Disruption of sugar nucleotide clearance is a therapeutic vulnerability of cancer cells.

Identifying metabolic steps that are specifically required for the survival of cancer cells but are dispensable in normal cells remains a challenge1. Here we report a therapeutic vulnerability in a sugar nucleotide biosynthetic pathway that can be exploited in cancer cells with only a limited impact on normal cells. A systematic examination of conditionally essential metabolic enzymes revealed that UXS1, a Golgi enzyme that converts one sugar nucleotide (UDP-glucuronic acid, UDPGA) to another (UDP-xylose), is essential only in cells that express high levels of the enzyme immediately upstream of it, UGDH. This conditional relationship exists because UXS1 is required to prevent excess accumulation of UDPGA, which is produced by UGDH. UXS1 not only clears away UDPGA but also limits its production through negative feedback on UGDH. Excess UDPGA disrupts Golgi morphology and function, which impedes the trafficking of surface receptors such as EGFR to the plasma membrane and diminishes the signalling capacity of cells. UGDH expression is elevated in several cancers, including lung adenocarcinoma, and is further enhanced during chemoresistant selection. As a result, these cancer cells are selectively dependent on UXS1 for UDPGA detoxification, revealing a potential weakness in tumours with high levels of UGDH.

UDP-glucose 6-dehydrogenase knockout impairs migration and decreases in vivo metastatic ability of breast cancer cells.

Dysregulated metabolism is a hallmark of cancer that supports tumor growth and metastasis. One understudied aspect of cancer metabolism is altered nucleotide sugar biosynthesis, which drives aberrant cell surface glycosylation known to support various aspects of cancer cell behavior including migration and signaling. We examined clinical association of nucleotide sugar pathway gene expression and found that UGDH, encoding UDP-glucose 6-dehydrogenase which catalyzes production of UDP-glucuronate, is associated with worse breast cancer patient survival. Knocking out the mouse homolog Ugdh in highly-metastatic 6DT1 breast cancer cells impaired migration ability without affecting in vitro proliferation. Further, Ugdh-KO resulted in significantly decreased metastatic capacity in vivo when the cells were orthotopically injected in syngeneic mice. Our experiments show that UDP-glucuronate biosynthesis is critical for metastasis in a mouse model of breast cancer.

Integrative Metabolic Pathway Analysis Reveals Novel Therapeutic Targets in Osteoarthritis.

In osteoarthritis (OA), impairment of cartilage regeneration can be related to a defective chondrogenic differentiation of mesenchymal stromal cells (MSCs). Therefore, understanding the proteomic- and metabolomic-associated molecular events during the chondrogenesis of MSCs could provide alternative targets for therapeutic intervention. Here, a SILAC-based proteomic analysis identified 43 proteins related with metabolic pathways whose abundance was significantly altered during the chondrogenesis of OA human bone marrow MSCs (hBMSCs). Then, the level and distribution of metabolites was analyzed in these cells and healthy controls by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), leading to the recognition of characteristic metabolomic profiles at the early stages of differentiation. Finally, integrative pathway analysis showed that UDP-glucuronic acid synthesis and amino sugar metabolism were downregulated in OA hBMSCs during chondrogenesis compared with healthy cells. Alterations in these metabolic pathways may disturb the production of hyaluronic acid (HA) and other relevant cartilage extracellular matrix (ECM) components. This work provides a novel integrative insight into the molecular alterations of osteoarthritic MSCs and potential therapeutic targets for OA drug development through the enhancement of chondrogenesis.

Cascade synthesis of uridine-5'-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes.

BACKGROUND: Enzymatic glycan synthesis has leapt forward in recent years and a number of glucuronosyltransferase (EC 2.4.1.17) have been identified and prepared, which provides a guide to an efficient approach to prepare glycans containing glucuronic acid (GlcA) residues. The uridine 5'-diphosphate (UDP) activated form, UDP-GlcA, is the monosaccharide donor for these glucuronidation reactions. RESULTS: To produce UDP-GlcA in a cost-effective way, an efficient three-step cascade route was developed using whole cells expressing hyperthermophilic enzymes to afford UDP-GlcA from starch. By coupling a coenzyme regeneration system with an appropriate expression level with UDP-glucose 6-dehydrogenase in a single strain, the cells were able to meet NAD+ requirements. Without addition of exogenous NAD+, the reaction produced 1.3 g L-1 UDP-GlcA, representing 100% and 46% conversion of UDP-Glc and UTP respectively. Finally, an anion exchange chromatography purification method was developed. UDP-GlcA was successfully obtained from the cascade system. The yield of UDP-GlcA during purification was about 92.0%. CONCLUSIONS: This work built a de novo hyperthermophilic biosynthetic cascade into E. coli host cells, with the cells able to meet NAD+ cofactor requirements and act as microbial factories for UDP-GlcA synthesis, which opens a door to large-scale production of cheaper UDP-GlcA.

