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.
Schulte
A E
AE
Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden, 2300 RA, The Netherlands. A.E.Schulte@STM.TUDelft.nl
van der Heijden
R
R
Verpoorte
R
R
D002478:Cells, Cultured; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D019684:Magnoliopsida / Q000201:enzymology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D011770:Pyruvate Kinase
D007770:L-Lactate Dehydrogenase; D017853:Phosphotransferases (Alcohol Group Acceptor); C021796:mevalonate kinase; D011770:Pyruvate Kinase; D017856:Phosphotransferases (Phosphate Group Acceptor); C022371:phosphomevalonate kinase