Biochemical characterization of soluble acid and alkaline invertases from shoots of etiolated pea seedlings.
Soluble invertase was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, DEAE-Sepharose column, Con-A- and Green 19-Sepharose affinity columns, hydroxyapatite column, ultra-filtration, and Sephacryl 300 gel filtration. The purified soluble acid (SAC) and alkaline (SALK) invertases had a pH optimum of 5.3 and 7.3, respectively. The temperature optimum of two invertases was 37 degrees C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the two purified enzymes were examined. Tris-HCl and HgCl(2) did not affect SAC activity, whereas 10 mM Tris-HCl and 0.05 mM HgCl(2) inhibited SALK activity by about 50%. SAC and SALK were inhibited by 4.8 mM and 0.6 mM CuSO(4) by 50%, respectively. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The Kms of SAC and SALK were determined to be 1.8 and 38.6 mM, respectively. The molecular masses of SAC shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were 22 kDa and 45 kDa. The molecular mass of SALK was 30 kDa. Iso-electric points of the SAC and SALK were estimated to be about pH 7.0 and pH 5.7, respectively.
Kim
Donggiun
D
Department of Biological Science, Silla University, Busan, 617-736, Korea.
Park
So Yun
SY
Chung
Youngjae
Y
Park
Jongbum
J
Lee
Sukchan
S
Lee
Taek-Kyun
TK
D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D018532:Pisum sativum / Q000201:enzymology*; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018520:Plant Shoots / Q000201:enzymology*; D036226:Seedlings / Q000201:enzymology*; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D043324:beta-Fructofuranosidase / Q000378:metabolism
D010940:Plant Proteins; D013395:Sucrose; D043324:beta-Fructofuranosidase