An anion-exchange radioassay for glucose 6-phosphate phosphatase: use in topological studies with endoplasmic reticulum vesicles.
A simple procedure is presented for the enzymatic preparation of [2-3H]mannose 6-phosphate (Man 6-P) with purified yeast hexokinase and unlabeled ATP. The enzymatically synthesized [2-3H]Man 6-P is utilized as the radiolabeled substrate in a new rapid assay for glucose 6-phosphate (Glc 6-P) phosphatase. The principle of the assay procedure is that the unreacted substrate, [2-3H]Man 6-P, is retained by the anion-exchange resin, AG 1-X8 (acetate), while the enzymatic product, [2-3H]-mannose, is eluted directly into a scintillation counting vial. When Glc 6-P phosphatase activity associated with mouse liver endoplasmic reticulum (ER) vesicles is assayed by the new chromatographic assay, the same characteristic latency and properties are observed, as determined by the commonly used colorimetric assay of inorganic phosphate produced. The anion-exchange radioassay described should be useful for a variety of topological studies on enzymes associated with membrane vesicles derived from liver and kidney ER.
Rush
J S
JS
Department of Biochemistry, University of Kentucky College of Medicine, A. B. Chandler Medical Center, Lexington 40536.
Waechter
C J
CJ
D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology; D001921:Brain / Q000201:enzymology; D016466:CHO Cells; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange / Q000379:methods; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005952:Glucose-6-Phosphatase / Q000032:analysis* / Q000378:metabolism; D006593:Hexokinase; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008297:Male; D008360:Mannosephosphates / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008811:Mice, Inbred DBA; D008862:Microsomes, Liver / Q000201:enzymology*; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis* / Q000378:metabolism; D011865:Radioisotope Dilution Technique; D051381:Rats; D014316:Tritium
D008360:Mannosephosphates; D014316:Tritium; C027693:mannose-6-phosphate; D006593:Hexokinase; C023553:mannose-6-phosphatase; D010744:Phosphoric Monoester Hydrolases; D005952:Glucose-6-Phosphatase