Functional characterization of the UDP-xylose biosynthesis pathway in Rhodothermus marinus.

UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS) are the two enzymes responsible for the biosynthesis of UDP-xylose from UDP-glucose. Several UGDs from bacterial sources, which oxidize UDP-glucose to glucuronic acid, have been found and functionally characterized whereas only few reports on bacterial UXS isoforms exist. Rhodothermus marinus, a halothermophilic bacterium commonly found in hot springs, proved to be a valuable source of carbohydrate active enzymes of biotechnological interest, such as xylanases, mannanases, and epimerases. However, no enzymes of R. marinus involved in the biosynthesis or modification of nucleotide sugars have been reported yet. Herein, we describe the cloning and characterization of two putative UGD (RmUGD1 and RmUGD2) and one UXS (RmUXS) isoform from this organism. All three enzymes could be expressed in recombinant form and purified to near homogeneity. UPLC- and NMR-based activity tests showed that RmUGD1 and RmUXS are indeed active enzymes, whereas no enzymatic activity could be detected by RmUGD2. Both RmUGD1 and RmUXS showed a temperature optimum of 60 °C, with almost no loss of activity after 1 h exposure at 70 °C. No metal ions were required for enzymatic activities. Zn(2+) ions strongly inhibited both enzymes. RmUGD1 showed higher salt tolerance and had a higher pH optimum than RmUXS. Furthermore, RmUGD1 was inhibited by UDP-xylose at higher concentrations. By coupling recombinant RmUXS and RmUGD1, UDP-xylose could be successfully synthesized directly from UDP-glucose. The high activity of the herein described enzymes make RmUGD1 and RmUXS the first thermo-tolerant biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose.

Down-regulation of UDP-glucuronic acid biosynthesis leads to swollen plant cell walls and severe developmental defects associated with changes in pectic polysaccharides.

UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.

A fluoro analogue of UDP-α-D-glucuronic acid is an inhibitor of UDP-α-D-apiose/UDP-α-D-xylose synthase.

UDP-2F-glucuronic acid was synthesized and analyzed as a mechanistic probe to investigate the ring contraction step catalyzed by UDP-d-apiose/UDP-d-xylose synthase (AXS).

Reconstruction of de novo pathway for synthesis of UDP-glucuronic acid and UDP-xylose from intrinsic UDP-glucose in Saccharomyces cerevisiae.

UDP-D-glucuronic acid and UDP-D-xylose are required for the biosynthesis of glycosaminoglycan in mammals and of cell wall polysaccharides in plants. Given the importance of these glycans to some organisms, the development of a system for production of UDP-D-glucuronic acid and UDP-D-xylose from a common precursor could prove useful for a number of applications. The budding yeast Saccharomyces cerevisiae lacks an endogenous ability to synthesize or consume UDP-D-glucuronic acid and UDP-D-xylose. However, yeast have a large cytoplasmic pool of UDP-D-glucose that could be used to synthesize cell wall beta-glucan, as a precursor of UDP-D-glucuronic acid and UDP-D-xylose. Thus, if a mechanism for converting the precursors into the end-products can be identified, yeast may be harnessed as a system for production of glycans. Here we report a novel S. cerevisiae strain that coexpresses the Arabidopsis thaliana genes UGD1 and UXS3, which encode a UDP-glucose dehydrogenase (AtUGD1) and a UDP-glucuronic acid decarboxylase (AtUXS3), respectively, which are required for the conversion of UDP-D-glucose to UDP-D-xylose in plants. The recombinant yeast strain was capable of converting UDP-D-glucose to UDP-D-glucuronic acid, and UDP-D-glucuronic acid to UDP-D-xylose, in the cytoplasm, demonstrating the usefulness of this yeast system for the synthesis of glycans. Furthermore, we observed that overexpression of AtUGD1 caused a reduction in the UDP-D-glucose pool, whereas coexpression of AtUXS3 and AtUGD1 did not result in reduction of the UDP-D-glucose pool. Enzymatic analysis of the purified hexamer His-AtUGD1 revealed that AtUGD1 activity is strongly inhibited by UDP-D-xylose, suggesting that AtUGD1 maintains intracellular levels of UDP-D-glucose in cooperation with AtUXS3 via the inhibition of AtUGD1 by UDP-D-xylose.

