Purification and characterization of an alkaline invertase from shoots of etiolated rice seedlings |
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Authors: | CHUNG-LIANG LIN HUI-CHIAO LIN AI-YU WANG & HSIEN-YI SUNG |
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Institution: | Department of Agricultural Chemistry, National Taiwan University No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan; Author for correspondence (tel +886 2 23634729;fax +886 2 23634729;e-mail;). |
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Abstract: | One alkaline invertase and two acid invertase activities were detected in the shoots of etiolated rice ( Oryza sativa ) seedlings. The alkaline invertase (AIT) was purified to homogeneity through steps of ammonium sulphate fractionation, concanavalin A-Sepharose affinity chromatography (non-retained), DEAE-Sephacel chromatography and preparative electrophoresis. The pH optimum of AIT was 7.0 and the molecular mass, determined by gel filtration, was 240 kDa. It is apparently a homotetrameric enzyme (subunit molecular mass 60 kDa). The isoelectric point was 4.4 by isoelectric focusing. The best substrate of the enzyme was sucrose, with a K m of 2.53 mM. The enzyme also hydrolysed raffinose, but not maltose or lactose, so it is a β-D-fructofuranosidase. It gave negative glycoprotein staining. Of the hydrolysis products, fructose was a competitive inhibitor and glucose was a non-competitive inhibitor. Treatment with an alkaline phosphatase could activate AIT, whereas other proteins such as BSA, concanavalin A and urease had no effect on the enzyme activity. The enzyme activity was inhibited by Tris, thiol reagents and heavy metal ions. |
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Keywords: | Oryza sativa (rice) shoots alkaline invertase purification characterization |
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