A kinetic method is described for the determination of γ-glutamyl transpeptidase activity in serum. Optimal conditions for the reaction were ascertained, normal values in milliunits per milliliter at 25° established, and the reliability of the method examined. γ-Glutamyl-p-nitroanilide in ammediol-HCl buffer at pH 8.2 is employed as substrate. The method requires only 0.1 ml. of serum and, using an automatic cuvet positioner and recorder as well as an automatic diluter, more than 60 determinations can be carried out in 1 hr. On the basis of a large number of determinations, the method was shown to be technically excellent and clinically valuable.
To establish optimum conditions for creatine kinase (EC 2.7.3.2) activity measurement with the creatine phosphate in equilibrium creatine reaction, we re-examined all kinetics factors relevant to an optimal and standardized enzyme assay at 30 and 25 degrees C. We determined the pH optimum in vaious buffers, considering the effect of the type and concentration of the buffer, as well as the influence of various buffer anions on the activity. The relation between activity and substrate concentration was shown and the apparent Michaelis constants of creatine kinase for creatine phosphate and ADP were evaluated. We tested the effect on creatine kinase measurement of the concentration of substrates (glucose and NADP+) in the auxillary and indicator reactions, especially the influence of the added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes on the lag phase, at different temperatures. The NADP+ concentration proved to be the factor limiting the duration of constant reaction rate. We studied the inhibition of creatine kinase and adenylate kinase by AMP and established a convenient AMP concentration. For reactivation of creatine kinase, N-acetyl cysteine as sulfhydryl compound was introduced. Finally, we examined the relationship between activity and temperature.
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