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Preparation and Properties of β-Glucuronidase
Abstract
This chapter discusses the preparation and properties of β–glucuronidase. All mammalian tissues contain a group-specific enzyme that catalyzes the hydrolysis of the biosynthetic β-D-glucopyranosiduronic acids of all types to the aglycons and D-glucuronic acid. This enzyme is commonly known as “β-glucuronidase,” a term that will be employed in the following pages. In the same way, for the sake of brevity, β-D-glucopyranosiduronic acids will often be referred to as β-glucuronides. Enzymes that decompose β-glucuronides have been obtained from diverse sources other than the mammalian cell. β-Glucuronidase finds its major application at the present time as a reagent for the hydrolysis of urinary steroid D-glucuronides, for which purpose it has certain advantages over mineral acids, and the rapid changes in tissue β-glucuronidase activity induced in vivo by certain steroid and pituitary hormones offer possibilities for the rapid measurement of the hormones. The enzyme might perhaps be employed with advantage as a hydrolytic agent in carbohydrate chemistry. Most of the assay methods depend upon colorimetric determination of the amount of aglycon liberated from a specified concentration of substrate at a fixed pH, the results being read from a standard curve. Unit β-glucuronidase activity is then almost universally expressed as that which liberates 1 μg. of aglycon in 1 hr. at 38°; a similar type of unit is employed when the liberation of D-glucuronic acid is followed.
... However, one of the problems associated with the use of the bacterial gus gene as a reporter is the presence of 'endogenous GUS' activity in some species. Endogenous GUS activity has been reported in various organisms including: 1bacterial species, such as the enterobacterium E. coli and Shigella, nonenterobacterial such as Bacteroides and Clostridium (Hawkesworth et al., 1971), Streptococcus, Staphylococcus, Corynebacterium (Levvy and Marsh, 1959), Alcaligenes (Jefferson, 1989) and many soil bacteria (Ritz et al., 1994); 2 -Some tissues of vertebrates such as kidney, liver and spleen (Kyle et al., 1992), human breast milk (Alonso et al., 1991;Ince et al., 1995); 3 -Invertebrates such as snails (Levvy and Marsh, 1959), nematodes (Jefferson, 1985;Sebastiano et al., 1986) and insects (Langley et al., 1983;Levvy and Marsh, 1959), housefly (Musca domestica) and various locusts (Levvy and Marsh, 1959) and 4 -Plant species. In plants, endogenous GUS activity was first described in Arabidopsis by Jefferson et al. (1987). ...
... However, one of the problems associated with the use of the bacterial gus gene as a reporter is the presence of 'endogenous GUS' activity in some species. Endogenous GUS activity has been reported in various organisms including: 1bacterial species, such as the enterobacterium E. coli and Shigella, nonenterobacterial such as Bacteroides and Clostridium (Hawkesworth et al., 1971), Streptococcus, Staphylococcus, Corynebacterium (Levvy and Marsh, 1959), Alcaligenes (Jefferson, 1989) and many soil bacteria (Ritz et al., 1994); 2 -Some tissues of vertebrates such as kidney, liver and spleen (Kyle et al., 1992), human breast milk (Alonso et al., 1991;Ince et al., 1995); 3 -Invertebrates such as snails (Levvy and Marsh, 1959), nematodes (Jefferson, 1985;Sebastiano et al., 1986) and insects (Langley et al., 1983;Levvy and Marsh, 1959), housefly (Musca domestica) and various locusts (Levvy and Marsh, 1959) and 4 -Plant species. In plants, endogenous GUS activity was first described in Arabidopsis by Jefferson et al. (1987). ...
... However, one of the problems associated with the use of the bacterial gus gene as a reporter is the presence of 'endogenous GUS' activity in some species. Endogenous GUS activity has been reported in various organisms including: 1bacterial species, such as the enterobacterium E. coli and Shigella, nonenterobacterial such as Bacteroides and Clostridium (Hawkesworth et al., 1971), Streptococcus, Staphylococcus, Corynebacterium (Levvy and Marsh, 1959), Alcaligenes (Jefferson, 1989) and many soil bacteria (Ritz et al., 1994); 2 -Some tissues of vertebrates such as kidney, liver and spleen (Kyle et al., 1992), human breast milk (Alonso et al., 1991;Ince et al., 1995); 3 -Invertebrates such as snails (Levvy and Marsh, 1959), nematodes (Jefferson, 1985;Sebastiano et al., 1986) and insects (Langley et al., 1983;Levvy and Marsh, 1959), housefly (Musca domestica) and various locusts (Levvy and Marsh, 1959) and 4 -Plant species. In plants, endogenous GUS activity was first described in Arabidopsis by Jefferson et al. (1987). ...
The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed (Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24°C, 38°C and 55°C) were investigated. To avoid this problem in microspores, two incubation temperatures (38°C and 55°C) at different periods after GUS assay (4, 24 and 48h) and in the presence of 1mM potassium ferricyanide and 1mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1mM potassium ferricyanide and 1mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55°C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2μM gibberellic acid.
... GUS enzymes have been found in animals, microbes and plants, and their characterization and role is subject of intensive studies covering a wide range of disciplines from experimental biology to human healthcare. GUS activity has been reported in prokaryotes [1,2], as well as in invertebrates such as insects, nematodes, gastropods, and in virtually all tissues of vertebrates [2,3]. In mammals, glucuronidation is a major detoxification process by which metabolites are efficiency excreted from the body, unless hydrolyzed by intestinal GUS enzymes: by uncoupling glucuronides in the intestine, GUS deconjugates potential toxins, increasing the formation of carcinogens and promoting enterohepatic recirculation of toxic compounds [4,5]. ...
... GUS enzymes have been found in animals, microbes and plants, and their characterization and role is subject of intensive studies covering a wide range of disciplines from experimental biology to human healthcare. GUS activity has been reported in prokaryotes [1,2], as well as in invertebrates such as insects, nematodes, gastropods, and in virtually all tissues of vertebrates [2,3]. In mammals, glucuronidation is a major detoxification process by which metabolites are efficiency excreted from the body, unless hydrolyzed by intestinal GUS enzymes: by uncoupling glucuronides in the intestine, GUS deconjugates potential toxins, increasing the formation of carcinogens and promoting enterohepatic recirculation of toxic compounds [4,5]. ...
β-Glucuronidases (GUS) are histochemically and fluorometrically detectable enzymes that cleave the glycosidic bond of glucuronides in living organisms. Previously thought to be absent in plants, endogenous GUS activity has recently been demonstrated ubiquitous, and its function is being widely investigated in model plants. Further, the GUS gene from Escherichia coli is the most widely used reporter gene in plant transformation experiments.Fluorometric assay of GUS activity is universally performed using fluorogenic substrate 4-methyl-umbelliferyl-β-d-glucuronide (MUG) by discontinuous measurement, based on the general notion that basification of the solution following enzymatic cleavage is necessary to detect the fluorophore 4-methyl-umbelliferone (MU). In this report, we analyze MU and MUG spectroscopic characteristics at different pHs, and show that MU is a fluorescent compound also at pHs below its pKa, although with a different excitation spectrum compared to the ionized form. On the basis of this evidence, we show that MUG is a suitable substrate to perform continuous measurement of GUS activity at the pH optima of plant endogenous and E. coli GUS enzymes. An efficient and straightforward method which greatly improves the procedure to assess GUS activity is described, and insights are given on the principles leading to optimal setting of instrumental parameters.
... Emodinol, a new triterpene identified in P. emodi (7), inhibits the β-glucuronidase activity. The overexpression of β-glucuronidase may cause liver cancer and damage the liver (27). The Paeonia root contains secondary metabolites with 17 monoterpenoid glucosides, 11 galloyl glucose, 5 flavonoids, 6 phenolic compounds (Figure 3) (28). ...
