Spectrophotometric and radioenzymatic determination of ribose 5-phosphate

The present work describes an assay which is higly specific or fibose 5-phosphate. The method is based on the following three-stage enzymatic conversion: (1) ribose 5-phosphate ⇌ ribose 1-phosphate associated with the conversion of adenine to inosine, or radioenzymatically by measuring the radioactivity (phosphopentomutase); (2) ribose 1-phosphate + adenine ⇌ adenosine + Pi (adenosine phosphorylase); (3) adenosine + H2O → inosine + NH3 (adenosine deaminase). Ribose 5-phosphate may be detemined either directly following the change in absorbance at 265 nm associated with the conversion of adenine to inosine, or radioenzymatically by measuring the radioactivity of inosing formed form [8-14C]adenine, after chromatrographic separation on polyethy-eneimine-cellulose. The spectrophotometric assay was used to follow ribose 5-phosphate formation and ribose 1-phosphagte consumption catalyzed by phosphopentomutase. Further, the ability of alkaline phospatase, 5′-nucleotidase and crude extract of Bacillus cereus cells to act on ribose 5-phosphate was tested. The radioenzymatic assay was proved useful in determining the levels of ribose 5- phosphate in rat tissues.