A radiometric method for the determination of NADH in subpicomole amounts.
A radiometric method has been devised for the determination of small quantities of NADH formed in preceding dehydrogenase reactions. In a coupled enzymatic reaction, phosphoglycerate kinase (PGK) catalyzes the transfer of [32P]orthophosphate from [gamma-32P]ATP to 3-phosphoglycerate; the intermediate, 1,3-[1-32P]diphosphoglycerate, is dephosphorylated by glyceraldehyde-3-phosphate dehydrogenase (GAP-DH). [32P]Orthophosphate is released proportionally to NADH and can be measured after adsorption of [gamma-32P]ATP to activated charcoal. With this method, 0.2 pmol of NADH are detectable in the presence of a 10(4)-fold excess of NAD over NADH.
Weber
G
G
Institute of Pharmacology, Freie Universität Berlin, F.R.G.
Rosenthal
W
W
Oberdisse
E
E
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D005988:Glyceric Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008832:Microchemistry; D009243:NAD / Q000032:analysis* / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011874:Radiometry
D004163:Diphosphoglyceric Acids; D005988:Glyceric Acids; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D009243:NAD; C015891:glycerate 1,3-biphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); C005156:3-phosphoglycerate; D000255:Adenosine Triphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010735:Phosphoglycerate Kinase