Characterization of ATPase activities in rice root plasmalemma vesicles isolated by two-phase partitioning |
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Authors: | R Ros A Sanz J Segura I Picazo |
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Institution: | Depart. de Biologia Vegetal, Facultat de Ciències Biològiques, Univ. de València, E-46100 Burjassot (Valencia), Spain. |
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Abstract: | Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants ( Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two-phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate-sensitive K+, Mg2+-ATPase activity that was 18 times higher in the upper (PEG-rich) phase than in the lower (Dextran-rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 m M KCl and 3 m M ATP with apparent Km (ATP) and Vmax of 0.75 m M and 79 μmol (mg protein)?1 h?1, respectively. With 50 m M KCl and 7 m M ATP a pH optimum of 6.5, an apparent Km (ATP) of 6.3 m M and Vmax of 159 μmol (mg protein)?1 h?1 were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca2+ but insensitive to azide and nitrate. |
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Keywords: | ATPase K+ stimulation marker enzyme Oryza sativa pH plasmalemma rice |
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