The purification of a detergent‐soluble glucose‐6‐phosphatase from rat liver

A highly active and soluble glucose‐6‐phosphatase has been purified to near homogeneity from rat liver. Successful purification has been initiated by covalent labeling of the enzyme in native rat liver microsomes with pyridoxal 5′‐phosphate and NaBH 4 , followed by solubilization of the microsomes with Triton X‐100, chromatography on phenyl‐Sepharose, hydroxyapatite, DEAE‐Sephacel and a second chromatography step on hydroxyapatite. The final enzyme preparation obtained was approximately 700‐fold purified over the activity of starting microsomes. As judged by SDS/PAGE the purified glucose‐6‐phosphatase is composed of a single protein with a molecular mass of 35 kDa. The present work demonstrates that the purified glucose‐6‐phosphatase must be arranged in the native microsomal membrane so that it is accessible to pyridoxal 5′‐phosphate from the cytoplasmic side.