Studies on Nucleotidases in Plants. Isolation and Properties of the Monomeric Form of the Crystalline and Homogeneous Mung Bean Nucleotide Pyrophosphatase

Abstract

Mung bean nucleotide pyrophosphatase isolated in a crystalline and homogeneous form as a dimer with a molecular weight of 65000 was converted by AMP into a tetramer. The tetramer was enzymatically active with altered kinetic properties. This conversion of the dimeric form by AMP to a tetrameric one was prevented by treating the dimer with p-hydroxymercuribenzoate. The molecular weight of the p-hydroxymercuribenzoate-treated enzyme was determined to be 32700 by a combination of Stokes' radius (2.4 nm) and sedimentation velocity (s20, w= 1.9 S), by thin-layer gel chromatography on superfine Sephadex G-200 and by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The monomer obtained by treatment of the native enzyme with p-hydroxymercuribenzoate was isolated by passage of the dissociated enzyme through a column of Biogel P-200. The monomer was optimally active at 37 °C, whereas the dimer and tetramer were active at 49 °C. All the three enzyme forms were maximally active at pH 9.4. The Km and V (measured as rate of FAD hydrolysis per mg protein) for FAD of the three enzyme forms were for the monomer, 0.5 mM and 7.0 μmol min−1, for the dimer, 0.25 mM and 3.3 μmol min−1 and for the tetramer, 0.58 mM and 2.5 μmol min−1, respectively. The time course of the reaction of the monomer was linear and comparable to the initial fast rate of the dimer. The monomer was not converted to a tetramer or a dimer on the addition of AMP; and it was irreversibly inhibited by urea and EDTA. ATP and ADP were noncompetitive inhibitors of the monomer.