Isolation of the catalytically competent small subunit of ribulose bisphosphate carboxylase/oxygenase from spinach under an extremely alkaline condition |
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Authors: | A Incharoensakdi T Takabe T Takabe T Akazawa |
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Affiliation: | 1. Donnelly Centre for Cellular & Biomolecular Research, Department of Molecular Genetics, University of Toronto, Ontario M5S 3E1, Canada;2. Research Institute for Sport & Exercise Sciences, Liverpool John Moores University, Liverpool L3 3AF, United Kingdom |
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Abstract: | A method for isolating the small subunit (B) of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from spinach leaf using an alkaline buffer (pH 11.2) in combination with sucrose gradient centrifugation is described. Although the yield of isolated subunit B (ca. 20%) was comparable to that previously described (ca. 25%) using the acid precipitation method [Andrews, T.J. and Lorimer, G.H. (1985) J. Biol. Chem. 260: 4632-4636], the isolated subunit B in this report suffered less denaturation (ca. 30%) as estimated from kinetic analysis of its reassembly with large subunit (A) derived from Aphanothece halophytica. Studies on the kinetic properties of the reassembled enzyme molecules suggested that spinach subunit B does not influence the affinity of the enzyme for substrate CO2. The catalytic core (A8) of spinach RuBisCO could not be isolated in the native form. |
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