Purification and characterization of an alkaline invertase from shoots of etiolated rice seedlings

One alkaline invertase and two acid invertase activities were detected in t he shoots of etiolated rice (Oryza sativa) seedlings. The alkaline invertas e (AIT) was purified to homogeneity through steps of ammonium sulphate frac tionation, concanavalin A-Sepharose affinity chromatography (non-retained), DEAE-Sephacel chromatography and preparative electrophoresis. The pH optim um 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 su bstrate of the enzyme was sucrose, with a K-m of 2.53 mM. The enzyme also h ydrolysed raffinose, but not maltose or lactose, so it is a beta-D-fructofu ranosidase. It gave negative glycoprotein staining. Of the hydrolysis produ cts, fructose was a competitive inhibitor and glucose was a non-competitive inhibitor. Treatment with an alkaline phosphatase could activate AIT, wher eas other proteins such as BSA, concanavalin A and urease had no effect on the enzyme activity. The enzyme activity was inhibited by Tris, thiol reage nts and heavy metal ions.