J Integr Plant Biol. ›› 2010, Vol. 52 ›› Issue (6): 536-548.DOI: 10.1111/j.1744-7909.2010.00937.x

• Metabolism and Biochemistry • Previous Articles     Next Articles

Biochemical Characterization of Soluble Acid and Alkaline Invertases from Shoots of Etiolated Pea Seedlings

Donggiun Kim1, So Yun Park2, Youngjae Chung3, Jongbum Park1, Sukchan Lee4* and Taek-Kyun Lee2*   

  1. 1Department of Biological Science, Silla University, Busan, 617-736, Korea
    2South Sea Environment Research Department, Korea Ocean Research and Development Institute, Geoje, 656-830, Korea
    3Department of Life Science and Biotechnology, Shin Gyeong University, Hwaseong 445-741, Korea
    4Department of Genetic Engineering, Sungkyunkwan University, Suwon, 440-746, Korea
  • Received:2009-10-08 Accepted:2010-01-31 Published:2010-03-16
  • About author:* *Authors for correspondence Tel: +82 55 639 8630; Fax: +82 55 639 8639; E-mail: tklee@kordi.re.kr Tel: +82-31-290-7866; Fax: +82-31-290-7870; E-mail: sukchan@skku.ac.kr

Abstract:

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 °C. The effects of various concentrations of Tris-HCl, HgCl2, and CuSO4 on the activities of the two purified enzymes were examined. Tris-HCl and HgCl2 did not affect SAC activity, whereas 10 mM Tris-HCl and 0.05 mM HgCl2 inhibited SALK activity by about 50%. SAC and SALK were inhibited by 4.8 mM and 0.6 mM CuSO4 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 D, Park SY, Chung Y, Park J, Lee S, Lee TK (2010) Biochemical characterization of soluble acid and alkaline invertases from shoots of etiolated pea seedlings. J. Integr. Plant Biol. 52(6), 536–548.

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