Since ancient times, people have used medicinal plants as a source of medications to treatand prevent diseases. Paeonia species are important therapeutic plants in Ayurvedic, Unani,and Traditional Chinese Medicine. This study aims to provide updated information on theethnobotany, phytochemistry, and pharmacological activities of Paeonia species discovereduntil now. Using the keywords “Paeonia”, “geographical distribution”, “ethnopharmacologyand traditional values”, “phytochemistry”, “antioxidant”, “anti-inflammatory”, “antimicrobial”,“cardiovascular diseases”, and “anticancerous properties”, the published reports from 2001 to2022 were retrieved using Google Scholar, Science Direct, PubMed, and Scopus databases. Atotal of 156 published articles were studied after meeting the qualifying criteria. Out of these,52 articles were studied for phytochemistry, ethnopharmacological and traditional uses.Paeonia emodi is used to treat hypertension, asthma, convulsions, epilepsy, bronchitis, ascites,uterine abnormalities, and a variety of skin ailments. Bioactive compounds like triterpenes,monoterpene glucosides, phenols, tannins, emodinol, benzoic acid, paeonin A and B, steroids,several secondary metabolites like paeoniflorin and paeonol, and several minerals areabundant in the Paeonia species. In recent studies, Paeonia emodi has been shown to possesspharmacological properties like antioxidant, antibacterial, anti-inflammatory, insecticidal,and anti-tumor activities. Convincing data supports the traditional ethnomedicinal claimsof the plant; the abundant phytocompounds of the plant are attributed to its broad spectrumof pharmacological activities. In order to understand the molecular mechanisms underlyingthe action of the bioactive ingredients in drug development processes and to investigate theirpotential at the clinical level, more research is required.
... Emodinol, a new triterpene identified in P. emodi (7), inhibits the β-glucuronidase activity. The overexpression of β-glucuronidase may cause liver cancer and damage the liver (27). The Paeonia root contains secondary metabolites with 17 monoterpenoid glucosides, 11 galloyl glucose, 5 flavonoids, 6 phenolic compounds (Figure 3) (28). ...
Since ancient times, people have used medicinal plants as a source of medications to treat and prevent diseases. Paeonia species are important therapeutic plants in Ayurvedic, Unani, and Traditional Chinese Medicine. This study aims to provide updated information on the ethnobotany, phytochemistry, and pharmacological activities of Paeonia species discovered until now. Using the keywords "Paeonia", "geographical distribution", "ethnopharmacology and traditional values", "phytochemistry", "antioxidant", "anti-inflammatory", "antimicrobial", "cardiovascular diseases", and "anticancerous properties", the published reports from 2001 to 2022 were retrieved using Google Scholar, Science Direct, PubMed, and Scopus databases. A total of 156 published articles were studied after meeting the qualifying criteria. Out of these, 52 articles were studied for phytochemistry, ethnopharmacological and traditional uses. Paeonia emodi is used to treat hypertension, asthma, convulsions, epilepsy, bronchitis, ascites, uterine abnormalities, and a variety of skin ailments. Bioactive compounds like triterpenes, monoterpene glucosides, phenols, tannins, emodinol, benzoic acid, paeonin A and B, steroids, several secondary metabolites like paeoniflorin and paeonol, and several minerals are abundant in the Paeonia species. In recent studies, Paeonia emodi has been shown to possess pharmacological properties like antioxidant, antibacterial, anti-inflammatory, insecticidal, and anti-tumor activities. Convincing data supports the traditional ethnomedicinal claims of the plant; the abundant phytocompounds of the plant are attributed to its broad spectrum of pharmacological activities. In order to understand the molecular mechanisms underlying the action of the bioactive ingredients in drug development processes and to investigate their potential at the clinical level, more research is required.
... Thus it seems unlikely that the probiotic effect we report here would be the consequence of the hydrolysis of endogenous β-glucuronides, as suggested by Gilissen et al. (1998), resulting in the release of -glucuronate that is an additional source of dietary carbohydrates. GUS activity was reported in Drosophila melanogaster (Langley et al., 1983) and in Musca domestica (Levvy & Marsh, 1959), but never investigated in aphids. Ingested GUS, however, could directly interfere with the aphid metabolism, enhancing the glycosaminoglycans degradation. ...
Transgenesis developed in the last 20 years offers new possibilities for crop protection. The transgenic process, however, requires the use of marker fusion genes to select and visualize the transformed tissues. Although the expression products of these marker genes are stably expressed in crops, little attention has been given to assess the eventual risks of these recombinant proteins on phytophage populations. Three independent transgenic potato (Solanum tuberosum) clones from the cultivar Désirée (DG5, DG18, and DG20) carrying the commonly used nptII-gus gene construct and exhibiting different β-glucuronidase activity (0.843 ± 0.011, 0.576 ± 0.096, and 0.002 ± 0.000 pmol min-1.mg-1, respectively) were evaluated to determine the impact of the encoded proteins on the behaviour, development, reproduction, and demography of the peach-potato aphid, Myzus persicae, under laboratory-controlled light and temperature. Our results revealed that the transgenic event can alter aphid physiology or behaviour. Experiments showed a probiotic effect of one transgenic line, the DG5, resulting in reduced prereproductive period and mortality, and enhanced daily fecundity, which was expressed in a greater population growth potential (rm = 0.205 vs. rm = 0.174 of the control). In contrast, aphids fed with the DG18 line exhibited reduced adult survival and reproductive period but no alteration of their demographic parameters (rm = 0.176). Finally, no physiological alteration was induced in aphids fed on a DG20 diet (rm = 0.170). Behavioural experiments conducted in a 4-choice olfactometer demonstrated that insects were significantly more attracted by the odour of transgenic DG18 potato plant than that of Désirée non-transformed plant, spending twice as much time in the DG18 plant odour. The two other transformed clones (DG5 and DG20) were as attractive as the non-transformed cultivar. It is concluded that the β-glucuronidase expression in potato plants might be responsible for the probiotic effect measured on the feeding aphids, whereas alteration of the foliage odour would result from a pleiotropic effect.
... Liver cancer is also suspected to be related to the over expression of this enzyme. Many β-glucuronidase inhibitor such as 8-hydroxytriceine glucuronide, scoparic acid A and C have already been isolated from different plants and some are used clinically [26][27][28]. ...
... Besides, heparanase in animals (vertebrates and invertebrates) are known to have an acidic pH optimum for its activity. The human heparanase has a pH optimum between 3.5 and 5.0 (Levvy and Marsh, 1959; Dutton, 1980). In Caenorhabditis elegans, the GUS has its optimum activity at pH 5.0 (Sebastiano et al., 1986) and between pH 3.0 and 5.5 in Drosophila (Langley et al., 1983 ). ...
We have chosen rice as a model crop to ascertain endogenous β-glucuronidase (family 79) activity and to differentiate the same from Escherichia coli based β-glucuronidase (family-2). The investigation dwells on characterizing endogenous β-glucuronidase (GUS) activity during the early stages of seed germination and from rice callus. Also, similar studies were made from homozygous transgenic rice line expressing E. coli GUS under the control of glyoxalase I promoter. Endogenous GUS activity was detected in plumules, shoots and calli of rice, nevertheless, showing differential response to pH. Further, endogenous GUS in rice was specifically inhibited by saccharic acid 1,4-lactone (SL) but, the E.coli β-glucuronidase remain unaffected, indicating distinct biochemical properties of family-2 and family-79 β-glucuronidase.
... The pH of the water-soluble layer was adjusted to 5.0 with 1 N acetic acid and each of the samples divided. One-half was treated with B-glucuronidase (10 000 units) while the other half was treated with aryl sulfatase (250 units) and 19.2 mg of D-saccharic acid-l,4-1actone to inhibit /3-glucuronidase activity in the aryl sulfatase (Levvy, 1952). After mixing for 2 min, the samples were incubated at 37°C with shaking for 24 h. ...
