Assay for Erythrocyte Adenosine Monophosphate Deaminase (AMP Aminohydrolase, EC 3.5.4.6) and Effects of Certain Blood Preservatives on this Enzyme

A method was developed to assay adenosine monophosphate (AMP) deaminase (AMP aminohydrolase, EC 3.5.4.6) in erythrocytes. Determination of AMP deaminase is complicated by the fact that, while AMP is disappearing under the influence of this enzyme, it is also being formed through the breakdown of adenosine triphosphate and adenosine diphosphate. We have found that hemolysis is followed by fairly rapid conversion of erythrocytic adenosine triphosphate and adenosine diphosphate to AMP. In this system, AMP deaminase activity was found to follow first order kinetics, the half-time being approximately 1 hr. at 37 C. The levels of the various intermediates were followed by ion-exchange chromatography to eliminate any complications or assumptions related to enzyme assays or substrate levels. Of the various additives used or under study in blood preservation, inorganic phosphate displayed a most remarkable inhibitory effect on AMP deaminase. This effect was related to the concentration of added phosphate. Adenine also inhibited the reaction. This inhibition was maximal at 1 mM and less at 0.5 or 2 mM. It appears that the ability of adenine to inhibit AMP deaminase plays an important role in its activity in preserving red blood cells. Inosine and dipyridamole (an adenosine deaminase inhibitor) appeared to have only slight effect on the activity of AMP deaminase.