Microplate Enzyme-Coupled Assays of Mevalonate and Phosphomevalonate Kinase fromCatharanthus roseusSuspension Cultured Cells

Abstract

A microplate assay for mevalonate and 5-phosphomevalonate kinase activities has been developed using an enzyme-coupled system of pyruvate kinase and lactate dehydrogenase. Mevalonate and 5-phosphomevalonate kinase activities were measured in crude and partially purified enzyme preparations fromCatharanthus roseussuspension-cultured plant cells. The assay was validated with respect to protein and substrate concentration. Mevalonate and 5-phosphomevalonate kinase showed Michaelis–Menten kinetics with respect to ATP and their specific substrates; the apparentKmvalues of mevalonate kinase for ATP and mevalonate were 210 and 65 μM, respectively, and of 5-phosphomevalonate kinase for ATP and 5-phosphomevalonate were 0.41 and 0.4 mM, respectively. Considering mevalonate kinase, the relative standard deviation of enzyme activity within a determination (n= 3) is always less than 2.5% and in between determinations (n= 9) is less than 2%. The method can be used in a continuous assay as well as in a discontinuous assay.