Analytical Differentiation of Purine and Pyrimidine Nucleotides Determination of ADP, ATP, and Sum of GTP + ITP in Biological Material

Abstract

This chapter presents the determination of adenosine-5′-diphosphate (ADP), adenosine-5′-triphosphate (ATP), and the sum of guanosine-5′-triphosphate (GTP) and ionosine-5′-triphosphate (ITP) in biological material. The specific enzymatic determinations of ATP, for example by using luciferase, have been known for some time; however, in the past, ATP has been determined mainly by two non-specific methods: (1) with hexokinase (HK) and glucose-6-phosphate dehydrogenase and (2) with phosphoglycerate kinase (PGK) and glyceraldehyde phosphate dehydrogenase (GAPDH). HK and PGK act on the other nucleoside triphosphates as well as on ATP. Only myokinase (MK) is specific for ADP and ATP. When nucleoside triphosphates are converted non-specifically into the diphosphates by any kinase, only ADP, out of all the diphosphates, can be specifically converted by myokinase into ATP and AMP. This chapter describes a method in which the creatine kinase (CK) reaction is used for the specific determination of the ADP originally present in the sample. ATP inhibits the F-6-PK reaction, but this inhibition can be eliminated by the addition of A-5-MP.