Purification and characterization of mouse kidney beta-glucuronidase.

Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.     

Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography.

Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants.     

Purification and characterization of rat liver microsomal beta-glucuronidase.

Beta-Glucuronidase (EC 3.2.1.31) has been isolated from rat-liver microsomes by a novel chromatographic method employing antibody to rat preputial gland beta-glucuronidase coupled to Sepharose. The purified enzyme, homogeneous by several methods, was purified some 1700-fold. The microsomal beta-glucuronidase has been characterized with respect to catalysis, stability, and molecular weight. The purified enzyme is a tetramer of 290 000 daltons. Comparative studies with lysosomal beta-glucuronidase indicate that while these two enzymes are electrophoretically distinct, they are catalytically and immunologically identical and have indistinguishable molecular dimensions. The results suggest that microsomal and lysosomal beta-glucuronidase are charge isomers.     

Purification and chemical characterization of papain-solubilized histocompatibility-2 antigens from mouse liver.

A large scale purification of histocompatibility-2 (H-2) antigens from mouse liver is described. The antigens were solubilized by a limited papain digestion of a crude preparation of liver membranes (strain A/J) and purified by ion exchange chromatography, gel filtration, affinity chromatography, and isoelectric focusing. The overall degree of purification of H-2Kk was 1,300-fold and that of H-2Dd was 1,500-fold; approximately 8 mg of purified H-2a antigens were obtained from 1 kg of liver. The purification was followed by a sensitive radioimmunoassay in which H-2a-containing fractions were used to inhibit the binding of 125I-labeled H-2a to appropriate antisera. H-2Dd and H-2Kk co-purified through all the steps but the concentration of H-2Kk was 2- to 3-fold higher than that of H-2Dd in the liver homogenate as well as in the purified H-2 preparation. beta 2-microglobulin was initially present in a 3- to 10-fold excess over H-2 in the liver homogenate, but the purified H-2 preparation contained approximately 2 mol of alloantigenic heavy chain/mol of beta 2-microglobulin. Isoelectric focusing and disc-gel electrophoresis showed a charge heterogeneity of H-2, with a mean isoelectric point of pH 4.9. Electrophoresis on sodium dodecyl sulfate gels showed one band. Denaturing conditions were required to remove beta 2-microglobulin and small amounts of impurities from H-2. The amino acid sequence of the first 27 residues of the isolated heavy chains was determined.     

Purification and immunological quantification of rat liver lysosomal glycosidases.

Although lysosomal enzyme activities are known to vary in response to numerous physiological and pharmacological stimuli, the relationship between lysosomal enzyme activity and enzyme concentration has not been systematically studied. Therefore we developed radioimmunoassays for two lysosomal glycosidases in order to determine lysosomal enzyme concentration. beta-Galactosidase and beta-glucuronidase were purified from rat liver 2780-fold and 1280-fold respectively, by using differential centrifugation, affinity chromatography, ion-exchange chromatography and molecular-sieve chromatography. Polyclonal antibodies to these enzymes were raised in rabbits, and two radioimmunoassays were established. Antibody specificity was shown by: (i) selective immunoprecipitation of enzyme activity; (ii) identical bands of purified enzyme on SDS/polyacrylamide-gel electrophoresis and immunoelectrophoresis; (iii) single immunoreactive peaks in molecular-sieve chromatography experiments. Sensitivities of the assays were such that 15 ng of beta-galactosidase and 45 ng of beta-glucuronidase decreased the ratio of bound to free radiolabel by 50%; minimal detectable amounts of immunoreactive enzymes were 2 ng and 10 ng respectively. The assays were initially used to assess the effects of physiological perturbations (i.e. fasting and age) on enzyme concentrations in rat liver; these experiments showed that changes in enzyme concentrations do not always correlate with changes in enzyme activities. This represents the first report of radioimmunoassays for lysosomal glycosidases. The results suggest that these radioimmunoassays provide useful technology for the study of regulatory control mechanisms of the concentrations of lysosomal glycosidases in mammalian tissues.     