The inositol oxygenase gene family of Arabidopsis is involved in the biosynthesis of nucleotide sugar precursors for cell-wall matrix polysaccharides.

The nucleotide sugar UDP-glucuronic acid (UDP-GlcA) is the principal precursor for galacturonic acid, xylose, apiose and arabinose residues of the plant cell-wall polymers. UDP-GlcA can be synthesized by two different functional pathways in Arabidopsis involving either UDP-glucose dehydrogenase or inositol oxygenase as the initial enzyme reaction to channel carbohydrates into a pool of UDP sugars used for cell-wall biosynthesis. The genes for the enzyme myo-inositol oxygenase (MIOX) were analyzed in Arabidopsis. They represent a small gene family containing four members. The transcription of all those members indicates a transient and organ-specific gene expression pattern in growing plant tissues as analyzed by RT-PCR and in promoter::GUS reporter gene lines. Two isoforms (MIOX1, MIOX2) are expressed in almost all tissues of the plant, whereas the expression of MIOX4 and MIOX5 is largely restricted to flowers, particularly maturing pollen. T-DNA insertion lines in MIOX genes were isolated; however, single knock-outs show growth phenotypes similar to the wild type. The monosaccharide composition of the cell wall in these mutants is not significantly changed compared to wild type plants. However, the incorporation of 3H-inositol into wall polymers of seedlings is greatly impaired in the mutant lines (Delta)MIOX1 and (Delta)MIOX2, which are the only isoforms that are expressed in seedlings.

Biosynthesis of UDP-GlcA, a key metabolite for capsular polysaccharide synthesis in the pathogenic fungus Cryptococcus neoformans.

UDP-glucose dehydrogenase catalyses the conversion of UDP-glucose into UDP-GlcA, a critical precursor for glycan synthesis across evolution. We have cloned the gene encoding this important enzyme from the opportunistic pathogen Cryptococcus neoformans. In this fungus, UDP-GlcA is required for the synthesis of capsule polysaccharides, which in turn are essential for virulence. The gene was expressed in Escherichia coli and the 51.3-kDa recombinant protein from wild-type and five mutants was purified for analysis. The cryptococcal enzyme is strongly inhibited by UDP-xylose and NADH, has highest activity at pH 7.5 and demonstrates Km (app) values of 0.1 and 1.5 mM for NAD+ and UDP-glucose respectively. Its activity was significantly decreased by mutations in the putative sites of NAD+ and UDP-glucose binding. Unlike previously reported eukaryotic UDP-glucose dehydrogenases, which are hexamers, the cryptococcal enzyme is a dimer.

Molecular characterization of Streptococcus pneumoniae type 4, 6B, 8, and 18C capsular polysaccharide gene clusters.

Capsular polysaccharide (CPS) is a major virulence factor in Streptococcus pneumoniae. CPS gene clusters of S. pneumoniae types 4, 6B, 8, and 18C were sequenced and compared with those of CPS types 1, 2, 14, 19F, 19A, 23F, and 33F. All have the same four genes at the 5' end, encoding proteins thought to be involved in regulation and export. Sequences of these genes can be divided into two classes, and evidence of recombination between them was observed. Next is the gene encoding the transferase for the first step in the synthesis of CPS. The predicted amino acid sequences of these first sugar transferases have multiple transmembrane segments, a feature lacking in other transferases. Sugar pathway genes are located at the 3' end of the gene cluster. Comparison of the four dTDP-L-rhamnose pathway genes (rml genes) of CPS types 1, 2, 6B, 18C, 19F, 19A, and 23F shows that they have the same gene order and are highly conserved. There is a gradient in the nature of the variation of rml genes, the average pairwise difference for those close to the central region being higher than that for those close to the end of the gene cluster and, again, recombination sites can be observed in these genes. This is similar to the situation we observed for rml genes of O-antigen gene clusters of Salmonella enterica. Our data indicate that the conserved first four genes at the 5' ends and the relatively conserved rml genes at the 3' ends of the CPS gene clusters were sites for recombination events involved in forming new forms of CPS. We have also identified wzx and wzy genes for all sequenced CPS gene clusters by use of motifs.