4 isomeric cyclopenta-derivatives of benz[e]anthracene (benz[a]aceanthrylene, benz[j]aceanthrylene, benz[l]aceanthrylene, and benz[k]acephenanthrylene) were examined for their ability to morphologically transform C3H10T1/2CL8 mouse-embryo fibroblasts. All of these polycyclic aromatic hydrocarbons studied except benz[k]acephenanthrylene transformed C3H10T1/2CL8 cells to both type II and type III foci in a concentration-dependent fashion. Benz[j]aceanthrylene was the most active, equivalent in activity to benzo[a]pyrene on a molar basis, in producing dishes of cells with transformed foci (94% at 1.0 μg/ml). Benz[e]aceanthrylene, and benz[l]aceanthrylene produced 58% and 85% of the dishes with foci respectively at 10 μg/ml. Metabolism studies with in C3H10T1/2CL8 cells in which unconjugated, glucuronic acid conjugated, and sulfate conjugated metabolites were measured indicated that the dihydrodiol precursor to the bay-region diol-epoxide, 9,10-dihydroxy-9,10-dihydrobenz[j]aceanthrylene, was the major dihydrodiol formed (55%). Smaller quantities of the cyclopenta-ring dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenz[j]aceanthrylene (14%), and the k-region dihydrodiol, 11,12-dihydroxy-11,12-dihydrobenz[j]aceanthrylene (5%) were also formed. Similar studies with indicated that the k-region dihydrodiol, 7,8-dihydroxy-7,8-dihydrobenz[l]aceanthrylene was the major metabolite formed (45%). The cyclopenta-ring dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenz[l]aceanthrylene and 4,5-dihydroxy-4,5-dihydrobenz[l]aceanthrylene were formed in minor amounts (< 6%). Therefore, metabolism at the cyclopenta-ring of B(j)A and B(l)A is a minor pathway in C3H10T1/2CL8 cells in contrast to previously reported studies with cyclopenta[cd]pyrene in which the cyclopenta-ring dihydrodiol was the major metabolite. These results suggest that routes of metabolic activation other than oxidation at the cyclopenta-ring such as bay region or k-region activation may play an important role with these unique polycyclic aromatic hydrocarbons in C3H10T1/2CL8 cells.
... Thus it seems unlikely that the probiotic effect we report here would be the consequence of the hydrolysis of endogenous β-glucuronides, as suggested by Gilissen et al. (1998), resulting in the release of -glucuronate that is an additional source of dietary carbohydrates. GUS activity was reported in Drosophila melanogaster (Langley et al., 1983) and in Musca domestica (Levvy & Marsh, 1959), but never investigated in aphids. Ingested GUS, however, could directly interfere with the aphid metabolism, enhancing the glycosaminoglycans degradation. ...
Transgenesis developed in the last 20 years offers new possibilities for crop protection. The transgenic process, however, requires the use of marker fusion genes to select and visualize the transformed tissues. Although the expression products of these marker genes are stably expressed in crops, little attention has been given to assess the eventual risks of these recombinant proteins on phytophage populations. Three independent transgenic potato (Solanum tuberosum) clones from the cultivar Désirée (DG5, DG18, and DG20) carrying the commonly used nptII-gus gene construct and exhibiting different β-glucuronidase activity (0.843 ± 0.011, 0.576 ± 0.096, and 0.002 ± 0.000 pmol min−1.mg−1, respectively) were evaluated to determine the impact of the encoded proteins on the behaviour, development, reproduction, and demography of the peach-potato aphid, Myzus persicae, under laboratory-controlled light and temperature. Our results revealed that the transgenic event can alter aphid physiology or behaviour. Experiments showed a probiotic effect of one transgenic line, the DG5, resulting in reduced prereproductive period and mortality, and enhanced daily fecundity, which was expressed in a greater population growth potential (rm = 0.205 vs. rm = 0.174 of the control). In contrast, aphids fed with the DG18 line exhibited reduced adult survival and reproductive period but no alteration of their demographic parameters (rm = 0.176). Finally, no physiological alteration was induced in aphids fed on a DG20 diet (rm = 0.170). Behavioural experiments conducted in a 4-choice olfactometer demonstrated that insects were significantly more attracted by the odour of transgenic DG18 potato plant than that of Désirée non-transformed plant, spending twice as much time in the DG18 plant odour. The two other transformed clones (DG5 and DG20) were as attractive as the non-transformed cultivar. It is concluded that the β-glucuronidase expression in potato plants might be responsible for the probiotic effect measured on the feeding aphids, whereas alteration of the foliage odour would result from a pleiotropic effect.
... Some novel GUS or GUS-like genes were also isolated and characterized from plants, such as Scutellaria baicalensis Georgi [45]. GUS-like enzymes were known to be present in microorganisms [2,3,46,47], animals [48,49] and plants [11,13,36,39,50], such as Scutellaria baicalensis [51], rye [52], and maize [53]. Sudan and his colleagues demonstrated that GUS is ubiquitously present in plants [36]. ...
A β-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable β-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo β-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic β-glucuronidase activity, which is a welcome improvement in its use as a reporter.
A new triterpenoid, glaucinoic acid (2α, 3β, 19α, 24-tetrahydroxyolean-12-en-30-oic acid) (1) along with several known compounds, arjunic acid (2), arjungenin (3), sericoside (4), and friedelin (5) were isolated from the stem barks of Terminalia glaucescens. These compounds showed β-glucuronidase inhibitory activity. The structures were identified on the basis of spectroscopic techniques.
ZusammenfassungDie Verteilung der β-N-Acetylhexosaminidase und der β-Galactosidase im Genitaltrakt des Hahnes wurde untersucht.Die höchste Aktivität an beiden Enzymen wies der Nebenhoden auf. Der Hoden zeigte die niederste Aktivität an β-N-Acetylhexosaminidase, der Ductus deferens die niederste β-Galactosidase-Aktivität.SummaryDistribution of β-N-acetylhexosaminidase and β-galactosidase in the genital tract of the cockerel (Gallus domesticus)The distribution of these two glycosidases in the reproductive organs of the cockerel was studied.The highest activity of both enzymes was in the epididymis. The testis showed the lowest activity of β-acetylhexosaminidase and the ductus deferens the lowest activity of β-galactosidase.RésuméRépartition de la β-N-acétylhexosaminidase et de la β-galactosidase dans les organes génitaux du coq (Gallus domesticus)La répartition de la β-N-acétylhexosaminidase et de la β-galactosidase a été recherchée dans le tractus génital du coq.La plus grande activité des deux enzymes s'est manifestée dans l'épididyme. Le testicule présenta la plus basse activité de β-N-acétylhexosaminidase et le canal déférent la plus faible activité de β-galactosidase.ResumenDistribución de la β-N-acetilhexosaminidasa y β-galactosidasa en el tracto genital del gallo domésticoSe examinó la distribución de la β-N-acetilhexosaminidasa y de la β-galactosidasa en el tracto genital del gallo. La actividad máxima en ambos enzimas se registró en el epidídimo. El testículo presentaba la actividad mínima de β-N-acetilhexosaminidasa, el conducto deferente la actividad más baja de β-galactosidasa.
