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Schulte, A.E. et al.

Schulte, van der Heijden, Verpoorte,

Microplate enzyme-coupled assays of mevalonate and phosphomevalonate kinase from Catharanthus roseus suspension cultured cells.

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 from Catharanthus roseus suspension-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 apparent Km values of mevalonate kinase for ATP and mevalonate were 210 and 65 microM, 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.[1]

References

Microplate enzyme-coupled assays of mevalonate and phosphomevalonate kinase from Catharanthus roseus suspension cultured cells. Schulte, A.E., van der Heijden, R., Verpoorte, R. Anal. Biochem. (1999) [Pubmed]

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