For the determination of 5'-ribonucleotide phosphohydrolase (EC 3.1.3.5; 5'-Nase) in rat liver, a radiochemical double-labelling assay was developed. [14C]-labelled AMP which is hydrolyzed to [14C]-adenosine by 5'-Nase activity is added to crude liver homogenates. After 30 min, the process is stopped and [2^-3H]-adenosine added to estimate the loss of [14C]-adenosine during separation by ion exchange column chromatography. The enzymatic reaction was found to be linear in correlation with the enzyme content and the incubation time. The specificity of the reaction was evaluated by addition of β-glycerophosphate which acts as a competitive inhibitor to eliminate the catalytic effect of non-specific phosphatases, and addition of α, β-methylene adenosine 5'-diphosphate, a specific inhibitor of 5'-Nase; both cause an almost complete suppression of enzyme activity.

Keywords:
5'-Ribonucleotide phosphohydrolase (EC 3.1.3.5), Radiochemical double-labelling assay, Liver homogenates, Circadian variation, β-Glycerophosphate, α,β-Methylene adenosine 5 '-diphosphate
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1979
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