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.
Laboratory of Metabolism, NIAAA, Rockville, Maryland 20852, USA.
G A
GA
Cook
W E
WE
O'Brien
H G
HG
Wood
M T
MT
King
R L
RL
Veech
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