A. Martinezmartinez et al., BIOCHEMICAL-PROPERTIES OF 5'-NUCLEOTIDASE FROM MOUSE SKELETAL-MUSCLE, Biochimica et biophysica acta. Protein structure and molecular enzymology, 1386(1), 1998, pp. 16-28
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.