Structure and Function of the Golgi Complex in Rice Cells (II. Purification and Characterization of Golgi...

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[prime]-diphosphate was 10 to 20% of IDP. The Km values for IDP, UDP, and GDP were 0.48, 0.50, and 0.67 mM, respectively, and Vmax 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 GDP. 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.