Purification and characterization of a 28 kDa cytosolic inhibitor of cholesteryl ester hydrolases in rat testis.
A 28 kDa inhibitory protein was purified from rat testis 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 purification. 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 X 10(-9) mols/L, which probably underestimates the actual concentration by an order of magnitude. This is greater than the minimum concentration required for in vitro inhibition (10(-9) mols/L), consistent with a physiological role for this protein.
Hines
D S
DS
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298-0614, USA.
Wee
S
S
Grogan
W M
WM
D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D000818:Animals; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D007120:Immunochemistry; D066298:In Vitro Techniques; D007526:Isoelectric Point; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology; D051381:Rats; D002787:Sterol Esterase / Q000037:antagonists & inhibitors*; D013696:Temperature; D013737:Testis / Q000378:metabolism*
D000596:Amino Acids; D004791:Enzyme Inhibitors; D002787:Sterol Esterase; D000645:Ammonium Sulfate