Rapid separation and quantitation of 3′,5′-cyclic nucleotides and 5′-nucleotides in phosphodiesterase reaction mixtures using high-performance liquid chromatography

D. Martin Watterson*, David B. Iverson, Linda J. Van Eldik

*Corresponding author for this work

Research output: Contribution to journalArticle

24 Scopus citations

Abstract

A simple, rapid high-performance liquid-chromatography system for the fractionation and direct quantitation of substrates and products in crude phosphodiesterase reaction mixtures is described. Phosphate buffers and a pellicular anion exchange resin are used at ambient temperature. The method is sensitive, measuring picomoles of products with ultraviolet detection and femtomoles with isotopic measurement, and offers several advantages over the more popular batch sorption and manual methods for measuring phosphodiesterase activity. The time required for analysis, less than 8 min for single substrate reaction mixtures, is a fraction of that required with other chromatographic systems, and precision is ±5%. Results of studies with an activatable form of phosphodiesterase demonstrate the accuracy, precision and utility of the procedure for biomedical analyses.

Original languageEnglish (US)
Pages (from-to)139-146
Number of pages8
JournalJournal of Biochemical and Biophysical Methods
Volume2
Issue number3
DOIs
StatePublished - Mar 1980

Keywords

  • HPLC
  • calmodulin
  • cyclic nucleotides
  • phosphodiesterase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry

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