Rapid three-step purification of a hepatic neutral cholesteryl ester hydrolase which is not the pancreatic enzyme.
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 (Mr = 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.
Ghosh
S
S
Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0614.
Grogan
W M
WM
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D002787:Sterol Esterase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*
D000596:Amino Acids; D001647:Bile Acids and Salts; D002787:Sterol Esterase