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Purification and characterization of an alkaline invertase from shoots of etiolated rice seedlings
Author(s) -
LIN CHUNGLIANG,
LIN HUICHIAO,
WANG AIYU,
SUNG HSIENYI
Publication year - 1999
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1046/j.1469-8137.1999.00416.x
Subject(s) - invertase , isoelectric point , chemistry , biochemistry , chromatography , molecular mass , concanavalin a , enzyme , size exclusion chromatography , isoelectric focusing , affinity chromatography , in vitro
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.