Purification and characterization of retinyl ester hydrolase as a member of the non-specific carboxylesterase supergene family.
Hepatic retinyl ester hydrolase (REH) activity was isolated from porcine and human liver and characterized, and some of its properties were compared with those of other retinyl-ester-splitting enzymes. Sequence analysis revealed that the REH proteins are structurally similar to non-specific carboxylesterases and distinct from bile salt-activated lipases and cholesterol esterases. Pig REH, a 64-kDa protein, hydrolyzed retinyl palmitate at a rate of 595 nmol x h(-1) x mg(-1) protein in the presence of 100 mM Chaps with an apparent Km value for retinyl palmitate of 27.5 microM. The pH optimum was 7.0-9.2. Its human counterpart has a molecular mass of 65 kDa and a pH optimum near 6.5. In the presence of Chaps, pig REH activity was stimulated up to 1.7-fold by various non-ionic detergents. The ranking order of retinyl palmitate cleavage initiated by the stimuli was n-dodecylglucoside > octanoyl-N-methylglucamide > n-octyglucoside > n-dodecylmaltoside > Triton X-114 > Triton X-100. Porcine REH was effectively inhibited by alpha-tocopherol and bis-(4-nitrophenyl) phosphate [(Np)2P]. The structural, immunological and catalytic features, pH dependence, and the effect of (Np)2P on enzyme activity of pig REH are similar to those reported for the non-specific carboxylesterase ES-4. However, ES-4 differed from REH in molecular mass and the requirement of Chaps or Chaps-like detergents as cofactor. Judging from these results, pig REH may be a non-specific carboxylesterase isoform.
Schindler
R
R
Department of Human Nutrition and Food Science, Christian-Albrechts-Universität zu Kiel, Germany. ruhsch@aol.com
Mentlein
R
R
Feldheim
W
W
D000595:Amino Acid Sequence; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D005810:Multigene Family; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine
D003902:Detergents; D002265:Carboxylic Ester Hydrolases; C022523:retinyl esterase