Direct observation of the NAD glycohydrolase reaction in human erythrocytes using NMR spectroscopy

Summary The hydrolysis of NAD by the extracellular membrane-associated enzyme NAD glycohydrolase was shown to be readily followed in concentrated suspensions of human erythrocytes using¹H spin-echo nuclear magnetic resonance spectroscopy (NMR). The maximal rate of the reaction was determined and the inhibitory effect of nicotinamide was confirmed by direct NMR observation. In addition, arginine, ergothioneine and iodoacetate did not influence the reaction rate.³¹P NMR analyses of reaction media from whole cells showed that no extraneous degradation of NAD occurred and the only phosphate-containing product was ADP-ribose.The work was supported by the Australian National Health and Medical Research Council