title: Procedures for the isolation and quantification of the intermediates of the mevalonic acid pathway.
abstract: 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.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
authors.forename: D
authors.initials: D
authors.lastname: McCaskill
authors.forename: R
authors.initials: R
authors.lastname: Croteau
mesh_terms: 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
chemical_list: D002250:Carbon Radioisotopes; D011106:Polyisoprenyl Phosphates; D014316:Tritium; D000105:Acetyl Coenzyme A; D014508:Urea; C002963:geranylgeranyl pyrophosphate; D008798:Mevalonic Acid