Purification and properties of bovine liver plasma membrane 5' nucleotidase.
5'-Nucleotidase from bovine liver plasma membranes has been extracted by the zwitterionic detergent sulfobetaine 14, and purified to apparent homogeneity. Two affinity chromatographies on concanavalin-A-Ultrogel and 5' AMP-Sepharose 4B followed by AcA-54-Ultrogel filtration resulted in a purification of 16000 times relative to the homogenate. Sodium dodecyl sulphate gel electrophoresis indicates that the apparent molecular weight of the subunit is 70000. Cross-linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows a band with an apparent molecular weight of 140000 indicating that the enzyme is a dimer. 5'-Nucleotidase is a glycoprotein and its activity is inhibited to different degrees by various lectins, indicating a direct interaction with the enzyme. The purified enzyme shows a sevenfold greater affinity for AMP than the membrane-bound enzyme. The optimum activity of the purified enzyme occurs at pH 7.5 while the membrane-bound enzyme showed a wide range of pH optimum (7.5-8.3). An Arrhenius plot of the membrane-bound enzyme shows a break at 28 degrees C, which disappears in the purified enzyme. The enzyme was inhibited by EDTA, and this inhibition was reversed by divalent cations. This, as well as other evidence, indicates that the enzyme contains a highly bound metal cation, perhaps Mn2+ or Mg2+.
D015720:5'-Nucleotidase; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D037102:Lectins / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009708:Nucleotidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature; D014462:Ultrafiltration
D002413:Cations, Divalent; D037102:Lectins; D004492:Edetic Acid; D009708:Nucleotidases; D015720:5'-Nucleotidase