A study is reported of the reactivities of the disaccharides isolated after deamination of beef-lung heparin and reduction of the products by sodium borotritide: 2,5-anhydro-O-(α-l-idopyranosyluronic acid sulfate)-d-mannitol sulfate, SIMS; 2,5-anhydro-O-(α-l-idopyranosyluronic acid)-d-mannitol sulfate, IMS; 2,5-anhydro-O-(α-l-idopyranosyluronic acid sulfate)-d-mannitol, SIM; and 2,5-anhydro-O-(β-d-glucopyranosyluronic acid)-d-mannitol sulfate, GMS. Results for the non-sulfated disaccharides IM and GM, prepared by desulfation of SIMS and GMS, are also reported. SIMS and SIM were inert to purified α-l-iduronidase, showed unexpected resistance to periodate oxidation, and lost sulfate rapidly in 50mm hydrochloric acid at 100°. Hydrolysis of IM and of IMS was catalyzed by α-l-iduronidase, and of GM and GMS by β-d-glucuronidase; the radioactive products were identified as 2,5-anhydro-d-mannitol (aM) and its sulfate (aMS). The products SIM and IMS obtained by deamination of heparin and desulfation of SIMS (the major deamination product) are apparently identical. In heparin partially desulfated by methanolic hydrogen chloride, residual sulfate groups were mostly linked to l-iduronic acid residues. Chemical, chromatographic, and electrophoretic methods that are valuable for separation and characterization of the disaccharides are described.
Luteolin 3′,4′-di-O-β-d-glucuronide is the major flavonoid in the liverwort Lunularia cruciata. It is accompanied by small amounts of luteolin 3′-O-β-d-glucuronide. Both are new natural products and the former appears to be a unique example of a 3′,4′-diglycosylated flavonoid. Luteolin 4′-O-β-d-glucuronide was isolated as a hydrolysis product of the diglucuronide.
Zusammenfassung Es wird eine Methode zur Bestimmung vonß-Glucuronidase-Aktivität in Milch und anderem eiweißreichem Material (Zentrifugenschlamm) unter Verwendung von Phenolphthalein-ß-d-glucuronid als Substrat beschrieben. Das Enzym wurde in üblicher Weise charakterisiert, nämlich durch die Proportionalität zwischen Enzymmenge-Aktivität und den Einflüssen durch Reaktionszeit, Substratmenge, pH-Wert und Reaktionstemperatur.
Summary Copper ist a moderate inhibitor towards lacto-ß-glueuronidase. The enzyme is uncompetitively inhibited at copper concentrations of 8,5 · 10-4 mol Cu++/l. The inhibitory intensity of copper can be increased by ascorbic acid.
Steady state rate equations have been developed for various mechanisms involving enzyme, hydrogen ion, substrate and inhibitor. By differentiating these equations with respect to hydrogen ion concentration and applying Descartes Rule of signs to the resulting equations it has been possible to ascertain which of these enzyme reaction mechanisms can result in double pH optima. The following three general situations can give rise to double pH optima: (i) the presence of two distinct isoenzymes with different pH optima; (ii) formation of active enzymesubstrate complexes by two ionic species of the enzyme which differ by at least two protons; (iii) the presence of an ampholyte inhibitor, only one ionic species of which can combine with the enzyme. Properties of a system of category (iii) are graphically illustrated by numerical substitution in the appropriate rate equations.
Cats differ from rats in that they do not excrete steroid glucuronides in the urine and there is evidence that they have diminished capacity for synthesizing them. Guinea pigs, which synthesize them readily, also do not excrete them in the urine. It has been postulated that this absence of glucuronides in urine might result from an increased rate of hydrolysis of glucuronides by β-glucuronidase. Therefore, tissue and serum β-glucuronidase levels have been measured for cats and guinea pigs, comparing their activities with each other and with those of the rat, run as reference standards in each assay. Enzymic activities were measured in adult animals, and as a function of maturation.There exists a marked difference in the activity of β-glucuronidase in the adrenal gland, liver, spleen, and kidney of the two mammals, the cat and the rat, whose capacities for the formation of glucuronides are not the same and in which the biosynthesis and metabolism of steroids are quite different. Activities in the four organs are closer together for the cat and guinea pig than for either of these animals compared to the rat. The activities of β-glucuronidase in the liver, spleen, and kidney of the rat are two to three times higher than in the same organs of the cat; the adrenal gland of the cat exhibits twice the β-glucuronidase activity observed in the rat adrenal.Changes in β-glucuronidase activity with age were determined in the cats and guinea pigs. In the cat, whose capacity for the formation of glucuronides seems to be low, a progressive decline of β-glucuronidase activity was observed from the very young animals to adults; the decline is extraordinarily manifested in the liver and also observed in the adrenal gland and the spleen.
Harmol glucuronide has been synthesized in vitro using uridine diphosphate glucuronic acid (UDPGA) and guinea pig liver microsomes. It was separated from the unreacted aglycone by column chromatography on Sephadex G-25. The glucuronide isolated in this was was chromatographically pure and it was hydrolyzable by commercial preparations of bovine liver, bacterial, and molluscan β-glucoronidase. Both harmol and glucuronic acid were demonstrated in the hydrolyzate. Boiled saccharate of concentration above 0.001 M inhibited the enzymic hydrolysis completely. This enzymically synthesized glucuronide was used as a substrate in the measurement of β-glucuronidase present in liver, kidney, spleen, heart, brain, and serum of a number of experimental animals and in human serum.
The chapter discusses later work on glycosiduronic acids and their derivatives from chemical point of view. As a consequence of the biological importance and general interest in the chemistry of Dglucosiduronic acids, the chapter is mainly concentrated around those conjugates having D-glucuronic acid as the sugar component. The nature of protective groups on the sugar component is of the utmost importance in the synthesis of many glycosiduronic acids. Successful synthesis of a glycosiduronic acid sensitive to alkaline or acidic conditions, or both, depends largely on the conditions under which the protecting groups of the sugar moiety can be removed after formation of the glycosidic bond. There has been considerable interest in the preparation of glucopyranuronate intermediates protected with groups readily removable under mild and non-hydrolytic conditions. The formation of β-D-glucosiduronic acids by enzymic conjugation is, in vertebrates, the most widespread of conjugation mechanisms, in which hydroxyl groups in almost any type of foreign compound are potentially capable of conjugating with D-glucuronic acids.
A kinetic analysis of the stepwise alternating action of β–glucuronidase and β–acetylglucosaminidase on oligosaccharides and dextrins derived from hyaluronic acid was undertaken, for better definition of the contribution of this process to hyaluronate catabolism. Production of monosaccharide from larger dextrins by action of either enzyme is powerfully inhibited by electrolyts. In the study, as in mammalian tissues, β–glucuronidase is present in excess so that the concentration of β–acetylgucosaminidase is rate controlling in the action on dextrin substrates. For this action, Vmax shows limited variation with ionic strength or molecular weight of substrate. At ionic strength 0. 03, but not 0. 18, Km decreases some 100–fold for increase of molecular weight from 2, 000 to 15, 000. It is specifically this decrease in Km that accounts for the prominent electrolyte inhibition observed with larger dextrins. The extremely low values of Km are attributed to multiple ionic enzyme–substrate interactions at sites remote from the catalytic center. The previously reported stimulation by electrolyte of the action of β–glucuronidase and β–acetylglucosaminidase on aryl glycosides, studied briefly, is apparently unrelated to the electrolyte effects seen with dextrins. The catabolic contribution of β–glucuronidase and β–acetyl glucosaminidase appears to be restricted to hydrolysis of the smaller oligosaccharides produced by action of hyaluronidase, since, for any reasonable assumptions regarding cellular environment, the extent of their action on polymeric hyaluronate or larger dextrins must be limited.
Glucuronides of drugs often accumulate during long term therapy. The hydrolysis of glucuronides can be catalysed by β-glucuronidase, an enzyme expressed in many tissues and body fluids in humans. The possible contribution of β-glucuronidase to drug disposition in humans has not been assessed in a systematic manner, but this enzyme may be able to release, locally or systemically, the active or inactive parent compound from drug glucuronides, thereby modifying the disposition and action of these drugs.