Mechanism of cadmium-decreased glucuronidation in the rat.

In isolated rat hepatocytes, cadmium (0-200 microM) decreased the overall glucuronidation of both isopropyl N-(3-chloro-4 hydroxyphenyl)carbamate (4-hydroxychlorpropham, 4-OHCIPC) and 4-nitrophenol in a concentration-dependent manner. In contrast, in native rat liver microsomes, glucuronidation of 4-OHCIPC was increased by cadmium through activation of microsomal 4-OHCIPC glucuronosyl transferase. In addition, in rat microsome incubations, the net amount of 4-OHCIPC glucuronide was also indirectly increased by cadmium through a reduction in the activity of beta-glucuronidase. As the effect of cadmium on the activity of 4-OHCIPC glucuronosyl transferase could not account for the decrease in glucuronide formation in intact hepatocytes, the influence of cadmium on the availability of UDP-glucuronic acid (UDPGA) was investigated further. In isolated rat hepatocytes, cadmium depleted the UDPGA content in a dose-dependent manner without a change in the UDP glucose (UDPG) content. Cadmium did not increase the breakdown of UDPGA by microsomal UDPGA pyrophosphatase but strongly decreased (30-66%) the synthesis of the cofactor in the cytosol by inhibiting UDP-glucose dehydrogenase (UDPGDH). Cadmium (10-50 microM) was found to inhibit the purified enzyme from bovine liver (EC 1.1.1.22) non-competitively. In vivo in the absence of a substrate undergoing glucuronidation, cadmium administration, 1.5 and 2.5 mg Cd/kg i.v., to normally fed rats resulted in a 15 and 30% decrease of hepatic UDPGA, respectively. However, in the liver, neither the NAD+/NADH ratio nor the UDPG content was significantly changed following cadmium treatment. Both in vitro and in vivo results support the conclusion that in intact cells the reduction in overall 4-OHCIPC glucuronidation caused by cadmium was due to a decrease in UDPGA availability which results from the inhibiting effect of cadmium on UDPGDH.

Mechanism of decreased acetaminophen glucuronidation in the fasted rat.

The mechanism by which an acute fast decreases the glucuronidation of hepatotoxic doses of acetaminophen in the rat was examined. Fasting did not depress the level of the enzyme, glucuronyl transferase, or the basal level of the co-substrate, UDP-glucuronic acid (UDPGA). Administration of a hepatotoxic dose of acetaminophen rapidly depleted UDPGA levels in both fed and fasted rats to the same nadir. Fed and fasted rats differed in that the rate of repletion of UDPGA levels was markedly slower in fasted rats. The total hepatic levels of UDP-glucose dehydrogenase and its cofactor, NAD+, were not decreased by fasting. In fasted rats, hepatic levels of the UDPGA precursor, UDP-glucose, were approximately 60% those of fed rats both before and after a hepatotoxic dose of acetaminophen. In fed rats, acetaminophen induced a marked depletion of hepatic glycogen levels and a dramatic increase in blood glucose levels. Acetaminophen induced a similar marked increase in blood glucose levels in fasted rats in spite of the fact that they lacked hepatic glycogen. It is concluded that the fasting-induced decrease in the glucuronidation of hepatotoxic doses of acetaminophen results from decreased production of UDPGA. The decreased synthetic capacity for UDPGA does not appear to be due to the inability of the liver to produce glucose units per se, but rather to the fasting-induced altered activities of the enzymes of carbohydrate metabolism which, in turn, alter the fate of glucose-6-phosphate derived from gluconeogenesis.

Effects of adenosine on glucuronidation and uridine diphosphate glucuronic acid (UDPGA) synthesis in isolated rat hepatocytes.

Dibutyryl cyclic adenosine 3':5'-monophosphate (DBcAMP) has been shown to inhibit glucuronidation of p-nitrophenol in a concentration-dependent manner in isolated rat hepatocytes. Adenosine (ADO) also decreased glucuronidation in a similar fashion. The effects of adenosine were examined on the variables controlling glucuronidation in intact cells. The addition of adenosine was without effect on either glucuronyltransferase or beta-glucuronidase. Adenosine decreased uridine diphosphate glucuronic acid (UDPGA) levels by 62% and, subsequently, inhibited glucuronidation by 41% in isolated rat hepatocytes. Since the synthesis of UDPGA requires NAD+ for the dehydrogenation of UDP-glucose, alterations in the redox state could account for the decrease in intracellular UDPGA levels. The effects of ADO (500 microM) on lactate and pyruvate content and redox state were examined in rat hepatocytes. ADO caused a 2.1-fold increase in lactate levels and a 2.65-fold increase in the [lactate]/[pyruvate] ratio. The NAD+/NADP ratio, therefore, was decreased by 63% in the presence of ADO. Carbohydrate reserve also affects UDPGA levels; thus, graded concentrations of glucose (5.5, 25, and 50 mM) were added to cells incubated with ADO. At 5.5 mM glucose, ADO caused a 61% decrease in glucuronide formation, while at concentrations of 25 and 50 mM glucose, the inhibition was diminished by 53 and 47% respectively. ADO appears to have decreased the synthesis of UDPGA by decreasing the NAD+/NADH ratio, thus inhibiting UDP-glucose dehydrogenase. Carbohydrate reserve also appears to be involved in the inhibition of glucuronidation mediated by ADO.

Hepatic UDP-glucose and UDP-glucuronic acid synthesis rates in rats during a reduced energy state.

Hepatic synthesis rates of UDP-glucose and UDP-glucuronic acid were determined in rats. Two high pressure liquid chromatographic methods were developed to quantitate and isolate UTP, UDP-glucose, and UDP-glucuronic acid from perchloric acid extracts of rat liver. The specific activities of UTP, UDP-glucose, and UDP-glucuronic acid were determined in liver samples obtained from rats killed by cervical dislocation at various times after [6-14C]orotic acid administration. Synthesis rates were calculated from the rate of change in specific activities of the compound of interest and its immediate precursor and the concentration of the compound of interest. Synthesis rates of UDP-glucose and UDP-glucuronic acid were 102 +/- 9 and 99 +/- 1 nmol X min-1 X g of liver-1, respectively. UDP-glucuronic acid synthesis apparently accounts for most of the UDP-glucose produced during a period (8 a.m.-10 a.m.) when glycogen synthesis is low. The effect of an ethionine-induced reduction of energy state on these basal synthesis rates was examined. UDP-Glucose and UDP-glucuronic acid synthesis rates were decreased by approximately 80%. In summary, the hepatic synthesis rates of UDP-glucose and UDP-glucuronic acid are approximately 100 nmol X min-1 X g of liver-1, and a reduced energy state can decrease these synthesis rates in vivo.

Regulatory mechanisms of UDP-glucuronic acid biosynthesis in cultured human skin fibroblasts.

Kinetic parameters and regulatory properties of UDPGDH extracted from cultured human skin fibroblasts were determined and compared with those of UDPGDH from cornea and epiphysial-plate cartilage. Fibroblast enzyme showed an affinity for UDPG 7 times higher than cartilage enzyme and 42 times higher than cornea enzyme. UDP-xylose acted as a co-operative allosteric inhibitor, but under the same experimental conditions fibroblast enzyme was significantly less inhibited. These results were in agreement with the different GAG production of the cells we studied. Fibroblast UDPGDH activity was regulated by the NAD/NADH ratio and it was also affected by modifications of extracellular matrix composition. A significant increase of UDPGDH affinity for UDPG was observed after the treatment of the monolayers with Chase ABC.

Hepatic UDP-glucuronic acid regulation during acetaminophen biotransformation in rats.

