Procedures for the isolation and quantification of the intermediates of the mevalonic acid pathway.
Procedures for the isolation and analysis of all 11 intermediates of the mevalonic acid pathway from acetyl-CoA through geranylgeranyl pyrophosphate were developed. Both acid-labile and base-labile metabolites are simultaneously extracted with good recoveries into 7 M urea at neutral pH and low temperature to minimize hydrolytic and degradative enzyme losses, and the extract is partially purified by adsorption and desorption in high yield from an anion-exchange membrane. With the use of internal standards, the pathway intermediates are subsequently separated and quantified by reversed-phase ion-pair HPLC with on-line radiodetection. Permeable secretory cells specialized for monoterpene biosynthesis were isolated from peppermint (Mentha x piperita) leaves and employed as a model system to test the analytical protocols by examining the incorporation of [14C]pyruvate, [14C]mevalonate, and [3H]isopentenyl pyrophosphate as precursors. This simple new method should be readily adaptable to a wide range of cell and tissue types that can be administered basic metabolic precursors, and should allow measurements of both flux and steady-state levels of the intermediates of the mevalonic acid pathway.
McCaskill
D
D
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
Croteau
R
R
D000105:Acetyl Coenzyme A / Q000302:isolation & purification / Q000378:metabolism; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D002623:Chemistry Techniques, Analytical / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008798:Mevalonic Acid / Q000302:isolation & purification / Q000378:metabolism*; D059828:Plant Cells; D010944:Plants / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000302:isolation & purification / Q000378:metabolism; D012015:Reference Standards; D014316:Tritium; D014508:Urea
D002250:Carbon Radioisotopes; D011106:Polyisoprenyl Phosphates; D014316:Tritium; D000105:Acetyl Coenzyme A; D014508:Urea; C002963:geranylgeranyl pyrophosphate; D008798:Mevalonic Acid