Rat epididymal alpha-D-mannosidase: purification, carbohydrate composition, substrate specificity, and antibody production.

Two alpha-D-mannosidases have previously been identified in rat epididymis. This communication reports the purification and characterization of the "acid" alpha-D-mannosidase. The enzyme was purified over 1000-fold to near homogeneity by acetone and (NH4)2SO4 precipitation followed by ion-exchange and hydroxylapatite chromatography. The molecular weight of the enzyme was estimated to be 220,000 by gel filtration. Polyacrylamide gel electrophoresis of the native enzyme under two conditions of buffer and pH showed a single band when stained for protein while electrophoresis under denaturing conditions resulted in bands of apparent Mr 60,000 and 31,000. The enzyme is a glycoprotein containing about 5.6% hexose. In addition to mannose (3.1%) and glucosamine (2.0%), the enzyme also contained small amounts of glucose, fucose, and galactose. Chemical analysis indicated the absence of sialic acid. The substrate specificity of the purified enzyme was investigated using linear and branched mannose-containing oligosaccharides. The enzyme cleaved linear oligosaccharides [Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc and Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc] very efficiently. However, little or no activity was observed toward high mannose oligosaccharides (Man9GlcNAc through Man5GlcNAc) or the branched trimannosyl derivative Man3GlcNAc. This specificity is very similar to that observed with rat kidney lysosomal alpha-D-mannosidase. Additional evidence that the epididymal enzyme is essentially a lysosomal alpha-D-mannosidase is the fact that polyclonal antibody prepared against the purified epididymal enzyme cross-reacted with lysosomal alpha-D-mannosidase from several rat tissues and with acidic alpha-D-mannosidase of a human cell line, results suggesting that the antibody will be useful in studying the biosynthesis and turnover of lysosomal alpha-D-mannosidases in at least two species.