Acetaminophen (AA) glucuronidation is capacity limited in several species after administration of high doses and previous data indicate that this phenomenon is due probably to a decrease in the concentration of the reaction cosubstrate UDP-glucuronic acid in liver. The rate-limiting determinant in UDP-glucuronic acid synthesis during AA glucuronidation is not known. The objective of the present study was to determine whether UDP-glucuronic acid synthesis during AA biotransformation is restricted by the supply of UDP-glucose or is limited by UDP-glucose dehydrogenase activity. Adult male Sprague-Dawley rats were injected with 600 mg/kg i.p of AA and liver was obtained 30, 60, 120 and 240 min later for quantitation of UDP-glucose, glycogen and UDP-glucuronic acid. AA was found to decrease markedly UDP-glucuronic acid concentration in liver 30, 60 and 120 min after injection (28, 52 and 58% of control values, respectively). In contrast, hepatic UDP-glucose levels were not altered after 30 min, but were decreased to 55 and 68% of control values 60 and 120 min after AA administration. Glycogen concentrations were decreased at the 30-min time interval only (78% of control). Therefore, maximal depletion of UDP-glucuronic acid occurred when UDP-glucose levels were not affected. UDP-glucose dehydrogenase is subject to product inhibition by NADH and UDP-glucuronic acid and it is possible that NADH accumulates during rapid utilization of UDP-glucuronic acid. Consequently, the effects of AA on cytosolic NADH/NAD ratios in liver were examined by determining the lactate/pyruvate ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of inhibitors of mitochondrial energy production on hepatic glutathione, UDP-glucuronic acid, and adenosine 3'-phosphate-5'-phosphosulfate concentrations.

The hepatic conjugation of xenobiotics with sulfate, glucuronic acid, and glutathione is decreased in vitro by compounds that impair cellular energy production. The proposed mechanism is that depletion of ATP in metabolically compromised cells causes a decreased synthesis of the co-substrates, adenosine 3'-phosphate 5'-phosphosulfate (PAPS), UDP-glucuronic acid, and glutathione. This proposal was examined in vivo by quantitating hepatic adenine nucleotides and co-substrates in rats treated with the following inhibitors of mitochondrial ATP production: rotenone, antimycin A, carbonyl cyanide m-chlorophenylhydrazone, and 2,4-dinitrophenol. Hepatic ATP levels 30 min after administration of the inhibitors were about 30% of control. Hepatic energy charge (ATP + 0.5 X ADP)/(ATP + ADP + AMP) was significantly reduced by each inhibitor. Unexpectedly, UDP-glucuronic acid, PAPS, and glutathione concentrations were not reduced at 30 or 60 min after administration of the inhibitors. Thus, it does not appear possible to deplete hepatic ATP in vivo by means of mitochondrial inhibitors to the extent necessary to affect basal levels of UDP-glucuronic acid, PAPS, and glutathione. PAPS levels increased after administration of 2,4-dinitrophenol. This was shown to be a property shared with phenolic inhibitors of phenol sulfotransferase.

UDP-glucuronyltransferase-catalyzed deconjugation of bilirubin monoglucuronide.

Bilirubin monoglucuronide is rapidly deconjugated when incubated with UDP and rat liver microsomal preparations at pH 5.1. The following evidence was found that this reaction is catalyzed by UDP-glucuronyltransferase: (i) unconjugated bilirubin and UDP-glucuronic acid were identified as the reaction products; (ii) Gunn rat microsomal preparations lack bilirubin UDP-glucuronyltransferase deficiency and do not catalyze the deconjugation reaction, and (iii) neither saccharo-1,4-lactone, a beta-glucuronidase inhibitor, nor butylated hydroxytoluene, an inhibitor of spontaneous isomerisation, affect the rate of the deconjugation reaction. Deconjugation appears to be the reverse of UDP-glucuronyltransferase-catalyzed glucuronidation. The conditions for the reverse reaction differ in the following aspects from those of the forward reaction: (i) nucleotide triphosphates stimulate the reverse reaction probably allosterically; (ii) UDP-N-acetylglucosamine stimulates the forward reaction but has no effect on the reverse reaction; (iii) the optimal pH for the reverse reaction is pH 5.1 and for the forward reaction is pH 7.8, and (iv) Mg++ ion is not required for the reverse reaction but stimulates the forward reaction. Detergents stimulate both reactions. Stimulation of the reverse reaction by nucleotide triphosphates and detergents is mutually independent and additive which suggests different mechanisms of action. Deconjugation reactions may become important during parenchymatous liver disease when, as a result of anaerobic glycolysis, intracellular pH decreases. Elevated levels of unconjugated bilirubin in the serum of patients with parenchymatous liver disease may be a sign of sick liver cells rather than decreased UDP-glucuronyltransferase activity.