Purification and characterization of a 28 kDa cytosolic inhibitor of cholesteryl ester hydrolases in rat testis

A 28 kDa inhibitory protein was purified from ratestis cytosol by sequential 40–65% ammonium sulfate precipitation, cation exchange chromatography, anion exchange chromatography, and preparative SDS‐polyacrylamide gel electrophoresis. The heat‐stable, trypsin‐labile protein exhibited nonenzymatic, concentration‐dependent inhibition of testicular and pancreatic cholesteryl ester hydrolases at all stages of putification. Copurifying at each stage was a 26.5 kDa protein which comprised 25% of the mass of the two proteins. Polyclonal antibodies raised to either or both 28 kDa and 26.5 kDa proteins by direct injection of excised electrophoretic bands cross‐reacted with both proteins on western blots, immunoprecipitated both proteins, and neutralized inhibitory activity. Amino acid compositions of the individual proteins electroeluted from SDS‐polyacrylamide gels were different from those of other surface‐active proteins of similar molecular weights. Both proteins exhibited identical pl of 4.8 on chromatofocusing columns and two‐dimensional gel electrophoresis. Although the subcellular distribution of the 28 kDa protein is unknown, its testicular cytosolic concentration, calculated from the purified protein mass, was 8×10−9 mols/L, which probably underestimates the actual concentration by an order of magnitude. This is greater than the minimum concentration required forin vitro inhibition (10−9 mols/L), consistent with a physiological role for this protein.