Based on the information available on the localisation, expression and variability of β-glucuronidase, the concept of β-glucuronidase-mediated drug metabolism is outlined in this article using examples from the literature. Since some issues surrounding the β-glucuronidase-mediated deconjugation of drug glucuronides still need to be clarified in humans, additional data from animal models supporting this concept have been included. Moreover, as β-glucuronidase has already been proven to be useful in tumour specific bioactivation of glucuronide prodrugs of anticancer agents, we also focus on anticancer prodrug approaches utilising β-glucuronidase. This review summarises the role of β-glucuronidase in drug disposition and drug targeting in humans.
Opossum and rabbit sperm sonicates dispersed the mouse cumulus oophorus in vitro at the same rate on a per sperm basis, despite much higher activities of the glycosidic acrosomal enzymes N-acetylhexosaminidase (350 ×) and arylsulphatase (40 ×) in the opossum preparation. Activities of another glycosidase, hyaluronidase, and the protease acrosin where similar in sperm extracts from both species. However, specific inhibitors of N-acetylhexosaminidase (iodoacetate) and arylsulphatase (SO42−, PO43−) markedly reduced the rate of cumulus dispersal by rabbit sperm sonicates. These findings suggest that, while hyaluronidase action probably represents the rate-limiting (slow) step, several glycosidases act together to disperse the cumulus and in the passage of the fertilizing spermatozoon through it in eutherian mammals. The likely role of these acrosomal enzymes in the marsupial, where the ovulated oocytes lack a cumulus oophorus, is more uncertain.
1.(1) A micro-method for the estimation of β-glucuronidase in human vaginal fluid based on total protein is described.2.(2) A linear relationship of β-glucuronidase with vaginal fluid concentration in the presence of Triton X-100 is shown.3.(3) Difficulties in obtaining vaginal fluid β-glucuronidase in a fully soluble form are described. The only treatments found to release fully the enzyme into solution are the non-ionic detergents Triton X-100 and Nonidet P.40 in combination with 0.9% NaCl.4.(4) Occasional inhibition of β-glucuronidase activity after its release into the soluble state is noted. This was shown to be reversible on subsequent treatment of the 0.1% Triton−0.9% NaCl suspension with water.5.(5) A brief comparison of β-glucuronidase solubility in other tissue homogenates is made.
Uptake, metabolism, and elimination of 2′,5-dichloro-4′-nitrosalicylanilide (Bayer 73) was studied in rainbow trout (Salmo gairdneri) exposed to 14C Bayer 73 in water. Transfer of trout to fresh water after exposure to Bayer 73 for 12 h resulted in the disappearance of 14C from most tissues after 48–72 h. A 24-h 14C bile to water ratio of 10,000:1 indicated a high degree of biliary concentration. A single polar 14C metabolite was purified from bile. Analysis by analytical thin-layer chromatography, β-glucuronidase hydrolysis, acid hydrolysis, infrared spectroscopy, and mass spectrometry indicated that the material was the glucuronide conjugate of Bayer 73.
Morphine is converted to morphine 3-β-D-glucuronide (M3G) by the UDP-glucuronosyltransferase Ugt2b1 in the endoplasmic reticulum (ER) of rat liver. Because of its luminal localization, UGT activity requires UDP-glucuronate import and glucuronide export across the ER membrane. The former transport is generally considered to be rate limiting and to explain the latency of UGT activities in intact microsomal vesicles. However, some observations indicate that the release of bulky glucuronides, such as M3G, might also be rate limiting for glucuronidation. This assumption was tested by characterizing the transport of M3G and its distribution between the intra- and extravesicular spaces during synthesis in rat liver microsomes. The amount of vesicle-associated M3G was measured using rapid filtration and LC-MS measurement. Our results reveal a remarkable accumulation of newly synthesized M3G in the microsomal lumen above the equilibrium. The transport showed a linear concentration-dependence in a wide range (5-200 μM). Therefore, the build-up of high (about 20 μM) luminal M3G concentration could adjust the rate of release to that of synthesis (44.85 ± 4.08 pmol/min/mg protein) during the conjugation of 100 μM morphine. These data can explain earlier findings indicative of separate intracellular pools of M3G in rat liver. Accumulation of bulky glucuronides in the ER lumen might also play an important role in their targeting and in the control of biliary excretion. © 2012 BioFactors, 2013.
β-Glucuronidase (EC 3.2.1.31), From Patella vulgala, was immobilized on a pellicular, polyethyleneimine-derivatized nylon net. Several types of nylon nets were used in order to ensure the best relationship between activity of the immobilized enzyme and net characteristics. No differences were observed between the two most used reagents for nylon activation by alkylation under standard working conditions. The influence of attachment by several protein aminoacidic side chains was determined, and enzyme immobilization involving lysyl and tyrosyl protein residues showed better activity. Coupling conditions for these derivatives have been optimized by studying the influence of pH, temperature and reaction time on derivative activity. The saturation profiles obtained for these derivatives showed a high protein content, 30mg/g, for a non-porous support. Characterization of these derivatives against pH, ionic strength and temperature was also studied, and no differences were found between soluble and immobilized activity-profiles apart from those found when thermal stability studies were performed. High stability towards intermittent use was found when assayed against p-nitrophenylglucuronide as substrate. After 18 months of use a loss of activity of only 22 and 33% for derivatives through lysyl and tyrosyl residues, respectively, was observed.
Abstract— Activities of β-glucuronidase were measured microchemically in the rat within cortical layers and subcortical white matter of somatosensory and visual cortex and the dorsal hippocampus. Distributions were related to histological composition, densities of myelinated fibers, and lysosome content as indicated by acid phosphatase staining. Three zones of relatively high activities were noted. The first corresponded to the pia-arachnoid and has been related to lysosmal particles within pericytes and macrophages of the meninges and in the pial cells. A second peak appeared in layer V and correlated well with the presence of neuronal lysosomes as detected by histochemical reaction. A third contribution was related to the presence of myelinated fibre bundles and white matter. Data from the literature and from unpublished results were cited to support the conclusion that nonlysosomal sources of enzyme in white matter included a major component from particles sedimenting with the microsomal fraction and a small component from myelin.
This chapter discusses the synthesis of mammalian glycosidases and their inhibition by aldonolactones. To consider the inhibitory action of aldonolactones mainly in connection with mammalian glycosidases provides an opportunity to discuss the assay of a group of enzymes with certain features in common, notably their ubiquity in mammalian tissues and their potential action on mucosubstances. Aldonolactone inhibition, which is always of a competitive character, usually reflects the substrate specificity of a glycosidase. β-Glucuronidase hydrolyzes all known biosynthetic β-glucuronides, whether the aglycon is alkyl, aryl, or alicyclic, and whether it is etheror ester-linked, and hydrolyzing synthetic β-galacturonides. Like β-glucuronidase, the β-N-acetylglucosaminidase enzyme attacks oligosaccharides derived from hyaluronie acid, removing terminal nonreducing N-acetylglucosamine residues. Natural substrates for the mammalian enzyme are still unknown, but mannose is a common component of glycoproteins, and terminal nonreducing mannose residues can be split from glycopeptides with almond emulsin. Rat epididymis is the richest known source of the mammalian enzyme; however the preparation is not free from other glycosidases of comparable activity.
The earlier preparation of cyclohexylammonium (phenyl α-l-idopyranosid)-uronate has been improved, and (4-methylumbelliferyl α-l-idopyranosid)uronic acid (14), a more sensitive substrate for α-l-iduronidase, has been synthesized by an analogous route. Zinc chloride-catalyzed condensation of 4-methylumbelliferone with 1,2,3,4,6-penta-O-acetyl-α-l-idopyranose (4) in 1,2-ethanediol diacetate gave crystalline 4-methylumbelliferyl 2,3,4,6-tetra-O-acetyl-α-l-idopyranoside (7). O-Deacetylation and catalytic oxidation gave 14, characterized as a cyclohexylammonium salt. The starting material 4 was prepared, in 21 % yield from l-glucose, by conversion of the intermediate 1,2,3,4,6-penta-O-acetyl-β-l-glucopyranose to 2,3,4,6-tetra-O-acetyl-β-l-glucopyranosyl chloride and acetoxonium ion rearrangement, as described for the D-series.
