Purification and characterization of a neutral, bile salt-independent retinyl ester hydrolase from rat liver microsomes. Relationship To rat carboxylesterase ES-2.
A neutral, bile salt-independent retinyl ester hydrolase (NREH) has been purified from a rat liver microsomal fraction. The purification procedure involved detergent extraction, DEAE-Sepharose ion exchange, Phenyl-Sepharose hydrophobic interaction, Sephadex G-100 and Sephacryl S-200 gel filtration chromatographies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolated enzyme has an apparent molecular mass of approximately 66 kDa under denaturing conditions on SDS-PAGE. Analysis of the amino acid sequences of four peptides isolated after proteolytic digestion revealed that the enzyme is highly homologous with other rat liver carboxylesterases. In particular, the sequences of the four peptides of the NREH (60 amino acids total) were identical to those of a rat carboxylesterase expressed in the liver (Alexson, S. E. H., Finlay, T. H., Hellman, U., Svensson, L. T., Diczfalusy, U., and Eggertsen, G. (1994) J. Biol. Chem. 269, 17118-17124). Antibodies against this enzyme also react with the purified NREH. Purified NREH shows a substrate preference for retinyl palmitate over triolein and did not catalyze the hydrolysis of cholesteryl oleate. With retinyl palmitate as substrate, the enzyme had a pH optimum of 7 and showed apparent saturation kinetics, with half-maximal activity achieved at substrate concentrations (Km) of approximately 70 microM.
Sun
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Department of Biochemistry, Medical College of Pennsylvania-Hahnemann School of Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19129, USA.
Alexson
S E
SE
Harrison
E H
EH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001647:Bile Acids and Salts / Q000378:metabolism*; D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004224:Diterpenes; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D000084562:Retinyl Esters; D014801:Vitamin A / Q000031:analogs & derivatives / Q000378:metabolism
D000906:Antibodies; D001647:Bile Acids and Salts; D004224:Diterpenes; D000084562:Retinyl Esters; D014801:Vitamin A; C014794:retinol palmitate; D002265:Carboxylic Ester Hydrolases; C022523:retinyl esterase