Determination of UTP and ATP pool sizes in human tonsillar lymphocytes by using Escherichia coli RNA polymerase.
The present paper describes a rapid, specific and sensitive method for quantitating ribonucleoside triphosphates (ATP and UTP) in cell extracts. The principle of the method is based on the synthesis of a ribonucleotide polymer in the presence of UTP, ATP and poly(dA-dT) as template. A method for calculation is also described, making the determination of UTP and ATP pool sizes in the cells possible under the same experimental conditions. The calculation takes into account the isotope dilution effect caused by the intracellular ATP. Our experiments show that the neutralized perchloric acid soluble fraction of human tonsillar lymphocytes contains no inhibitors for the RNA polymerase test. According to our results, this cell extract contains 80 pmol of UTP and 340 pmol of ATP per mug RNA.
D000255:Adenosine Triphosphate / Q000032:analysis*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000032:analysis*; D008433:Mathematics; D008722:Methods; D014066:Palatine Tonsil / Q000032:analysis*; D013997:Time Factors; D014500:Uracil Nucleotides / Q000032:analysis*
D014500:Uracil Nucleotides; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases