Rapid three-step purification of a hepatic neutral cholesteryl ester hydrolase which is not the pancreatic enzyme

Abstract

A rat liver cytosolic cholesteryl ester hydrolase (CEH) was purified 12,600-fold by ammonium sulfate precipitation, cation exchange chromatography and gel permeation high-performance liquid chromatography, with an overall yield of 20%. Its properties are compared to those of pancreatic CEH, with which it has sometimes been identified. Liver CEH exhibited a single silver stained band following SDS-polyacrylamide gel electrophoresis (M_r=66 kDa), was activated by 0.5–10 mM taurocholate but was strongly inhibited by higher levels of taurocholate, which activate pancreatic CEH. Whereas bile salts are known to induce formation of a hexamer of pancreatic CEH, in the current study, 0.5 mM taurocholate dissociated a multimeric form of liver CEH to monomer. Liver CEH did not coelute with pancreatic CEH from cation exchange and chromatofocusing columns, exhibited no immunoreactivity with anti-rat pancreatic CEH IgG in Western blots, was not inhibited by anti-rat pancreatic CEH IgG and had a different amino acid composition from pancreatic CEH. In contrast to liver CEH, which is known to be activated by protein kinases A and C, pancreatic CEH was unaffected by cofactors for protein kinase A and was inhibited by cofactors for protein kinase C.