Solubilization and purification of rat liver 5'-nucleotidase by use of a zwitterionic detergent and a monoclonal-antibody immunoadsorbent.
1. A variety of detergents were used to solubilize 5'-nucleotidase from rat liver plasma membranes. 2. The zwitterionic detergent Sulphobetaine 14 gave optimal solubilization by the criteria of release into a high-speed-centrifugation supernatant and the formation of the smallest and least polydisperse active enzyme observed on polyacrylamide-gel electrophoresis. 3. The Sulphobetaine 14-solubilized enzyme from rat liver was purified by using the conventional techniques of ion-exchange chromatography and gel filtration, or by an immunoaffinity step with a monoclonal antibody immunoadsorbent. 4. 5'-Nucleotidase was purified at least 12 000-fold relative to liver homogenate by the immunoaffinity purification scheme and had a specific activity in the range 285-340 mumol/min per mg of protein. The yield was in the range 9-16%. 5. The purified enzyme shows a major polypeptide band of apparent Mr 70 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and a minor band of apparent Mr 38 000. 6. A rational approach to the general problem of the purification of minor intrinsic membrane proteins is discussed, with the use of polyacrylamide-gel electrophoresis to determine the most appropriate detergent and monoclonal antibodies in subsequent immunoaffinity purification.
D015720:5'-Nucleotidase; D000818:Animals; D000911:Antibodies, Monoclonal; D001622:Betaine / Q000031:analogs & derivatives; D002462:Cell Membrane / Q000201:enzymology; D002498:Centrifugation; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D007164:Immunosorbents; D008099:Liver / Q000201:enzymology*; D009708:Nucleotidases / Q000302:isolation & purification*; D051381:Rats; D012995:Solubility
D000911:Antibodies, Monoclonal; D003902:Detergents; D007164:Immunosorbents; D001622:Betaine; D009708:Nucleotidases; D015720:5'-Nucleotidase