Co²⁺ - mediated time - and temperature - dependent activation of neutral α-D-mannosidase from monkey brain

Abstract

Neutral alpha-D-mannosidase from monkey brain was purified by Co²⁺-chelate affinity chromatography and immunoadsorbent affinity chromatography. The purified enzyme, with subunit Mr 45,000, was essentially homogeneous with only traces of two contaminant proteins as revealed by SDS/polyacrylamide-gel electrophoresis and AgNO3 staining. The purified enzyme, on preincubation with Co²⁺ at 37 degrees C or 60 degrees C followed by assay, showed a time-dependent enhancement in activity. The enhanced activity of the enzyme persisted even after removal of the Co²⁺. Bacitracin could partially prevent the activation. An aminopeptidase activity that was stimulated by Co²⁺ both at 37 degrees C and at 60 degrees C was present in the purified enzyme. After preincubation of the enzyme with Co²⁺ there was evidence for the release of amino acids, as revealed by t.l.c., but the Mr determined by SDS/polyacrylamide-gel electrophoresis was not appreciably altered. It is suggested that a Co²⁺-stimulated thermostable aminopeptidase, inseparable from the neutral mannosidase, may be involved in the stimulation of neutral mannosidase activity during its preincubation with Co²⁺.