Isopentenoid synthesis in isolated embryonic Drosophila cells: absolute, basal mevalonate synthesis rate determination.
Embryonic Drosophila cells (Kc cells) and [5-3H]mevalonate (less than or equal to 10 microM) were used to determine the absolute basal in vivo rate of total mevalonic acid synthesis/utilization. An absolute in vivo mevalonic acid synthesis rate of 0.69 nmol/h/mg total cell protein was measured. Absolute mevalonate utilization was obtained by correcting for the extent of endogenous dilution of exogenous [3H]mevalonate at isotopic equilibrium. Cellular [3H]farnesol specific radioactivity was used as representative of a rapidly turning over isopentenoid pool. Although our previous Kc cell study (Havel, C. M., Rector, E. R. II, Watson, J. A., 1986, J. Biol. Chem. 261, 10,150-10,156) demonstrated that greater than or equal to 40% of the metabolized [3H]mevalonate appeared as 3H-labeled media water, this report established that t,t-3,7,11-[3H]trimethyl-2,6,10-dodecatriene-1,12 dioic acid was also secreted. Media accumulation of the C15-alpha,omega-prenyl dioic acid and 3H2O was related directly to [3H]mevalonic acid availability. This is the first mevalonate carbon balance study reported for a eukaryotic organism. It was concluded that (i) Kc cells synthesized more mevalonate than needed for normal growth and essential isopentenoids and (ii) excess mevalonate carbon accumulated intra- and extracellularly as isopentenoid compounds distal to C5 products. Finally, this study emphasized the need to measure total mevalonate utilization and not mevalonate conversion to a single isopentenoid end product in carbon balance investigations.
Havel
C M
CM
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Watson
J A
JA
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004330:Drosophila / Q000196:embryology / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005204:Farnesol / Q000378:metabolism; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D008798:Mevalonic Acid / Q000378:metabolism*; D013729:Terpenes / Q000378:metabolism*; D014316:Tritium
D013729:Terpenes; D014316:Tritium; D005204:Farnesol; D006903:Hydroxymethylglutaryl CoA Reductases; D008798:Mevalonic Acid