Purification and properties of a mouse liver plasma-membrane glycoprotein hydrolysing nucleotide pyrophosphate and phosphodiester bonds.
1. A mouse liver plasma-membrane preparation was solubilized in an N-dodecylsarcosinate-Tris buffer, pH7.8, and the proteins and glycoproteins were separated by a rate-zonal centrifugation in sucrose-detergent gradients. 2. A peak of alkaline phosphodiesterase activity which sedimented ahead of the 5'-nucleotidase peak was associated with a major glycoprotein component of the plasma membrane. 3. The phosphodiesterase activity was then purified further by gel filtration and gave a single glycoprotein band after electrophoresis on polyacrylamide gels. The apparent molecular weight of the polypeptide at pH7.4 and 8.9 was 128000-130000 and was independent of the polyacrylamide concentration. Electrophoresis in gels containing deoxycholate showed that the protein band was coincident with phosphodiesterase activity. 4. After two-dimensional immunoelectrophoresis, with agarose containing rabbit anti-(mouse plasma-membrane) antiserum as second dimension, the enzyme showed one component which was also coincident with the phosphodiesterase activity. 5. An amino acid composition of the glycoprotein is presented. Carbohydrate analysis indicated the presence of glucosamine, neutral sugars and sialic acid. 6. The enzyme was also a nucleotide pyrophosphatase, as shown by a similar enrichment during purification of activity towards ATP, NAD(+), UDP-galactose and UDP-N-acetylglucosamine. The phosphodiesterase activity, measured by using dTMP p-nitrophenyl ester as substrate, was competitively inhibited by nucleotide pyrophosphate substrates. The enzyme showed little or no activity towards RNA, cyclic AMP, AMP, ADP and glycerylphosphorylcholine. 7. The significance of this enzyme activity in the plasma membrane is discussed.
D000596:Amino Acids / Q000032:analysis; D000606:Amino Sugars / Q000032:analysis; D000818:Animals; D001667:Binding, Competitive; D002241:Carbohydrates / Q000032:analysis; D002462:Cell Membrane / Q000201:enzymology*; D002501:Centrifugation, Zonal; D002850:Chromatography, Gel; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007122:Immunoelectrophoresis; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000302:isolation & purification*; D011817:Rabbits / Q000276:immunology
D000596:Amino Acids; D000606:Amino Sugars; D002241:Carbohydrates; D003902:Detergents; D006023:Glycoproteins; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases