title: Regulation of two rat liver microsomal carboxylesterase isozymes: species differences, tissue distribution, and the effects of age, sex, and xenobiotic treatment of rats.
abstract: The preceding paper described the purification of two rat liver microsomal carboxylesterases, designated hydrolases A and B, that have high affinity (Km approximately 25 microM) and low affinity (Km approximately 400 microM) for para-nitrophenylacetate, respectively. The present study describes the preparation and purification of polyclonal antibodies against these purified enzymes. Each antibody was subjected to immunoabsorption chromatography to remove antibodies against epitopes common to both hydrolases A and B. The resulting isozyme-specific antibodies were used to study the regulation of hydrolases A and B by Western immunoblotting and Ouchterlony immunodiffusion. Liver microsomes from mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and humans contained one or more proteins that were immunochemically related and similar in size (M(r) approximately 60 kDa) to hydrolase A and/or hydrolase B. These proteins were preferentially recognized by the antibody against hydrolase A, except for cat liver microsomal esterase, which was preferentially recognized by antibody against hydrolase B. In rats, the levels of hydrolases A and B in liver microsomes were coregulated as a function of age, sex, and xenobiotic treatment of rats. The levels of both enzymes were very low in 1- and 2-week-old rats, but increased abruptly at 3 weeks of age in both male and female rats. Treatment of mature male rats with 11 known microsomal enzyme inducers caused little (< 35%) or no induction of hydrolase A or B, whereas treatment of rats with beta-naphthoflavone, pregnenolone- 16 alpha-carbonitrile or dexamethasone suppressed the levels of both enzymes. The kinetic analysis of para-nitrophenylacetate hydrolysis described in the preceding paper identified a high-affinity esterase (Km 20-35 microM) in rat liver, testis, lung, prostate, and pancreas and identified a low-affinity enzyme (Km 300-800 microM) in liver, kidney, small intestine, lung, brain, spleen, and heart. Immunoblot analysis established that hydrolase A was present in liver, testis, lung, and prostrate at concentrations that accounted for the high-affinity esterase activity in these tissues. Hydrolase A was not detected in the pancreas, even though this tissue contained low levels of a high-affinity esterase. Hydrolase B was detected in liver and kidney at concentrations that accounted for the low-affinity esterase activity in these tissues. Hydrolase B was not detected in the other tissues examined, some of which (e.g., small intestine) contained high levels of a low-affinity esterase. These results indicate that hydrolases A and B are independently expressed in a wide variety of extrahepatic tissues in rats.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City 66160-7417.
authors.forename: E W
authors.initials: EW
authors.lastname: Morgan
authors.forename: B
authors.initials: B
authors.lastname: Yan
authors.forename: D
authors.initials: D
authors.lastname: Greenway
authors.forename: A
authors.initials: A
authors.lastname: Parkinson
mesh_terms: D000367:Age Factors; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002415:Cats; D006224:Cricetinae; D003580:Cytochromes / Q000378:metabolism; D004285:Dogs; D005260:Female; D006168:Guinea Pigs; D000882:Haplorhini; D006801:Humans; D005779:Immunodiffusion; D007158:Immunologic Techniques; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D011233:Precipitin Tests; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012737:Sex Factors; D013379:Substrate Specificity; D014018:Tissue Distribution; D014105:Tosyl Compounds / Q000494:pharmacology; D015262:Xenobiotics / Q000494:pharmacology*
chemical_list: D003580:Cytochromes; D014105:Tosyl Compounds; D015262:Xenobiotics; C003614:4-toluenesulfonyl fluoride; D002265:Carboxylic Ester Hydrolases