The 'enzyme-probe' method for characterizing metabolite pools. The use of NAD-glycohydrolase in human erythrocyte sonicate as a model system.
1. An approach for testing the homogeneity of metabolite pools is described. An alien enzyme that can attack the metabolite in question is introducted 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 of reactivity towards the probe-enzyme or can be divided into fractions of different reactivities. 2. The information obtainable from such experiments is illustrated by the case of human erythrocyte sonicate as model system with NAD-glycohydrolase as probe-enzyme. The nicotinamide adenine dinucleotide pool in the concentrated sonicate could be resolved into three fractions (I, II and III) with half-lives of about 1, 7 and 240 min, respectively. Fraction I is free NAD, fraction II is NAD bound to glyceraldehyde-3-phosphate dehydrogenase, and fraction III is coenzyme strongly bound to some, so far unidentified, protein. Sonicate glycolysis seems to require only fraction II and is unable to use fraction III under the experimental conditions applied. 3. The scope of application of the enzyme-probe method is discussed.
D004912:Erythrocytes / Q000378:metabolism* / Q000648:ultrastructure; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D007773:Lactates / Q000097:blood; D009243:NAD / Q000097:blood; D009244:NAD+ Nucleosidase / Q000378:metabolism; D010084:Oxidation-Reduction
D007461:Iodoacetates; D007773:Lactates; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D009244:NAD+ Nucleosidase