Antibody against mouse liver 5′-nucleotidase immunostains white matter in the adult mouse central nervous system

Abstract

An antiserum against rat liver 5′-nucleotidase has been shown to immunostain myelinated fibers and oligodendrocytes in the rat CNS, consistent with evidence for 5′-nucleotidase activity in rat brain myelin and oligodendrocytes (Cammer, Sacchi and Kahn, Devel. Brain Res., 1985, 20: 89–96). However, in the mouse CNS, in which myelin also has 5′-nucleotidase activity, that antiserum stained only blood vessels. To obtain an antibody against the mouse enzyme, 5′-nucleotidase was partly purified from mouse liver membranes by detergent extraction, heat treatment, affinity chromatography, acidification, and ammonium sulfate fractionation. The preparation, which was enriched about 110-fold in 5′-nucleotidase specific activity, compared to the starting extract, was electrophoresed on a preparative slab gel containing Triton X-100, a strip was stained histochemically for 5′-nucleotidase, and the material corresponding to the stained band was used to immunize a rabbit. The immune IgG fraction, but not the preimmune IgG, reacted with mouse brain homogenates. The immune serum gave consistently greater inhibition of 5′-nucleotidase activity in mouse liver homogenates, mouse brain myelin and mouse brain homogenates, but not rat brain or liver homogenates, compared to the preimmune serum. The immune serum, but not the preimmune serum, immunostained white matter in the normal adult mouse brain and spinal cord. The findings suggest that the mouse may have one isozyme of 5′-nucleotidase similar to that in rat with respect to subunit sizes but differing in primary structure at one or more antigenic sites and support previous observations of 5′-nucleotidase activity in myelin from mouse brains and spinal cords.