Isolation and kinetic studies of nucleoside diphosphokinase from human platelets and effects of cAMP phosphodiesterase inhibitors.
Nucleoside diphosphokinase (NDK) of human platelets has been purified by chromatography on Blue Sepharose CL-6B gel (purification factor of 950) and shown to be free of adenylate kinase, ATPase and adenylate cyclase. The molecular weight was 70,000 with subunits of 17,000. The pH optimum was 8.0 Km values for ATP and dTDP were determined in two ways using the pyruvate kinase-lactate dehydrogenase coupled enzyme assay. Values of 0.38 and 0.20 mM were obtained for ATP and 0.29 and 0.21 mM for dTDP. Km values for ADP (0.024 mM) and GTP (0.12 mM) were determined with the hexokinase-glucose-6-phosphate dehydrogenase coupled enzyme assay. These values are in agreement with those reported for NDK from other sources. Theophylline, which inhibits the NDK activity of intact platelets and platelet membrane preparations and inhibits the ADP-induced shape change of platelets, was shown to be a competitive inhibitor of both the free and phosphorylated forms of NDK with competitive inhibition constants (Kic) of 9.3 and 9.6 mM respectively. Papaverine, another cAMP phosphodiesterase inhibitor, which also inhibits the ADP-induced shape change of platelets, had no inhibitory effect on platelet NDK. It was concluded that the inhibitory effect of theophylline on the activity of the purified enzyme was due to the structural similarity between the methylxanthine and the adenine moiety of ADP.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009701:Nucleoside-Diphosphate Kinase / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D010208:Papaverine / Q000494:pharmacology; D010770:Phosphotransferases / Q000097:blood*; D013806:Theophylline / Q000494:pharmacology; D013942:Thymine Nucleotides / Q000378:metabolism
D013942:Thymine Nucleotides; C027923:thymidine 5'-diphosphate; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D013806:Theophylline; D010208:Papaverine; D010770:Phosphotransferases; D009701:Nucleoside-Diphosphate Kinase; D015105:3',5'-Cyclic-AMP Phosphodiesterases