Uridine diphosphate glucose dehydrogenase activity was normally highest in the liver, followed by the intestine and kidneys. The activity was significantly lower in the liver of fasted than of fed animals. ß-Glucuronidase activity was higher in the liver and intestine of fasted than of fed rats. Glucuronidation was low in the kidney and intestine compared with the liver. Fasting resulted in a slight decrease in this process in the intestine only. Release of phosphate from uridine diphosphate glucuronic acid during incubation was normally highest in the intestine, fasting having no detectable effect. The presence of ethylene diamine tetra-acetic acid in the assay mixture resulted in a marked increase in glucuronidation and a fall in the simultaneous release of phosphate, both these effects being particularly high in the gut. Uridine diphosphate glucuronyl-transferase activity and the release of phosphate in the presence of ethylene diamine tetra-acetic acid were higher in the liver of fasted than of fed rats. Addition of saccharo-1→4-lactone, an almost specific inhibitor of β-glucuronidase, was followed by an increase of glucuronidation in the liver but not in the kidney and intestinal homogenate and there was no detectable change in the release of phosphate. Activation was observable whether ethylene diamine tetra-acetic acid was present or not, but was less marked in the fasted liver. The role of uridine diphosphate glucose dehydrogenase, uridine diphosphate glucuronyltransferase, uridine diphosphate glucuronic acid pyrophosphatase and ß-glucuronidase in the metabolism of glucuronic acid and in the glucuronide production of fed and fasted animals is discussed.
Hyaluronidase, a matrix-degrading enzyme, was assayed in extracts from breast primary tumors and regional metastases using a pool of human sera as a standard. Optimal activities of tumor extracts and serum were found for concentrations of 0.15–0.20 M NaCl in pH 3.8–4.0 buffer. In evaluating contamination by serum due to vascular proliferation, we expressed our results as the ratio of the entire activity (mU/l extract) on serum albumin content of tumors (g/l). Median (interquartile range) activities were 9.02 (6.04–14.34) for primary tumors and 37.36 (24.06–99.63) mU/g albumin for metastases. The difference was significant. Zymographic analysis showed that 3 bands of activity were detected which corresponded to 68, 53 and 49 kDa for tumoral hyaluronidase. The same pattern was observed for cellular extracts of breast cancer cell line CAL51, demonstrating that hyaluronidase detected in tumor extracts had mainly a cellular origin. Our results suggest that hyaluronidase may be involved in the metastatic process. Int. J. Cancer 73:327–331, 1997. © 1997 Wiley-Liss, Inc.
Phenolphthalein-glucuronide is a commonly used glucuronide conjugate for beta-glucuronidase measurements. The quantity of
phenolphthalein liberated by beta-glucuronidase is measured spectrophotometrically. The detection limit of the quantity of
phenolphthalein using spectrophotometry is a few μg. In this study, a new radioanalytical technique for the measurement of
beta-glucuronidase was applied which is 106times more sensitive than the spectrophotometric technique. Radioiodinated phenolphthalein-glucuronide and phenyl-N-glucuronide
were used in this study in which the beta-glucuronidase levels of some tissue samples were measured.
This article reports the application of Hayashi's histochemical technique for -glucoronidase to mouse epididymis. A methodological study. which established optimal conditions for demonstrating the enzyme in this organ, is reported. The distribution pattern of -glucuronidase is described and correlated with previous data for -naphtyl acetate esterase. Differences between sites of granular and diffuse reaction product for these two enzymes are recorded. Possible interpretations of these findings in terms of intracellular localization of enzymes are discussed. Studies on different strains reveal regular differences in histochemical organization between mice of various genotypes. Histochemical data which imply androgen inducibility of -glucuronidase in mouse epididymis are preliminarily noted.
An improved indigogenic method for the detection of β-glucuronidase was devised. The recommended medium consists of 2.4×10−4 M 4-Cl-5Br-3-indolyl-β-D-glucuronide (dissolved in a small amount of dimethylformamide) in 0,1 M acetate buffer pH 5, 3.4×10−3 M potassium ferricyanide and 3.4×10−3 M potassium ferrocyanide. The activity of β-glucuronidase escapes from unfixed or acetone postfixed cold microtome sections into the incubation solution and this leakage cannot be entirely prevented by either the postfixation of sections in cold aldehyde fixatives, or in acetone-formaldehyde-chloralhydrate mixture, or by fixation of freeze-dried sections in isopropylalcohol. No leakage was ascertained from sections prepared from tissue blocks fixed for 24 hours in cold aldehydes. This procedure leads to 70–80% inactivation of enzyme activity in rat spleen, liver and kidney, however. The recommended concentration of ferri-ferrocyanide mixture inhibits the activity by 30% at an average. The overall staining intensity obtained with the improved method in various organs reflects well activities of β-glucuronidase as estimated biochemically in homogenates of these organs. The method enables to demonstrate the lysosomal localization. The distribution pattern obtained with it resembles that obtained with the simultaneous azo-coupling method using naphthol AS-BI-β-glucuronide and hexazonium-p-rosanilin.
The -glucuronidase (GUS) gene is to date the most frequently used reporter gene in plants. Marketing of crops containing this gene requires prior evaluation of their biosafety. To aid such evaluations of the GUS gene, irrespective of the plant into which the gene has been introduced, the ecological and toxicological aspects of the gene and gene product have been examined. GUS activity is found in many bacterial species, is common in all tissues of vertebrates and is also present in organisms of various invertebrate taxa. The transgenic GUS originates from the enterobacterial species Escherichia coli that is widespread in the vertebrate intestine, and in soil and water ecosystems. Any GUS activity added to the ecosystem through genetically modified plants will be of no or minor influence. Selective advantages to genetically modified plants that posses and express the E. coli GUS transgene are unlikely. No increase of weediness of E. coli GUS expressing crop plants, or wild relatives that might have received the transgene through outcrossing, is expected. Since E. coli GUS naturally occurs ubiquitously in the digestive tract of consumers, its presence in food and feed from genetically modified plants is unlikely to cause any harm. E. coli GUS in genetically modified plants and their products can be regarded as safe for the environment and consumers
Use of phenyl α-L-iduronide as a test substrate now makes it possible to show occurrence in rat liver lysosomes of an α-L-iduronidase. The enzyme can be shown to be distinct from the well studied β-glucuronidase. With the phenyl glycoside, measurements can be made of this relatively weak mammalian activity, whose occurrence could previously be inferred only indirectly from the slow degradation of dermatan sulfate derivatives by tissue extracts. Trials with appropriate aryl glycosides indicate absence of detectible α-D-glucuronidase, α-D-mannuronidase, α-D-galacturonidase, or β-L-iduronidase activities from lysosomal extracts.