Egasyn affects the processing of beta-glucuronidase in mouse liver.

Three differently modified forms of beta-glucuronidase are known to exist: a microsomal enzyme form (M) existing in tissues where egasyn, a second microsomal protein, is present; and an acidic (La; complex-type oligosaccharide) and a basic (Lb; non-complex type oligosaccharide) lysosomal form which occur in all mouse tissues. Lb predominates in tissues containing microsomal beta-glucuronidase, La in those lacking it. In pulse-labelling experiments using mouse strain C57BL/6 liver containing egasyn (Eg+/Eg+) and microsomal enzyme, about half of the newly synthesized beta-glucuronidase was processed to the microsomal enzyme form, which was evidently further processed to Lb, and about half directly to La. In contrast, in liver of the congenic line C57BL/6.YBR Es-1b Eg0 that lacks egasyn (Eg0/Eg0) and microsomal enzyme, most of the labelled beta-glucuronidase was processed to La, and only a minor portion to Lb. Newly synthesized enzyme appeared first in microsomal, then in light and heavy lysosomal fractions of Eg+/Eg+ liver. In Eg0/Eg0 liver, no labelled enzyme was measurable in the microsomes, but it appeared rapidly in both types of lysosomes. Taken together these findings indicate that the microsomal enzyme form serves as a precursor of Lb, and that La is synthesized independently. The apparent half-life of La is only two-thirds that of Lb; this fact accounts for the reduced beta-glucuronidase activity in Eg0/Eg0 liver, which contains La as the predominant form.     

The egasyn gene affects the processing of oligosaccharides of lysosomal beta-glucuronidase in liver.

The accumulation of the relatively large amounts of beta-glucuronidase in microsomal fractions of normal mice depends on formation of complexes with the protein egasyn. Unexpectedly, it was found that the egasyn gene also affects the processing of beta-glucuronidase, which is segregated to lysosomes. In egasyn-positive mice lysosomal beta-glucuronidase from liver has a mean pI of 5.9 with a minor proportion at pI 5.4, whereas in egasyn-negative mice the proportion of the two lysosomal forms is reversed. Combined experiments measuring susceptibility to neuraminidase and to endoglycosidase H and specific binding to Ricinus communis lectin-agarose columns showed that the alterations in isoelectric point were associated with a decrease in complex oligosaccharides of lysosomal beta-glucuronidase in egasyn-positive mice. Since this alteration occurs not only in a congenic strain carrying the Eg0 gene but also in several other inbred strains that are homozygous for this gene, it is considered to be a genuine effect of the Eg gene rather than other genes that might regulate oligosaccharide processing. Also, the alteration is likely to be a result of direct physical interaction of the egasyn protein and lysosomal beta-glucuronidase, since a second lysosomal enzyme, beta-galactosidase, which does not form complexes with egasyn, is unaffected. The results suggest a model in which egasyn not only causes accumulation of beta-glucuronidase in the microsomal compartment but also acts upon the precursor to lysosomal beta-glucuronidase to alter its interaction with trans-Golgi-apparatus processing enzymes.     

Elevated serum beta-glucuronidase reflects hepatic lysosomal fragility following toxic liver injury in rats.

The level of serum beta-glucuronidase increases in various pathological conditions, including liver disorders. The aim of this investigation was to study the changes in liver lysosomal membrane stability during experimentally induced hepatic fibrosis that may result in the elevation of serum beta-glucuronidase. Liver injury was induced by intraperitoneal injections of N-nitrosodimethylamine (NDMA) in adult male albino rats over 3 weeks. The progression of fibrosis was evaluated histopathologically as well as by monitoring liver collagen content. Lipid peroxides and beta-glucuronidase levels were measured in the liver homogenate and subcellular fractions on days 0, 7, 14, and 21 after the start of NDMA administration. Serum beta-glucuronidase levels were also determined. A significant increase was observed in beta-glucuronidase levels in the serum, liver homogenate, and subcellular fractions, but not in the nuclear fraction on days 7, 14, and 21 after the start of NDMA administration. Lipid peroxides also increased in the liver homogenate and the lysosomal fraction. The measurement of lysosomal membrane stability revealed a maximum lysosomal fragility on day 21 during NDMA-induced fibrosis. In vitro studies showed that NDMA has no significant effect on liver lysosomal membrane permeability. The results of this investigation demonstrated that lysosomal fragility increases during NDMA-induced hepatic fibrosis, which could be attributed to increased lipid peroxidation of lysosomal membrane. In this study, we also elucidated the mechanism of increased beta-glucuronidase and other lysosomal glycohydrolases in the serum during hepatic fibrosis.     

