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 NaBH4, 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.
Speth
M
M
Biochemisches Institut, Justus-Liebig-Universität Giessen, Federal Republic of Germany.
Schulze
H U
HU
D000818:Animals; D002845:Chromatography; D003902:Detergents; D005952:Glucose-6-Phosphatase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000378:metabolism; D051381:Rats; D012995:Solubility
D003902:Detergents; D011732:Pyridoxal Phosphate; D005952:Glucose-6-Phosphatase