Extraction of adenine nucleotides from cultured endothelial cells.
The goal of this work was to find a method suitable for the extraction of adenine nucleotides from cultured vascular endothelial cells. Extraction of cell monolayers with 80% methanol in water yielded extracts with a higher content of ATP than did extraction of cells with perchloric acid, trichloroacetic acid, or boiling water. The optimal extraction solution was 80% methanol with 0.5 mM ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or EDTA, heated to 70 degrees C immediately before use. Extraction of nucleotides by this solution was rapid and the recovery of exogenous ATP added during the extraction process was generally greater than 90%. An aqueous methanol or ethanol solution may be applicable for the extraction of nucleotides and other metabolites from cultured animal cells, dispersed cells, and frozen, powdered tissues.
D000227:Adenine Nucleotides / Q000302:isolation & purification*; D000255:Adenosine Triphosphate / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004285:Dogs; D004492:Edetic Acid; D004533:Egtazic Acid; D004727:Endothelium / Q000032:analysis*; D010472:Perchlorates; D013552:Swine
D000227:Adenine Nucleotides; D010472:Perchlorates; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D004492:Edetic Acid