REGULATION OF 2 RAT-LIVER MICROSOMAL CARBOXYLESTERASE ISOZYMES - SPECIES-DIFFERENCES, TISSUE DISTRIBUTION, AND THE EFFECTS OF AGE, SEX, ANDXENOBIOTIC TREATMENT OF RATS

The preceding paper described the purification of two rat liver micros omal carboxylesterases, designated hydrolases A and B, that have high affinity (K-m similar to 25 mu M) and low affinity (K-m similar to 400 mu M) for para-nitrophenylacetate, respectively. The present study de scribes the preparation and purification of polyclonal antibodies agai nst these purified enzymes. Each antibody was subjected to immunoabsor ption chromatography to remove antibodies against epitopes common to b oth hydrolases A and B. The resulting isozyme-specific antibodies were used to study the regulation of hydrolases A and B by Western immunob lotting and Ouchterlony immunodiffusion. Liver microsomes from mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and humans c ontained one or more proteins that were immunochemically related and s imilar in size (M(r) similar to 60 kDa) to hydrolase A and/or hydrolas e B. These proteins were preferentially recognized by the antibody aga inst hydrolase A, except for cat liver microsomal esterase, which was preferentially recognized by antibody against hydrolase B. In rats, th e 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 litt le (<35%) or no induction of hydrolase A or B, whereas treatment of ra ts with beta-naphthoflavone, pregnenolone-16 alpha-carbonitrile or dex amethasone suppressed the levels of both enzymes. The kinetic analysis of para-nitrophenylacetate hydrolysis described in the preceding pape r identified a high-affinity esterase (Km 20-35 mu M) in rat liver, te stis, lung, prostate, and pancreas and identified a low-affinity enzym e (K-m 300-800 mu M) in liver, kidney, small intestine, lung, brain, s pleen, and heart. Immunoblot analysis established that hydrolase a was present in liver, testis, lung and prostrate at concentrations that a ccounted for the high-affinity esterase activity in these tissues. Hyd rolase A was not detected in the pancreas, even though this tissue con tained low levels of a high-affinity esterase. Hydrolase B was detecte d in liver and kidney at concentrations that accounted for the low-aff inity 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 indica te that hydrolases A and B are independently expressed in a wide varie ty of extrahepatic tissues in rats. Based on enzyme kinetics, hydrolas es A and B were estimated to comprise similar to 1.5 and similar to 0. 5% of the liver microsomal protein from mature male rats. This estimat e was confirmed by immunochemical analysis. The extent to which hydrol ases A and B contribute to para-nitrophenylacetate hydrolysis by rat l iver microsomes was assessed by immunoprecipitating each enzyme from d etergent-solubilized microsomes. The results indicated that hydrolases A and B account for 50-65% and 35-50% of the microsomal esterase acti vity toward para-nitrophenylacetate, respectively. these results suppo rt our hypotheses that (1) high levels of hydrolase A are expressed in rat liver and testis (with low levels in lung and prostate); (2) high levels of hydrolase B are expressed in rat liver and kidney; and (3) hydrolases A and B are the only esterases in rat liver microsomes that contribute significantly to the hydrolysis of para-nitrophenylacetate . (C) 1994 Academic Press, Inc.