Acyl glucuronide conjugates of acidic drugs have been shown to be reactive metabolites capable of undergoing non-enzymic hydrolysis, rearrangement (isomerization via acyl migration) and covalent binding reactions with plasma protein. In an earlier study (King and Dickinson, Biochem Pharmacol45: 1043–1047, 1993), we documented formation of covalent adducts of diflunisal (DF), a salicylate derivative which is metabolized in part to a reactive acyl glucuronide (DAG), with liver, kidney, skeletal muscle and small and large intestine (in addition to plasma protein) of rats given the drug i.v. twice daily at 50 mg DF/kg for 7 days. The present study shows that covalent adducts of DF were also formed with urinary bladder tissue of these rats, achieving concentrations (ca. 5 μg DF equivalents/g tissue) higher than those found in the other tissues noted above. After cessation of dosing, the adduct concentrations declined with an apparent T value of ca. 20 hr. Adducts were also formed ex vivo in excised rat bladders in which DAG or a prepared mixture of its acyl migration isomers (iso-DAG) were incubated at pH 5.O, 6.5 and 8.0. After 8 hr incubation, the highest concentrations (ca. 11, μg DF equivalents/g) were produced with iso-DAG at pH 5.O, and the lowest (ca. 2.3 μg DF equivalents/g) with DAG at pH 5.0. However, a major competing reaction for DAG (at least at pH 5.0) was hydrolysis by β-glucuronidases originating from bladder tissue. By contrast, iso-DAG was quite resistant to such hydrolysis. The phenolic glucuronide conjugate, another important metabolite of DF, was hydrolysed only slowly. Similar results were obtained in fresh rat urine adjusted to pH 5.0. The results support covalent DF adduct formation in rat bladder originating from both DAG and iso-DAG as ultimate reactants, though the extent of binding is modulated by both urinary pH and β-glucuronidases.
Human serum β-glucuronidase has a high requirement of phenolphthalein β-d-glucosiduronic acid for its saturation as evidenced by the apparent Km value of 0.002 M. The digest containing 0.006 M substrate ensures an elevated linear rate as a function of serum concentration which permits the reduction of incubation time to 4 h. One can expect a variation of between 1.4 and 6.5% in replicate determinations depending on the enzyme level. The role of circulating saccharolactone, an endogenous β-glucuronidase inhibitor, was found to be minimal following the study of the effect of dialysis on enzyme activity. Serum values in normal individuals and in patients with diabetes, liver cirrhosis and cancer are reassessed and were found to be approximately twice the previously reported values. Conditions have also been recommended for the use of alternate substrates, i.e. β-d-glucosiduronic acids of p-nitrophenol and 8-hydroxyquinoline. Finally, factors of biochemical specificity, of integrity of subcellular organelles and genetics are dealt with in the evaluation of the significance of serum β-glucuronidase values.
1.1. Extracts of the hepatopancreas of the tunicate Pyura stolonifera contained a wide range of polysaccharidase activities and also hydrolysed a variety of aryl glycosides.2.2. Further kinetic studies indicated that the anomeric glycosides of d-glucoronic acid and of N-acetyl-d glucosamine were hydrolysed by different enzymes.3.3. The aryl α-glucoronidase also hydrolysed α-phosphate esters of d-glucuronic acid and could act as a transferase.
Cellulose ion-exchange chromatography was used to purify bovine liver β-glucuronidase by an improved method. An 18% yield of enzyme having a specific activity of approximately 2 × 105 Units per milligram was obtained. The purified enzvme appeared homogeneous by ion-exchange chromatography on several adsorbents, gel filtration, electrophoresis in starch gel and polyacrylamide gel, and ultracentrifugation. From sedimentation-equilibrium, a molecular weight of about 280,000 was calculated. With dry weight as a measure of protein, the E1cm1% at 280 mμ was 17. The amino acid composition was determined. These enzyme preparations contained 3–6% carbohydrate, and glucosamine was detected in acid hydrolyzates by ion-exchange chromatography. The purified enzyme contained less than one mole of phosphate per mole of enzyme. The Km with phenolphthalein glucuronide as a substrate was about 0.05 mm. A broad pH-activity curve with a single optimum at pH 4.8 was observed, and the enzyme was most stable between pH 4 and 7. The enzyme could be chromatographed on diethylaminoethyl-cellulose in the presence of 7.7 m urea, with full recovery of activity after dilution of the urea. It was also found to be markedly stable to autolysis in liver homogenates at 37 °.
Attention has been centered on the role and source of the enzyme beta-glucuronidase in physiological and pathological states. In earlier publications,1, 2 control values for vaginal fluid and uterine cervix tissues in health and disease have been presented. It has been demonstrated that the blind vaginal pouch, with the uterus and cervix ablated, is frequently rich in beta-glucuronidase activity. Two obvious possible sources of the enzyme in vaginal fluid, Trichomonas vaginalis and the presence of blood elements, were considered. An evaluation has been made of the significance of the Trichomonas growing in the vaginal fluid as a contributor to the enzyme content of vaginal fluid.³
The present study investigates the leucocytes and their relation to vaginal fluid beta-glucuronidase activity. Since previous data on human blood and lymph⁴ showed low enzyme activity in these substances, not exceeding 10 to 20 units per gram, the leucocytes,4, 5 averaging around 1,000 units per gram, remain the most probable extraneous source rich in beta-glucuronidase.
The reaction between methanol and glucuronolactone is catalyzed by cation exchange resin as well as by mineral acids. In addition to the γ-lactone of methyl β-glucofururonoside, a known product of the acid-catalyzed reaction, a second compound was isolated from the resin-catalyzed reaction; this was identified as the γ-lactone of methyl α-glucofuruonoside. Rotation data indicate that the latter compound undergoes mutarotation to the β-isomer as the reaction is prolonged.
The increasing interest in the beta-glucuronidase activity of body tissues and fluids in health and disease has made urgent the report of an adequate control series. A systematic study of vaginal fluid and cervical biopsy specimens has been undertaken in this laboratory on several groups of women in an attempt to establish the base line control levels for normal women. The findings and a discussion of the present concepts of their physiologic import are reported in this, the first of our series of papers on this subject. The method for collecting the specimens and the procedure for handling the material will be described in detail, together with an analysis of the results in a study of 500 nonpregnant, noncancerous women.1 Subsequent papers in this series will deal with additional aspects of the beta-glucuronidase activity of tissues, fluid and venous blood in physiologic and pathologic states in women.Beta-glucuronidase is
A large number of compounds with glycosidic linkages have been tested as substrates for the ,8-glucosidase of Stachybotrys atm. The enzyme appears to be specific for ,8-glucosides and all configurational alterations to the D-glucopyranose ring or substitutions in it lead to non-substrates; phenyl-fl-thioglucoside is a substrate, however. Aryl-,8-glucosides have a higher affinity for the enzyme than alkyl-,8-glucosides and no hydrolysis of cellobiose by the enzyme can be demonstrated. arrha-Substitution in aryl-fl-glucosides leads to a marked decrease in the affinity between enzyme and substrate.
Rabbit polymorphonuclear leucocytes contain an enzyme capable of hydrolyzing biosynthetic phenolphthalein mono-β-glucuronide. The concentration of the enzyme in the white cell is some 2000 times the concentration of the enzyme in the blood plasma. Under the conditions of study, the β-glucuronidase activity was proportional to the concentration of the enzyme. The effect of substrate concentration on the enzyme activity was studied and the Michaelis constant, Ks, determined. The course of the reaction was linear with time for the first 12 hr. and then fell off slightly during the next 12 hr. The optimum pH of the enzyme was 4.45 in either 0.2 M acetate or 0.2 M phthalate buffer. It was not inhibited by cyanide, azide, iodoacetate, fluoride, glycine, thiourea, urethane, arsanilic acid, acetophenone, o-cresol or m-cresol, in a final concentration of 0.01 M. The possible function of β-glucuronidase in rabbit polymorphonuclear leucocytes is discussed.
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RENEWED interest in the physiological function of the enzyme (β-glucuronidase was initiated by the report of Fishman (1940) that the feeding of glucuronidogenic substances to mice and dogs effected an increase in the β-glucuronidase activity of liver, kidney and spleen, while the enzyme concentration of the sex organs remained unchanged. However, the administration of estrogens to ovariectomized mice caused an increase in β-glucuronidase only in the uterus and possibly the vagina and conversely, ovariectomy resulted in a marked decrease in uterine β-glucuronidase activity (Fishman and Fishman, 1944; Fishman, 1947). It was concluded from these observations that β-glucuronidase catalyzes the synthesis of glucuronides in vivo and that the observed increases in enzyme activity represented an enzyme adaptation phenomenon in response to the presence of increased substrate. Fishman (1947) further postulated that β-glucuronidase plays a fundamental role in the physiological action of estrogenic hormones and that ...
