ASSAYS FOR 3 ENZYMES INVOLVED IN MEVALONIC ACID METABOLISM

Enzyme assays have been developed for mevalonate (MVA) kinase, mevalonate phosphate (MVAP) kinase and mevalonate pyrophosphate (MVAPP) anhydrodecarboxylase. The procedures involve radioactively labelled substrates and separation of the reaction products by anion exchange chromatography. The separation on Dowex 1-X2 in self-packed microcolumns is simple, inexpensive and results in good separation of the MVA derivatives from each other. Because separation of MVAPP from isopentenyl pyrophosphate (IPP) was not possible directly, samples or column fractions containing MVAPP and IPP simultaneously were dephosphorylated by alkaline phosphatase. The resulting MVA and isopentenol are then easily separated in the same system. The assays for all three enzymes not only allows the determination of activities in crude enzyme preparations but is also applicable to the in vitro determination of intermediate pools in the reaction sequence from MVA to IPP after using C-14-MVA as substrate. The major advantage is accuracy and ease of use. The utility of the methods was demonstrated for enzyme extracts from the higher plants Chenopodium and spinach as well as for the fungus Phycomyces.