Phosphomevalonate kinase is eluted with a linear gradient of 60-250 mM potassium phosphate buffer pH 7.5 containing 10 mM mercaptoethanol and 0. l mM EDTA (3 liters total volume) at a flow rate of 150 ml/ hr. The active fractions are pooled and the enzyme is precipitated with solid ammonium sulfate added up to 80% saturation. After centrifuging for 15 min at 16,000 g, the precipitate is resuspended in about 60 ml of 10 mM potassium phosphate buffer, pH 7.5, l0 mM mercaptoethanol, 100 mM KC1. At this stage, the enzyme is free of phosphatase and NADH oxidase activities. Phosphomevalonate kinase catalyzes the phosphorylation of phosphomevalonate (MVAP) by MgATP to pyrophosphomevalonate (MVAPP) and ADP. Only the 3R isomer of MVAP is phosphorylated and the reaction is reversible. The equilibrium constant is near unity at pH 7.5 and 30°. Percentage transformation of MVAP to MVAPP is calculated and the values obtained at different times (usually 1, 2, and 3 min) are used for initial velocity calculations. Other radioassay methods for phosphomevalonate kinase using paper and column chromatography to separate labeled substrates and products are described by Tchen.
Áreas temáticas de ASJC Scopus
- Biología molecular