Simple assay of 0.1–1.0 pmol of ATP, ADP, and AMP in single somatic cells using purified luciferin luciferase
✍ Scribed by Horst Spielmann; Ursula Jacob-Müller; Petra Schulz
- Publisher
- Elsevier Science
- Year
- 1981
- Tongue
- English
- Weight
- 555 KB
- Volume
- 113
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
The sensitivity of ATP determinations with crude firefly luciferin luciferase is limited by contaminating ATP converting enzymes, which cause a rapid decrease of the ATP level during the assay. Purified luciferase has the advantage of producing an almost constant light intensity proportional to the ATP concentration. Sensitivity and specificity of the ATP assay are, therefore, considerably increased when purified enzyme is used instead of crude extracts of the enzyme. ATP, 0.1 -1 .O pmol as well as higher amounts can be determined with commercial preparations of purified and stabilized luciferase. In ADP and AMP measurements with the luciferase assay, problems are arising from the enzymes required for the conversion to ATP, since they are frequently contaminated by low amounts of adenine ribonucleotides. Exclusion of contaminated enzymes and removal of ammonium sulfate from adenylate kinase were the only prerequisites for determinations of 0. 1 -1 .O pmol of ADP and AMP with purified luciferase. The application of the assay in determinations of ATP, ADP, and AMP in single preimplantation mouse embryos is described. measurements with crude enzyme extracts