Isolation and partial characterization of trehalose 6-phosphate synthase aggregates from Selaginella lepidophylla plants.
Trehalose 6-phosphate synthase was purified from Selaginella lepidophylla plants and three aggregates of the enzyme were found by molecular exclusion chromatography, ion exchange chromatography and electrophoresis. Molecular exclusion chromatography showed four activity peaks with molecular weights of 624, 434, 224 and 115 kDa. Ion exchange chromatography allowed three fractions to be separated with TPS activity which eluted at 0.35, 0.7 and 1 M KCl. Native PAGE of each pool had three protein bands with apparent M(r) 660, 440 and 200 kDa. Western blot results showed that anti-TPS antibody interacted with 115 and 67 kDa polypeptides; these polypeptides share peptide sequences as indicated by internal sequence data. The effects of pH and temperature on enzyme stability and activity were studied. For fractions eluted at 0.35 and 1.0 M KCl, the optimum pH is 5.5, while an optimum pH of 7.5 for 0.7 M fraction was found. The three fractions eluted from ion exchange chromatography were stable in a pH 5-11 range. Optimal temperatures were 25, 45 and 55 degrees C for 0.7, 0.35 and 1.0 M fractions, respectively. The 0.7 M KCl fraction showed highest stability in a temperature range of 25-60 degrees C, whereas the 0.35 M KCl fraction had the lowest in the same temperature range.
Márquez-Escalante
Jorge A
JA
Centro de Investigación en Alimentación y Desarrollo A.C., Cuahutémoc, Apdo. Postal 781, Chihuahua, Mexico.
Figueroa-Soto
Ciria G
CG
Valenzuela-Soto
Elisa M
EM
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D032503:Selaginellaceae / Q000145:classification / Q000201:enzymology*; D013696:Temperature
D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase