BIOCHEMICAL-PROPERTIES OF 5'-NUCLEOTIDASE FROM MOUSE SKELETAL-MUSCLE

Ecto-5'-nucleotidase (eNT) from mouse muscle has been purified after e xtraction with detergent followed by chromatography on concanavalin A- and AMP-Sepharose. Three fractions were recovered: UF was NT non-reta ined in immobilised AMP; F-I was bound enzyme eluted with beta-glycero phosphate, and F-II was bound NT released with AMP. eNT was 80 000-fol d purified in F-II, this fraction showing proteins of 74, 68 and 51 kD a 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 mi grated at 5.8S in Triton X-100-, or 4.4S in Brij 96-containing gradien ts. The hydrodynamic behaviour, concentration in Triton X-114, binding to phenyl-agarose, and sensitivity to phosphatidylinositol specific p hospholipase C revealed that enzyme forms in F-I or F-II were amphiphi lic dimers with linked phosphatidylinositol residues, whilst most of N T 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 mu M Zn2+ o r 100 mu M Mn2+. In assays with Tris buffer, NT showed a K-m for AMP o f 12 mu M, and was competitively inhibited by ATP or ADP. (C) 1998 Els evier Science B.V. All rights reserved.