The assay of ATP by the luciferin-luciferase method interference by a bacterial phosphatase enzyme stable to perchlorate treatment
✍ Scribed by J.A. Davison; G.H. Fynn
- Publisher
- Elsevier Science
- Year
- 1974
- Tongue
- English
- Weight
- 285 KB
- Volume
- 58
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Interference by a Bacterial Phosphatase Enzyme Stable to Perchlorate Treatment
In an investigation of the nucieotide pools in Bacillus brevis we have observed the presence of an unusual ATP-degrading enzyme. This enzyme appears to be stable to perchlorate precipitation and neutralisation procedures commonly used (14) in preparing extracts for ATP assay by the luciferin-luciferase assay. In view of the low levels of ATP being measured by this method, it seems important to establish whether the presence of this enzyme constit,utes a potential source of error in assay procedures of this type.
Methods
Bacillus brevis ATC 10068 was grown from a spore inoculum on an asparagine-glycerol medium under conditions of limiting 0, (5).
Cell-free ext,racts (105 S) were prepared as previously described (6). ATP extracts (ATP-X) were prepared from the cells by the method of Cole et al. (4). Two milliliters of cell suspension was injected into 0.4 ml of 36% ice-cold perchlorate and shaken vigorously. The samples were left for 10 min on ice before neutralisat.ion with 0.48 ml of 6 N KOH. The samples were then centrifuged and frozen at -15°C until required.
More concentrated extracts were prepared by first harvesting the cells by centrifugation at 20,OOOg for 2 min, followed by addition of appropriate volumes of 6% ice-cold perchlorate, and then treating as previously described ( 6).
ATPase assay. The frozen sample containing the enzyme was thawed, and a volume equivalent to 0.2 ml of the original growing culture was added to an incubation medium originally described by Chapman et al.
(2) viz: 3 mM MgCl,; 15 mM potassium phosphate buffer, pH 7.4; 1 mM EDTA; H,O to 0.9 ml.