Distribution of UDPglucuronosyltransferase in rat tissue ( tissue distribution / intracellular distribution / immunocytochemistry )

UDPglucuronosyltransferase [UDPglucuronate 13-D-glucuronosyltransferase (acceptor-unspecific), EC 2.4.1.17] is a group of enzymes with distinct but partially overlapping substrate specificity. A rabbit antiserum raised against one purified rat liver UDPglycuronosyltransferase isoform was specific for UDPglucuronosyltransferase and recognized all transferase isoforms by immunodiffusion or immunotransblot analysis. The transferase activity toward all substrates was immunoabsorbed from solubilized rat liver microsomes by IgG purified from the antiserum. The purified IgG was used for immunocytochemical localization of UDPglucuronosyltransferase in rat liver, jejunum, kidney, and adrenal gland. In the liver, UDPglucuronosyltransferase was present exclusively in hepatocytes and was uniformly distributed within all zones of the hepatic lobule. In the jejunum, the transferase was present exclusively in the epithelial cells and showed a progressive increase in concentration from the crypt to the villar tip. In the kidney, the greatest concentration of the transferase was observed in the epithelial cells of the proximal convoluted tubule. Adrenal medullary cells showed intense immunocytochemical staining; the zona glomerulosa and the zona reticularis of the adrenal cortex were more intensely stained than the zona fasciculata. By light microscopy, UDPglucuronosyltransferase was found in the endoplasmic reticulum and nuclear envelope of all the four organs; this was confirmed in the hepatocyte by electron microscopy. The transferase was not observed in mitochondria, Golgi apparatus, lysosomes, peroxisomes, and plasma membrane, even after 3to 4-fold induction of various substrate-specific UDPglucuronosyltransferase activities. UDPglucuronosyltransferase (UDPglucuronate ,B-Dglucuronosyltransferase, EC 2.4.1.17) is a group of membrane-bound enzymes that catalyze the transfer of glucuronic acid from uridine diphosphoglucuronic acid to a variety of metabolites, drugs, toxins, carcinogens, and other xenobiotics. The resulting glucuronides are generally less biologically active and more polar than the aglycone substrates, and they are readily excreted in bile and urine. Glucuronidation is an important pathway of detoxication (1); tissue and intracellular location of UDPglucuronosyltransferase may be important in the chemical defense against these toxins. We have previously isolated six UDPglucuronosyltransferase isoforms with distinct but partially overlapping substrate specificity from solubilized rat liver microsomes (2). A rabbit antiserum raised against one purified isoform was specific for UDPglucuronosyltransferase and recognized all isoforms of the enzyme. Using purified IgG from this antiserum, we have determined the tissue and intracellular location of UDPglucuronosyltransferase in rat liver, kidney, jejunal mucosa, and adrenal gland; this was correlated with the tissue distribution of the enzyme activity toward various substrates. MATERIALS AND METHODS Male Wistar rats (200-250 g) were purchased from Charles River Breeding Laboratories. Clofibrate was purchased from Ayerst Laboratories (New York); 3-methylcholanthrene was a gift of W. G. Levine. Horseradish peroxidase-conjugated staphylococcal protein A (pA-HRP) was purchased from E-Y Laboratories (San Mateo, CA). Assay of UDPglucuronosyltransferase Activity. Enzyme activity in tissue homogenates toward bilirubin (3), 4nitrophenol (4, 5), testosterone (6), androsterone (6), f3estradiol (6), and estrone (7) was assayed in the presence of 4mM glucaro-1,4-lactone (7) after activation with digitonin (2 mg/ml). For purified preparations, phosphatidylcholine liposomes (0.1 mg/ml) were added (5, 6). Purification of UDPglucuronosyltransferase Isoforms. Six UDPglucuronosyltransferase isoforms were purified from Emulgen 911-solubilized rat liver microsomes (5) by a combination of chromatofocusing (pH 9.4-6.0), affinity chromatography, and gel-permeation high-pressure liquid chromatography as described (2, 6). Preparation and Characterization of Antisera. An antiserum was raised in rabbits against purified UDPglucuronosyltransferase isoform III as described (5). IgG purified from the antiserum by affinity chromatography (8) was tested by double immunodiffusion (9) against solubilized rat liver microsomes or pure transferase isoforms (2) and by immunotransblot analysis against liver homogenates (50 ,ug of protein), microsomes (25 ,ug of protein), or purified transferase isoforms (1-2 ,g of protein) after electrophoresis on 10% NaDodSO4/polyacrylamide slabs (10). To evaluate the recognition of the functional forms of UDPglucuronosyltransferase by the antiserum, solubilized rat liver microsomal proteins (5 mg in 0.5 ml) were gently stirred with the specific IgG or IgG from nonimmunized rabbit serum (2 mg in 0.5 ml of 0.1 M Tris HCl, pH 7.4) or saline at 4°C for 16 hr. The immune complexes were removed after gentle mixing (8 hr at 4°C) with 0.5 ml of staphylococcal protein A-Sepharose slurry (binding capacity, 10 mg of rabbit IgG), followed by centrifugation (10,000 x g for 1 min). The protein ASepharose beads were washed twice with 0.5 ml of 0.1 M Tris-HCl containing 50 mM KCl; the supernatants were pooled and UDPglucuronosyltransferase activity for 4nitrophenol, 1-naphthol, 4-methylumbelliferone, testosterone, 13-estradiol, estrone, androsterone, and bilirubin was assayed. Enzyme Induction. Rats were injected with clofibrate (300 mg/kg, s.c.) for 7 days, or with 3-methylcholanthrene (40 mg per kg of body weight in olive oil, i.p.) once 4 days prior to sacrifice; UDPglucuronosyltransferase activity for 4-nitrophenol, testosterone, ,-estradiol, androsterone, estrone, and bilirubin in liver homogenates was assayed. Immunocytochemical and Microscopic Procedures. Tissues were fixed and processed as described (11). Nonfrozen sections were exposed to the anti-rat UDPglucuronosyltransferase (rabbit) IgG (0.07-0.7 mg per ml of phosphatebuffered saline); nonimmune rabbit IgG was used as control. Vibratome sections were prepared and examined by light and 2990 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 82 (1985) 2991