Purification and characterization of rat epididymal - fluid x - D - mannosidase : similarities to sperm plasma - membrane x - D - mannosidase

We have previously reported the occurrence and partial characterization of a novel a-D-mannosidase activity on rat sperm plasma membranes [Tulsiani, Skudlarek and OrgebinCrist (1989) J. Cell Biol. 109, 1257-1267]. Here, we report the presence of a similar a-D-mannosidase activity in a soluble form in rat epididymal fluid. The soluble enzyme was purified nearly 500-fold with 9-12o% recovery to a state approaching homogeneity using: (1) (NH4)2SO4 precipitation; (2) affinity chromatography on immobilized mannan and D-mannosamine; (3) ion-exchange (DE-52) column chromatography; (4) molecular-sieve chromatography. The enzyme was eluted from the final column (Sephacryl S-400) at an apparent molecular mass of 460 kDa. When resolved by SDS/PAGE (under denaturing conditions), the enzyme showed a major protein band (115 kDa) and few very minor bands. The polyclonal antibody raised against the major protein band was found to cross-react with the a-D-mannosidase activity present in epididymal fluid