Purification of human microsomal liver glucose-6-phosphatase system by affinity chromatography and immunodetection.

A multiple purification of phosphohydrolase (PH) and phosphotranslocase (PT) of the human liver microsomal glucose-6-phosphatase system has been obtained by a rapid two-step procedure using affinity chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the final products showed one major band each at 63 and 37 kDa for PH and PT respectively. The immunoblot analysis of SDS-PAGE of various purification steps for human liver using rabbit antibodies raised against the enzyme preparations also showed major bands at 63 and 37 kDa for PH and PT respectively. A major band at 260 kDa was observed by the Western blot of native PAGE of the enzyme preparation for PH. Cross-reacting materials at the positions of 63 and 37 kDa were detected only in liver, kidney and intestine. From five liver samples of patients suffering from type Ia glycogenosis there were diminished amounts of crossreacting materials at 63 kDa only in two samples. The uptake of glucose-6-phosphate has taken place in liposomes of Sepharose affinity purified products suggesting that this preparation may be a complex of PH and glucose-6-phosphate translocase.