Mannosidosis: characterization of the enzymatic defect as a kinetic mutation

Mannosidosis, an inborn error of glycoconjugate metabolism, is characterized enzymatically by the defective activities of the acidic α mannosidoses A and B and normal levels of the neutral C isozyme. Previously the authors reported the presence of residual α mannosidase A and B activities in tissues and fluids from an affected homozygote (Amer. J. hum. Genet., 1975, 27, 48A). To further characterize the molecular nature of these defective activities, hepatic α mannosidases A, B and C from the affected homozygote and normal age matched control tissue were isolated and purified by differential ammonium sulfate precipitation, oligo affinity chromatography on concanavallin A Sepharose, and chromatography on DEAE cellulose. The normal A, B and C isozymes were purified 150, 550 and 160 fold, respectively, by these procedures; the purified isozymes were easily resolved by electrophoresis on cellulose acetate gels at pH 7.0. The specific activities of the residual A and B activities purified from the mannosidosis liver were less than 1% of the respective normal levels; the B isozyme represented 80% of the residual activity allowing further physical and kinetic characterization. Compared to the purified normal B isozyme, the purified residual B isozyme appeared more electronegatively charged on DEAE cellulose, was more thermostable at pH 4.4 and more thermolabile at pH 6.5, was stimulated by Co⁺⁺ and Mn⁺⁺, cations which inhibited the normal isozyme, and had altered kinetic properties; the apparent K(m) was about 50 times higher (K(m) = 2.86 mM vs. 0.56 mM). These findings suggest that the enzymatic defect in this homozygote resulted from a single base substitution in the structural gene coding for a common subunit of the A and B isozymes which rendered the enzyme proteins kinetically defective.