Purification of bovine liver cytosolic 5'-nucleotidase. Kinetic and structural studies as compared to the membrane isoenzyme.
Cytosolic 5'-nucleotidase from bovine liver has been purified to homogeneity. Two affinity chromatographies on concanavalin A and 5'AMP-Sepharose columns result in a 12,000-fold purification. The sequential elution of glycoproteins from the concanavalin-A-Sepharose column with methyl alpha-D-glucoside and methyl alpha-D-mannoside greatly increases the degree of purification of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate shows two subunits having apparent molecular masses of 65 kDa and 57 kDa respectively, while only one band at 70 kDa is observed in the case of the membrane-bound 5'-nucleotidase. Both the Stokes radii, measured by gel exclusion HPLC, and the sedimentation coefficient, determined by density gradient ultracentrifugation, indicate that the cytosolic enzyme is a heterodimer of about 130 kDa. This contrasts with the membrane-bound 5'-nucleotidase which is a homodimer of 140 kDa. Moreover, the antibodies raised against the membrane 5'-nucleotidase inhibited the cytosolic form indicating that a common antigenic determinant(s) exists between the two isoenzymes. However, structural differences are revealed by immunoblotting. In the same way, the effect of lectins suggests that differences in the structure of the carbohydrate chains exist between the two isoenzymes. The purified cytosolic enzyme has lower affinity for the nucleotides than does the membrane enzyme. In addition, while ADP, [alpha,beta-CH2]ADP and ATP were strong competitive inhibitors of the membrane enzyme, ADP and ATP activate the cytosolic form and [alpha,beta-CH2]ADP has no effect. Moreover, two pH optima at 7.5 and 9.5 are observed in the cytosolic enzyme while only one at 7.5 occurred in the membrane form. Finally the exogenous cations, MgCl2 and MnCl2, are necessary for the maximal activity of the cytosolic but not of the membrane 5'-nucleotidase. All these observations indicate that the two isoenzymes are different.
Zekri
M
M
Laboratoire de Biochimie Médicale, U.E.R. de Médecine, Nantes, France.
Harb
J
J
Bernard
S
S
Meflah
K
K
D015720:5'-Nucleotidase; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D037102:Lectins / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D009708:Nucleotidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D037102:Lectins; D009708:Nucleotidases; D015720:5'-Nucleotidase