Biochemical properties of 5'-nucleotidase from mouse skeletal muscle.
Ecto-5'-nucleotidase (eNT) from mouse muscle has been purified after extraction with detergent followed by chromatography on concanavalin A- and AMP-Sepharose. Three fractions were recovered: UF was NT non-retained in immobilised AMP; F-I was bound enzyme eluted with beta-glycerophosphate, and F-II was bound NT released with AMP. eNT was 80000-fold purified in F-II, this fraction showing proteins of 74, 68 and 51 kDa after immunoblotting. NT in UF migrated at 6.7S after centrifugation in sucrose gradients with Triton X-100, the peak being split into two of 6.7S and 4.4S in gradients with Brij 96. Ecto-NT in F-I or F-II migrated at 5.8S in Triton X-100-, or 4.4S in Brij 96-containing gradients. The hydrodynamic behaviour, concentration in Triton X-114, binding to phenyl-agarose, and sensitivity to phosphatidylinositol-specific phospholipase C revealed that enzyme forms in F-I or F-II were amphiphilic dimers with linked phosphatidylinositol residues, whilst most of NT forms in UF were hydrophilic dimers. A zinc/protein molar ratio of 2.2 was determined for eNT in F-II. NT activity was decreased in assays made in imidazole buffer, and was partly restored with 10 microM Zn2+ or 100 microM Mn2+. In assays with Tris buffer, NT showed a Km for AMP of 12 microM, and was competitively inhibited by ATP or ADP.
Martínez-Martínez
A
A
Departamento de Bioquímica y Biología Molecular A, Universidad de Murcia, Apdo. 4021, E-30071 Murcia, Spain.
Flores-Flores
C
C
Campoy
F J
FJ
Muñoz-Delgado
E
E
Fini
C
C
Vidal
C J
CJ
D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D051379:Mice; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology*; D009708:Nucleotidases / Q000378:metabolism; D013379:Substrate Specificity; D015032:Zinc / Q000032:analysis
D002413:Cations, Divalent; D009708:Nucleotidases; C018775:adenosine monophosphatase; D015720:5'-Nucleotidase; D015032:Zinc