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 language | English (US) |
|---|---|
| Pages (from-to) | 139-146 |
| Number of pages | 8 |
| Journal | Journal of biochemical and biophysical methods |
| Volume | 2 |
| Issue number | 3 |
| DOIs | |
| State | Published - Mar 1980 |
Funding
Supported by funds from the Andrew Mellon Foundation, the Muscular Dystrophy Association, N.S.F. Grant No. PCM-7807966 and N.I,H. Grants RR-07065, GM 26383 and CA 06381. L.J.V.E. is recipient of N.S.F. Postdoctoral Fellowship No. 78-15643. We thank Mr. d. De dovin of Hewlett Packard for making available the HP 1081, and Dr. N.-H. Chua and Dr. Stanford Moore for their critical reading of the manuscript.
Keywords
- HPLC
- calmodulin
- cyclic nucleotides
- phosphodiesterase
ASJC Scopus subject areas
- Biophysics
- Biochemistry