
Friedman
M
M
D000595:Amino Acid Sequence; D000837:Anion Exchange Resins; D002846:Chromatography, Affinity; D002850:Chromatography, Gel / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D003208:Concanavalin A; D005964:Glucosyltransferases / Q000737:chemistry / Q000302:isolation & purification*; D007475:Ion Exchange Resins; D008689:Methacrylates; D008969:Molecular Sequence Data; D037121:Plant Lectins; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012117:Resins, Synthetic; D011198:Solanum tuberosum / Q000201:enzymology*
D000837:Anion Exchange Resins; D007475:Ion Exchange Resins; D008689:Methacrylates; D037121:Plant Lectins; D010940:Plant Proteins; D012117:Resins, Synthetic; D003208:Concanavalin A; C077921:Macro-Prep 50Q; D005964:Glucosyltransferases; C077995:solanidine glucosyltransferase