Purification of a form of mouse liver UDP glucuronosyltransferase which glucuronidates androgens

A form of UDP glucuronosyltransferase active in the glucuronidation of the androgens, testosterone, androsterone and dihydrotestosterone has been purified to apparent homogeneity as judged by sodium dodecylsulfate polyacrylamide gel electrophoresis from the livers of phenobarbital-treated C57BL/6N mice. This UDP glucuronosyltransferase is inactive towards estrone as substrate. Data from chromatofocusing and purification experiments suggest that testosterone and androsterone are glucuronidated primarily by this enzyme form and to a lesser extent by an enzyme form which has a slightly higher isoelectric point. However, this major form is only responsible for about half the capacity to glucuronidate dihydrotestosterone.     

Some molecular properties of rat-liver lysosomal beta-glucuronidase isoenzymes.

The isoenzymes of rat-liver lysosomal beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase (EC 3.2.1.31)) were inactivated at different rates at 0 degrees C in 3M guanidinium chloride solutions adjusted to pH 5.0 In 4 M urea buffered by 0.01 M glycylglycine, pH 7.0 isoenzymes I, III, and V were reversibly inhibited 80%. Sodium dodecyl sulfate (SDS), 0.1% in 0.01 M phosphate buffer, pH 7.0 irreversibly inhibited at 37 degrees C all five isoenzymes. Sedimentation analysis showed that loss of catalytic activity in these denaturing media is accompanied by dissociation into slower sedimenting subunits. SDS gel electrophoresis revealed that the isoenzymes are apparently tetramers made up of different proportions of subunits alpha, beta, and gamma having apparent molecular weights of 62,900, 60,200, and 58,700, respectively. The three subunits appear to be glycoproteins.     

Purification and immunochemical characterization of a low-pI form of UDP glucuronosyltransferase from mouse liver

A liver UDP glucuronosyltransferase (GT) enzyme from either phenobarbital- or 3-methylcholanthrene-treated C57BL/6N mice was isolated by phenyl-Sepharose, DEAE-ion exchange, and UDP hexanolamine chromatographic steps. This enzyme had a broad substrate specificity and was mainly responsible for the microsomal capacity to glucuronidate testosterone, 1-naphthol, and morphine. This UDP glucuronosyltransferase ( GTM1 ) appeared to be at least 95% homogeneous and had a subunit molecular weight of 51,000 using sodium dodecyl sulfate-polyacrylamide gel and two-dimensional gel electrophoreses. Antibodies prepared against the purified protein developed a single immunoprecipitin line by double-diffusion analysis with purified antigen and with solubilized microsomes from both control and drug-induced C57BL/6N and DBA/2N mice. A precipitin line was also observed with microsomal proteins which isoelectrofocused at approximately pH 6.7, but not with those which isoelectrofocused at approximately pH 8.5. GTM1 was, therefore, designated at low-pI form. Immunopurified antibody preferentially inhibited and immunoprecipitated GT activities toward testosterone, 1-naphthol, and morphine. To a lesser extent, activities toward phenolphthalein, 3-hydroxybenzo[a]pyrene, and estrone were inhibited while activities toward 4-nitrophenol and 4-methylumbelliferone were not affected. All activities, however, were immunoadsorbed in the presence of protein A-Sepharose. This observation can be explained by the following results. Immunoprecipitates from labeled microsomes contained primarily a 51,000-Da protein. When the immune complexes were adsorbed with protein A-Sepharose, a 54,000-Da protein as well as the expected 51,000-Da GTM1 was detected. This 54,000-Da protein was associated with the glucuronidation of 3-hydroxybenzo[a]pyrene and 4-nitrophenol, and was designated GTM2 .     

