A rapid, enzymatic assay for the measurement of inorganic pyrophosphate in animal tissues.
A simple, rapid enzymatic assay for the determination of inorganic pyrophosphate in tissue and plasma has been developed using the enzyme pyrophosphate--fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) which was purified from extracts of Propionibacterium shermanii. The enzyme phosphorylates fructose-6-phosphate to produce fructose-1,6-bisphosphate using inorganic pyrophosphate as the phosphate donor. The utilization of inorganic pyrophosphate is measured by coupling the production of fructose-1,6-bisphosphate with the oxidation of NADH using fructose-bisphosphate aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1), and glycerol-3-phosphate dehydrogenase (NAD+)(EC 1.1.1.8). The assay is completed in less than 5 min and is not affected by any of the components of tissue or plasma extracts. The recovery of pyrophosphate added to frozen tissue powder was 97 +/- 1% (n = 4). In this assay the change in absorbance is linearly related to the concentration of inorganic pyrophosphate over the curvette concentration range of 0.1 microM to 0.1 mM.
Cook
G A
GA
Laboratory of Metabolism, NIAAA, Rockville, Maryland 20852, USA.
O'Brien
W E
WE
Wood
H G
HG
King
M T
MT
Veech
R L
RL
D000818:Animals; D002623:Chemistry Techniques, Analytical / Q000379:methods; D011756:Diphosphates / Q000032:analysis* / Q000097:blood; D008099:Liver / Q000737:chemistry; D008297:Male; D009206:Myocardium / Q000737:chemistry; D010770:Phosphotransferases / Q000302:isolation & purification; D011424:Propionibacterium / Q000201:enzymology; D051381:Rats; D017208:Rats, Wistar
D011756:Diphosphates; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase