Purification and characterization of rat epididymal-fluid alpha-D-mannosidase: similarities to sperm plasma-membrane alpha-D-mannosidase.
We have previously reported the occurrence and partial characterization of a novel alpha-D-mannosidase activity on rat sperm plasma membranes [Tulsiani, Skudlarek and Orgebin-Crist (1989) J. Cell Biol. 109, 1257-1267]. Here, we report the presence of a similar alpha-D-mannosidase activity in a soluble form in rat epididymal fluid. The soluble enzyme was purified nearly 500-fold with 9-12% 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 alpha-D-mannosidase activity present in epididymal fluid (soluble) and detergent-solubilized spermatozoa from the rat and mouse. This result suggested that the soluble and membrane-bound enzyme activities shared a common antigenic site(s). The antibody was used to characterize further the alpha-D-mannosidase activity(ies) present in the rat epididymal fluid and rat sperm plasma membranes. Data from these studies show that the two forms are similar in (a) subunit molecular mass, (b) substrate specificity and (c) inhibitory effect of several sugars. These similarities suggest that the soluble and membrane-bound alpha-D-mannosidase activities are isoforms. Immunoprecipitation studies after solubilization of the testis and epididymal particulate fraction from sexually immature rats show that the testis (but not the epididymis) contains the immunoreactive alpha-D-mannosidase activity. This result and the fact that spermatozoa from the rat rete testis show alpha-D-mannosidase activity indicate that the sperm enzyme is synthesized in the testis during spermatogenesis.
Tulsiani
D R
DR
Center for Reproductive Biology Research, Vanderbilt University School of Medicine, Nashville, TN 37232-2633.
Skudlarek
M D
MD
Nagdas
S K
SK
Orgebin-Crist
M C
MC
D000818:Animals; D015153:Blotting, Western; D001826:Body Fluids / Q000201:enzymology; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000201:enzymology*; D007106:Immune Sera; D007700:Kinetics; D008297:Male; D008361:Mannosidases / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D013094:Spermatozoa / Q000201:enzymology*; D013379:Substrate Specificity; D043323:alpha-Mannosidase
D007106:Immune Sera; D008361:Mannosidases; D043323:alpha-Mannosidase