This chapter describes the purification procedure of nucleoside diphosphokinase enzyme from human erythrocytes. The term “nucleoside diphosphokinase” (NDP kinase) is applied to a family of enzymes that catalyze the transfer of the terminal phosphate groups of 5'-triphosphate nucleotides to 5'-diphosphate nucleotides, where N1 and N2 are purine or pyrimidine ribo- or deoxyribonucleosides. All nucleoside-diphosphate (NDP) kinases function through the formation of enzyme-bound high-energy phosphate intermediates. The early attempts to purify human erythrocytic NDP kinase involved the use of diethylaminoethyl (DEAE)-cellulose (phosphate) as the initial step to separate enzyme from hemoglobin. Further purification of this preparation yielded a homogeneous NDP kinase with a specific activity of about 70. Alternative methods for the measurement of NDP kinase activity include isotopic, staining, and coupled enzymic procedures. Any enzyme that functions through a high-energy phosphate-enzyme intermediate that has low specificity for nucleotide substrates, is capable of catalyzing the NDP kinase reaction.
ASJC Scopus subject areas
- Molecular Biology