Methods in Enzymology Vol.51 Purine and Pyrimidine Nucleotide Metabolism

This chapter describes the assay methodology of phosphoribosylpyrophosphate synthetase (PRPP) (ribose-5-phosphate pyrophosphokinase) from Salmonella typhimurium. PRPP synthetase can be conveniently assayed in most tissues by measuring the transfer of radioactivity from [γ-32P] adenosine triphosphate (ATP) to PRPP (and the products of the acid hydrolysis of PRPP) after the removal of unreacted ATP by charcoal adsorption. The procedure can be applied to crude cell extracts containing ATPase activities if the apparent activity of a control lacking ribose-5-phosphate is subtracted from each experimental measurement. The assay procedure can be used with a variety of crude cell extracts, so long as NaF is included and appropriate blanks are subtracted. The reaction of PRPP with orotate to form orotidylic acid and uridylic acid forms the basis of a spectrophotometric assay for PRPP synthetase, but this assay is not sufficiently sensitive for kinetic work. An alternative form of this assay in which release of 14CO2 from [7-14C] orotate is measured is also described in the chapter.

• Hardcover: 654 pages
• Publisher: Academic Press;
  
• Language: English
• ISBN-10: 0121819515
• ISBN-13: 978-0121819514
• Product Dimensions: 6 x 9 inches

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