Enzymatic treatment to eliminate the extracellular ATP for improving the detectability of bacterial intracellular ATP.
A novel and effective treatment of biological samples with a combination of adenosine phosphate deaminase and apyrase was developed for reducing extracellular ATP, which has been a major problem encountered in improving the sensitivity of assays for intracellular ATP by the firefly luciferin-luciferase (L-L) method. Under the enzymatic reaction conditions, ATP and the related adenosine derivatives were converted to IMP, which are not active to the L-L system. In the model system (3.2 x 10(-8) M ATP in 1% yeast extract solution) the treatment with adenosine phosphate deaminase resulted in the reduction of ATP to 1.3 x 10(-11) M, and the concomitant use of apyrase lowered the concentration to 3.3 x 10(-13) M. The treatment (0.05 U/ml of adenosine phosphate deaminase and apyrase) was applied to the detection of bacteria in broth by the L-L method, affording the detection of 42 colony-forming unit (CFU)/ml of Escherichia coli and 10 CFU/ml of Staphylococcus aureus in the broth.
Sakakibara
T
T
Research & Development Division, Kikkoman Corporation, Noda-shi, Chiba, Japan.
Murakami
S
S
Hattori
N
N
Nakajima
M
M
Imai
K
K
D000243:Adenosine Deaminase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000032:analysis* / Q000378:metabolism*; D001081:Apyrase / Q000378:metabolism*; D001419:Bacteria / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D005110:Extracellular Space / Q000737:chemistry; D007424:Intracellular Fluid / Q000737:chemistry; D013211:Staphylococcus aureus / Q000737:chemistry
D000255:Adenosine Triphosphate; D000243:Adenosine Deaminase; D001081:Apyrase