The reversible activation by Mn 2+ ions of the Ca 2+-requiring neutral proteinase of human erythrocytes

Abstract

Mn 2+ (50 μ m) satisfies the requirement for activity of the purified Ca 2+-dependent neutral proteinase from human erythrocytes. Unlike the activation by Ca 2+ [ E. Melloni et al. (1984) Biochem. Int. 8, 477–489 ], the effect of Mn 2+ is fully reversible and does not involve autodigestion of the native 80-kDa catalytic subunit. However, the native dimeric proenzyme (procalpain), which contains both the 80-kDa subunit and a smaller 30-kDa subunit, is not activated by Mn 2+ alone but also requires the presence of micromolar concentrations of Ca 2+. Under these conditions, 40% of the maximum activity is expressed without dissociation of the 80- and 30-kDa subunits. Mn 2+, but not micromolar Ca 2+, can also partially satisfy the metal requirement of the native 80-kDa subunit isolated after dissociation of the heterodimer. This activity is further enhanced by the addition of 5 μ m Ca 2+, which is ineffective in the absence of Mn 2+. After procalpain is converted to active calpain by incubation with Ca 2+ and substrate [ S. Pontremoli et al. (1984) Biochem. Biophys. Res. Commun. 123, 331–337 ] full activity is observed with 5 μ m Mn 2+, which now substitutes completely for Ca 2+. Activation of procalpain by Mn 2+ represents a new mechanism for modulation of the Ca 2+-dependent proteinase activity.