Localization of 5'-nucleotidase in bovine brain myelin fraction and myelin subfractions

Abstract

Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and 5'-nucleotidase (5'N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5'N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5'N activity maxima suggest that myelin may be a predominant locus of 5'N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5'N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5'N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (LUbrol WX) on 5'N activity were studied, and the results also suggest the intrinsic nature of 5'N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5'N activity, but not for the solubilization of this enzyme.