Purification and characterization of dipeptidyl peptidase IV in rat liver lysosomal membranes.

Dipeptidyl peptidase IV (m-DPP IV) in rat liver lysosomal membranes was purified about 50-fold over the lysosomal membranes with 38% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The enzyme amounts to about 3% of lysosomal membrane protein constituents. The purification procedures included: extraction of lysosomal membranes by Triton X-100, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme (M(r) 240,000) is composed of two identical subunits with an apparent molecular weight of 110,000. The enzyme contains about 12.4% carbohydrate and the carbohydrate moiety was composed of mannose, galactose, fucose, N-acetylglucosamine, and neuraminic acid in a molar ratio of 14:17:2:24:11. Susceptibility to neuraminidase and immunoreactivity of the enzyme in intact tritosomes were examined to study the topology of the enzyme in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the enzyme were not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock. This result indicated that both the oligosaccharide chains and the main protein portion of the enzyme are on the inside surface of the tritosomal membranes. Subcellular localization of DPP IV was determined by means of enzyme immunoassay, which indicated that bile canalicular membranes and lysosomal membranes are the major sites of localization, and DPP IV activity in lysosomes was separated into a membrane bound form (60%) and a soluble form (40%). Immunoelectron microscopy clearly confirmed that DPP IV occurs not only in the bile canalicular domain but also in the lysosomes of rat liver.     

Clearance of lysosomal hydrolases following intravenous infusion. The role of liver in the clearance of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase.

Certain highly purified forms of rat lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, are rapidly cleared from the circulation following intravenous infusion. Several lines of evidence are presented which indicate that the primary site of enzyme uptake is the liver. Clearance of the two enzymes was unaffected by nephrectomy, whereas it was abolished by evisceration. Tissue distribution experiments with native and [¹²⁵I]β-glucuronidase indicate the liver as the major, if not exclusive, site of enzyme uptake. Experiments with the isolated perfused liver showed clearance of certain enzyme preparations but not others. Those enzymes cleared by the isolated perfused liver were likewise cleared in vivo. Liver fractionation studies following infusion of large doses of β-glucuronidase revealed a rapid, short-lived increase in microsomal β-glucuronidase and a slower but larger increase in lysosomal β-glucuronidase. The results indicate that β-glucuronidase, N-acetyl-β-d-glucosaminidase, and probably other glycosidases are rapidly incorporated into the lysosomal compartment of liver.     

Effect of swainsonine on the processing and turnover of lysosomal beta-galactosidase and beta-glucuronidase from mouse peritoneal macrophages

The effect of swainsonine, an inhibitor of Golgi alpha-mannosidase II and lysosomal alpha-mannosidase, on the synthesis, processing, and turnover of two glycoproteins, lysosomal beta-galactosidase and lysosomal beta-glucuronidase, has been studied in cultured mouse peritoneal macrophages. No effect of the inhibitor on the relative rates of synthesis of the precursor form of either enzyme was observed. On the other hand, carbohydrate processing of beta-galactosidase and beta-glucuronidase was markedly altered by swainsonine, consistent with a blockage by the inhibitor of the removal of the alpha-1,3- and alpha-1,6-linked mannose residues which occurs in normal processing. In homogenates of both normal and swainsonine-treated cells, the precursor forms of the enzymes were found exclusively in the light membrane fraction on Percoll gradients and the mature forms exclusively in the lysosomal fractions indicating that translocation from Golgi to lysosomes and proteolytic processing in the lysosome were not impaired by the presence of abnormal oligosaccharide side chains. There was no detectable effect of swainsonine during a 4-day chase period on the total cellular turnover of these enzymes which involves two processes, secretion and degradation. In the absence of swainsonine, secretion represented about 40% of the total turnover of beta-galactosidase and about 50% with beta-glucuronidase. The presence of swainsonine increased these proportions to about 60 and 70%, respectively.