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 ( K m ∼25 μM) and low affinity ( K m ∼400 μM) 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 ∼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 β-naphthoflavone, pregnenolone-16α-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 ( K m 20-35 μM) in rat liver, testis, lung, prostate, and pancreas and identified a low-affinity enzyme ( K m 300-800 μM) 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. Based on enzyme kinetics, hydrolases A and B were estimated to comprise ∼1.5 and ∼0.5% of the liver microsomal protein from mature male rats. This estimate was confirmed by immunochemical analysis. The extent to which hydrolases A and B contribute to para-nitrophenylacetate hydrolysis by rat liver microsomes was assessed by immunoprecipitating each enzyme from detergent-solubilized microsomes. The results indicated that hydrolases A and B account for 50-65% and 35-50% of the microsomal esterase activity toward para-nitrophenylacetate, respectively. these results support 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.