RECENTLY one of us (Szego, 1952) reported that 17-hydroxycorticosterone (Compound F) and 11-dehydro, 17-hydroxy-corticosterone (Compound E) exhibited a marked inhibitory influence on the uterine water imbibition response (cf., Astwood, 1938) to simultaneously administered estrogen in the ovariectomized rat. The following additional steroids tested were negative in this regard: desoxycorticosterone acetate, testosterone, progesterone, and pregnenolone. Thus, considerable specificity in structural requirements was apparently involved in this evidence of steroid interaction.
The finding in this laboratory (Beyler and Szego, 1951) that the (β-glucuronidase activity of the preputial glands of the rat was many times greater than that reported for any other, including neoplastic, tissue and that this enzyme activity fluctuated in apparent response to altered levels of circulating hormones accompanying pregnancy (Beyler and Szego, 1954) led us to investigate the β-glucuronidase activity of these modified sebaceous glands in relation to exogenous steroid treatment. The observations to be reported below reveal that this enzymatic activity in preputial glands is strikingly sensitive to hormonal influence.
INTRODUCTION
THE effects of sex hormones on tissue β-glucuronidase activity in inbred mice were reported in a previous paper (Fishman & Farmelant, 1953). Among other findings, it was established that testosterone propionate produced a marked elevation in kidney β-glucuronidase in all inbred strains which were studied. In some preliminary histochemical experiments, it appeared that the enzyme was localized in both the lumina and in the epithelial cells lining the renal tubules. This suggested that some of the androgen-stimulated kidney β-glucuronidase may be excreted in the urine. The present paper deals with experiments designed to study this possibility.
METHODS AND EXPERIMENTAL PROCEDURES
Details may be found in a previous publication (Fishman and Farmelant, 1953) concerning types and source of animals used, the nature of the hormone treatment, types of controls, method of assay of β-glucuronidase activity, etc.
The routine of urine collection was as follows. Often at two times in the day, 10 A.M. and 4 P.M. and always once each day
In recent years, Morrow et al. (1949, 1950) reported that certain pure inbred strains of mice exhibited strikingly low concentrations of β-glucuronidase in the liver, spleen and kidney. Cohen and Bittner (1951) found that mammary tissue β-glucuronidase activity bore a relationship to the strain of mice employed, extremely low concentrations being found in the C3H strain. Among the interesting questions which these findings provoked is the one of whether or not tissue β-glucuronidase in these low-glucuronidase strains is responsive to extrinsic factors. It is known that certain glucuronidogenic drugs will produce an increase in β-glucuronidase activity in the non-sex organs (Fishman, 1940)) and that estrogen will restore uterine β-glucuronidase activity of the castrated mouse to normal levels Fishman and Fishman, 1944; Fishman, 1947) with no alteration in the non-sex organ glucuronidase. (Fishman, 1947; Harris and Cohen, 1951). An exception to this lack of effect in the non-sex organs was reported by Kerr,...
THE ability of testosterone to enhance kidney β-glucuronidase activity was observed in white Swiss and C3H mice (Fishman, 1951) and was later investigated in detail by Fishman and Farmelant (1953) in several strains of inbred mice. Testosterone propionate administration (7 mg. divided over 14 days) resulted in elevated kidney β-glucuronidase levels which were from four to thirteen-fold greater than control levels, depending on the strain employed in the study. Liver β-glucuronidase activity remained unchanged. Coincident with the rise in kidney β-glucuronidase activity, a marked excretion in the urine of β-glucuronidase was noted by Riotton and Fishman (1953). It is evidently desirable to arrive at some method of quantitating the activity of androgenic steroids on the basis of a biochemical analysis rather than by morphological or weight changes in a given target tissue. With regard to the renal β-glucuronidase response the following questions required consideration.
WHEN a peripheral nerve is cut, the portion of the nerve distal to the point of section undergoes a series of changes characteristic of Wallerian degeneration. The axon and, later, the myelin sheath are destroyed and there is an increase in the number of both Schwann cells and endoneurial cells (chiefly fibrocytes and macrophages) found along the course of the nerve1,2. We have now shown that in such a degenerating peripheral nerve there is a marked increase in the activity of the enzyme β-glucuronidase.
Upon dilution of highly purified β-glucuronidase, prepared from both calf spleen and liver, there occurs a considerable reduction of the specific enzyme activity, and the monomolecular reaction constant decreases immediately following dilution. This phenomenon is explained by the dissociation of the enzyme into inactive components. The dissociation of β-glucuronidase can be reversed by a number of substances which thus function as activators. Both dissociation and activation follow the Law of Mass Action. The following substances, in the order of decreasing potency, enhance β-glucuronidase activity: chitosan, protamine, cryst. bovine serum albumin, DNA from salmon milt, 1,10-diamino-n-decane, DNA from fish sperm, gelatin, cryst. chymotrypsin, thymus DNA, other α,ω-diaminopolymethylenes, spermine, spermidine, yeast ribonucleic acid, lysine and ornithine. The activation potency of DNA is essentially preserved after prolonged acid or alkaline hydrolysis, as well as after exhaustive enzymic degradation. Monoamino acids, and pure preparations of mononucleotides and monodesoxyribonucleotides failed to activate β-glucuronidase. The chemical nature of the activator and its activating potency are related to each other as follows: at least two basic groups per molecule are required; a greater number of basic groups per molecule produces no further effect; the presence of carboxyl groups decreases potency; the location of the basic groups in the molecule, and their distance from each other markedly influences potency, the size of the molecule being unimportant.
Catalytic, oxidation of 2-naphthyl β-p-glucopyranoside with molecular oxygen and platinum black yielded 2-naphthyl β-D-glucopyruronoside. Fusion of 2-naphthol with triacetyl-β-D-glucofururonolactone in the presence of p-toluenesulfonic acid yielded 2-naphthyl diacetyl-β-D-glucofururonolactone, which was converted to 2-naphthyl-β-D-glucofururonamide with dry ammonia and methanol. The amide reacted with sodium nitrite in 50% acetic acid to give 2-naphthyl β-D-glucofururonolactone rather than the acid. The pyranoside was readily hydrolyzed by β-D-glucuronidase whereas the furanoside was not. Therefore, substrates susceptible to β-D-glucuronidase activity require a pyranose ring.
Cation exchange resins have been shown to be effective catalysts for promoting the formation of glycosides from pentoses, hexoses, uronic acids and methylated sugars. These resins also catalyze the formation of acetone derivatives of sugars and methyl glycosides.
Phenyl β-D-glucopyruronoside was synthesized by the catalytic oxidation of phenyl β-D-glucopyranoside in the presence of platinum black. Its identity to the natural product was shown by its physical properties and its hydrolysis by β-D-glucuronidase. A previously reported synthesis by Neuberg and Neimann is discussed.
THE monoglucosiduronic acids of polyhydroxy-flavones, for example, chrysin, baicalein and scutellarein, that have been isolated from plant material, occur chiefly in the genus Scutellaria1. These have been shown to be beta-D-glucopyranuronides with a high affinity for animal tissue beta-glucuronidase2. Recently, a conjugate of apigenin with glucuronic acid has been found in the flowers of Erigeron annuus3. French beans (Phaseolus vulgaris) have been reported4 to contain a glycoside which on acid hydrolysis yielded glucuronic acid and a flavone that appeared to be quercetin; this has been confirmed and the compound has been found to be a substrate for beta-glucuronidase.