The 'Enzyme-Probe' Method for Characterizing Metabolite Pools The Use of NAD-Glycohydrolase in Human Erythrocyte Sonicate as a Model System. The Use of NAD-Glycohydrolase in Human Erythrocyte Sonicate as a Model System

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Abstract
An approach for testing the homogeneity of metabolite pools is described. An alien enzyme that can attack the metabolite in question is introduced into the system studied. By analyzing the time-course of decomposition of the metabolite it can be decided whether the pool is homogeneous in respect to reactivity towards the probe-enzyme or can be divided into fractions of different reactivities. Method is illustrated using human erythrocyte sonicate as model system with NAD-glycohydrolase (EC 3.2.2.5) as probe-enzyme. The NAD pool in the concentrated sonicate was resolved into 3 fractions (I, II and III) with half-lives of about 1, 7 and 240 min, respectively. Fraction I was free NAD, fraction II was NAD bound to glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and fraction III was coenzyme strongly bound to some so far unidentified protein. Sonicate glycolysis seemed to require only fraction II and was unable to use fraction III under the experimental conditions applied. The scope of application of the enzyme-probe method is discussed.