Measurement of plasma gamma-glutamyltransferase in clinical chemistry: kinetic basis and standardisation propositions.
The conditions for the measurement of gamma-glutamyltransferase (EC 2.3.2.2) activity of human plasma were studied at 30 degrees C using the kinetic technique of Szasz [3]. The optimum pH in Tris (hydroxymethylaminomethane) buffer and 2-amino-2-methyl-1.3-propanediol at a concentration of 100 mmol/1 are 8.0 and 8.1. The kinetic characteristics of human plasma gamma-glutamyltransferase were studied using gamma-L-glutamyl p-nitroanilide and its carboxyl derivative as donor substrates. Glycylglycine was chosen as the best acceptor of the gamma-glutamyl radical. Under these conditions, we have shown that the inhibition by the donor substrate was more important at acidic pH and vanished at alkaline pH. This inhibition was obviously related to the presence of the acceptor, but did not vary with glycylglycine concentration. At pH 8.0, by increasing the acceptor concentration some competition occurs at the donor binding site, as reported by other authors in relation to the known ping-pong bi-bi enzyme mechanism for the gamma-glutamyltransferase. Some displacement of donor substrate by increasing amounts of acceptor substrate could be observed at all pH values we studied. However, the influence of glycylglycine on the enzyme's maximum velocity and affinity for the donor substrate was also pH dependent. Studying the kinetic characteristics of the enzyme as a function of the pH suggests that the enzyme works with more than one active site at pH 7.5-8.0. Based on the results of this study, we propose measurement conditions for gamma-glutamyltransferase at 30 degrees C in routine clinical chemistry without preference in the choice of substrate.
D015055:1-Carboxyglutamic Acid / Q000031:analogs & derivatives; D000813:Anilides; D002021:Buffers; D005971:Glutamates; D005973:Glutamine / Q000031:analogs & derivatives; D006033:Glycylglycine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008722:Methods; D013379:Substrate Specificity; D005723:gamma-Glutamyltransferase / Q000097:blood*
D000813:Anilides; D002021:Buffers; D005971:Glutamates; D005973:Glutamine; D006033:Glycylglycine; D015055:1-Carboxyglutamic Acid; C013540:gamma-glutamyl-3-carboxy-4-nitroanilide; C001300:gamma-glutamine-4-nitroanilide; D005723:gamma-Glutamyltransferase