STRUCTURE AND FUNCTION OF THE GOLGI-COMPLEX IN RICE CELLS .2. PURIFICATION AND CHARACTERIZATION OF GOLGI MEMBRANE-BOUND NUCLEOSIDE DIPHOSPHATASE

Inosine diphosphatase bound to Golgi membranes was studied in rice (Oryza sativa L. cv Nipponkai) cells. The enzyme was solubilized with Triton X-100 from isolated rice Golgi membranes and was highly purified employing a series of chromatography steps in the presence of 20% glycerol and 0.1% Triton X-100. The apparent molecular mass of the enzyme was estimated by gel filtration column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 200 and 55 kD, respectively. The isoelectric point of the enzyme was determined to be 7.5. The optimal pH for the enzyme activity was around 7 and the enzyme required Mg2+ for hydrolyzing activity. IDP, UDP, and GDP were effective substrate for the purified rice Golgi membrane-bound inosine diphosphatase, whereas activity with ADP, CDP, and thymidine 5'-diphosphate was 10 to 20% of IDP. The K-m values for IDP, UDP, and CDP were 0.48, 0.50, and 0.67 mM, respectively, and V-max values were 1.85, 1.54, and 1.67 mu mol min(-1) mg(-1) respectively. These results indicate that the rice Golgi enzyme is a nucleoside diphosphatase that is specific for IDP, UDP, and CDP. Furthermore, this rice Golgi nucleoside diphosphatase stimulated the activity of glucan synthase I also localized in rice Golgi membranes. The results strongly support the view that this nucleoside diphosphatase is involved in regulation of beta-glucan synthesis in the plant Golgi complex.