Purification and Characterization of Golgi Membrane-Bound Nucleoside Diphosphatase from Suspension-Cultured Cells of Sycamore (Acer Pseudoplatanus L.)

Abstract

Inosine diphosphatase (IDPase) isoforms associated with Golgi membranes were studied in sycamore cell culture. These enzyme isoforms were solubilized with Triton X-100 and purified by chromatography using DEAE-Toyopearl and SOURCE-S columns. The isoforms were separated into two distinguishable fractions (peak 1 and 2) by SOURCE-S column chromatography. Furthermore the peak 1 contained at least two isoform bands detected by native-PAGE analysis. The apparent molecular sizes of these three isoforms were estimated by both gel filtration and SDS-PAGE to be 50 kDa, indicating that the Golgi membrane-bound IDPase has a monomeric structure. These IDPase isoforms required divalent cations (Ca²⁺, Mg²⁺, Co²⁺, Mn²⁺) for their hydrolyzing activity, and were inhibited by ATP. IDP, UDP, and GDP were effective substrates for these enzymes. It is clearly indicated that the sycamore Golgi membrane-bound IDPase is a nucleoside diphosphatase.