Stimulation of polyprenyl 4-hydroxybenzoate transferase activity by sodium cholate and 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate.
Polyprenyl 4-hydroxybenzoate transferase (Coq2p) plays a central role in ubiquinone biosynthesis. Coq2p mediates the conjugation of 4-hydroxybenzoate, the benzoquinone ring precursor, with the completed side chain. The activity is most easily assayed by measuring the rate of incorporation of 4-hydroxybenzoate as radiolabeled substrate into polyprenyl 4-hydroxybenzoate. The in vitro assay requires addition of a detergent into the reaction mixture to activate enzyme activity, and Triton X-100 is used for this purpose in the routine assay. We have found that both 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate and sodium cholate, but not sodium deoxycholate, lysophosphatidyl choline, or octylglucoside, significantly stimulate the activity over that measured with Triton X-100. High-performance liquid chromatography analysis of lipid extracts revealed that the increase of specific activity resulted in a similar increase in reaction product, this effect is due not merely to a better lipid extraction but also to the actual stimulation of enzyme activity. With our improved method, we were able to measure Coq2p activity with much greater sensitivity in both fresh and frozen/thawed mitochondria and in crude homogenates obtained from cultured cells. Our method will simplify evaluation of Coq2p activity in scarce biological materials, such as cells obtained from human tissue biopsies, and thus it will facilitate the biochemical characterization of ubiquinone deficiencies.
Burón
María I
MI
Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Campus Universitario de Rabanales, Edificio Severo Ochoa, Universidad de Córdoba, Spain.
Hermán
María D
MD
Alcaín
Francisco J
FJ
Villalba
José M
JM
D019883:Alkyl and Aryl Transferases / Q000187:drug effects*; D000818:Animals; D002478:Cells, Cultured; D002793:Cholic Acids / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003902:Detergents / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008930:Mitochondria, Liver / Q000201:enzymology; D017830:Octoxynol / Q000494:pharmacology; D010226:Parabens / Q000378:metabolism; D051381:Rats; D020358:Sodium Cholate / Q000494:pharmacology*; D014451:Ubiquinone / Q000096:biosynthesis*
D002793:Cholic Acids; D003902:Detergents; D008055:Lipids; D010226:Parabens; D014451:Ubiquinone; D017830:Octoxynol; D019883:Alkyl and Aryl Transferases; C021386:4-hydroxybenzoate polyprenyltransferase; C038193:4-hydroxybenzoic acid; D020358:Sodium Cholate; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate