Properties of a 5'-nucleotidase purified from mouse liver plasma membranes.
1. Extraction of a mouse liver plasma-membrane fraction with a detergent buffer, N-dodecylsarcosinate-Tris buffer (sarcosyl-Tris buffer), solubilized 90% of the protein and 70% of the 5'-nucleotidase activity. 2. The proteins of the sarcosyl-Tris buffer extract were fractionated by a rate-zonal centrifugation in a sucrose-detergent gradient. The major protein peak sedimented ahead of phospholipids, which mainly remained in the overlay. Glycoproteins were separated ahead of the protein peak. 3. The 5'-nucleotidase activity peak was associated with 5% of the protein applied to the gradient, and contained relatively few protein bands. 4. The 5'-nucleotidase was purified further by gel filtration on Sepharose and Sephadex columns equilibrated with sarcosyl-Tris buffer, to give a single glycoprotein band on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. The purified enzyme was lipid-free. 5. Electrophoresis in polyacrylamide gels in sarcosyl-Tris buffers showed that the enzymic activity was coincident with the protein band. 6. The molecular weight suggested for the enzyme activity by gel filtration or centrifugation in sucrose gradients was 140000-150000. Sometimes, a minor enzyme peak of lower molecular weight was obtained. 7. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate indicated that as the polyacrylamide concentration was increased from 5 to 15%, the apparent molecular weight of the enzyme decreased from 130000 to 90000. 8. The evidence that 5'-nucleotidase is composed of two active and similar, if not identical, glycoprotein subunits and the role of detergent in effecting the separation of membrane proteins and glycoproteins are discussed. 9. Substrate requirements, pH optima and the nature of inhibition by an analogue of adenosine diphosphate are reported.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D002501:Centrifugation, Zonal; D002845:Chromatography; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000166:cytology* / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D009708:Nucleotidases / Q000302:isolation & purification*; D010743:Phospholipids / Q000032:analysis; D011134:Polysaccharides; D011485:Protein Binding; D012521:Sarcosine; D012967:Sodium Dodecyl Sulfate; D013056:Spectrophotometry, Ultraviolet; D014462:Ultrafiltration
D006023:Glycoproteins; D010743:Phospholipids; D011134:Polysaccharides; D012967:Sodium Dodecyl Sulfate; D009708:Nucleotidases; D012521:Sarcosine