Purification and characterization of phosphopentomutase from rat liver

Abstract

Phosphopentomutase (α- d-glucose-1,6-bisphosphate:deoxy- d-ribose-1-phosphate phosphotransferase, EC 2.7.5.6) was purified 515-fold from rat liver utilizing the techniques of ammonium sulfate fractionation, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. The highly purified enzyme, analyzed by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate, was found to consist of two subunits of molecular weight 32 500 which associated to yield an active mol molecular weight 65 000. Three of the substrates tested were used by phosphopentomutase with relative reactivities: ribose 1-phosphate (1.0) > deoxyribose 1-phosphate (0.25) > glucose 1-phosphate (0.024). Phosphopentomutase was activated by Mg 2+, Mn 2+, cysteine and glucose 1,6-bisphosphate, although low levels of activity were detected in the absence of any of these compounds. Arsenate was found to be a non-competitive inhibitor with an apparent K i of 6 · 10 −3 M when ribose 1-phosphate was used as substrate and an apparent K i of 11 · 10 −3 M when deoxyribose 1-phosphate was used as substrate. In the absence of inhibitor and in the presence of optimal concentrations of activators, phosphopentomutase had an apparent K m of 8.3 · 10 −5 M with deoxyribose 1-phosphate and 1.3 · 10 −4 M with ribose 1-phosphate as substrate. The information in this paper clearly distinguishes mammalian phosphopentomutase and phosphoglucomutase as separate enzymes and represents the first extensive purification of a mammalian mutase with major specificity for pentose 1- and 5-phosphates.