CANDIDA-ALBICANS NUCLEOSIDE-DIPHOSPHATE KINASE - PURIFICATION AND CHARACTERIZATION

Soluble nucleoside-diphosphate kinase (NDP kinase) from Candida albica ns was purified to electrophoretic homogeneity and a partial sequence was determined, The enzyme was kinetically and physically characterize d. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pu rified enzyme revealed a single polypeptide of 17 kDa upon staining, b y immunodetection with heterologous and homologous antibodies, and by autoradiography of the phosphorylated enzyme. Furthermore, isoelectric focusing of the purified native enzyme showed a single acidic band (p i 4.5). These results together with a native molecular mass of 98 kDa suggest a hexameric native enzyme composed of identical subunits. Like NDP kinases from other sources, the catalysis involves a phosphoenzym e intermediate that is rapidly formed upon incubation of the enzyme wi th ATP. The transfer of phosphate from phosphoprotein intermediate to nucleoside diphosphates is equally fast. Kinetic experiments indicated that GTP and ATP had the lowest K-m compared to UTP, dTTP, and CTP, G DP acted as a preferred acceptor as assessed by kinetic measurements a s well as by competition experiments. Experimental data are presented indicating the existence of a membrane-associated NDP kinase. Prelimin ary characterization of this enzyme suggests that cytosolic and membra ne-associated NDP kinases are similar proteins. (C) 1995 Academic Pres s, Inc.