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An enzymatic method was devised for determination of free glucuronic acid by the use of glucuronolactone reductase from rat kidney. Free glucuronic acid in the range of 4 to 200 micrograms was determined quantitatively by this method even in the presence of neutral sugars and oligosaccharides of glycosaminoglycans. The interference due to 20 to 100 micrograms of aldohexose or aldopentose was less than 0.5% at the same concentration of free glucuronic acid. The activity of the enzyme towards substituted glucuronic acid was only 1% of that towards free glucuronic acid. The enzyme did not act on N-acetylhexosamines. The method was applied to a study of glycosaminoglycan degradation by lysosomal enzyme. When hyaluronic acid and chondroitin 4-sulfate were incubated for 24 h with a glycoprotein fraction obtained from rat liver lysosomes by concanavalin A agarose chromatography, free glucuronic acid was liberated (showing a sigmoid curve as a function of incubation time) and reached 21 and 10% of total glucuronic acid, respectively. Tetrasaccharide from chondroitin 4-sulfate was degraded to equimolar amounts of free glucuronic acid and sulfated trisaccharide.
The D-glucuronate product of myo-inositol oxygenase (EC 1.13.99.1) is efficiently reduced by NADPH in the presence of either purified D-glucuronate reductase (EC 1.1.1.19), or reductase that is part of a protein aggregate that also contains the oxygenase. This occurs despite the fact that the maximum concentration of D-glucuronate that could be formed by the oxygenase under the conditions used for the coupled enzyme experiments is 7 microM, and 10 microM externally supplied D-glucuronate (Km = 7.6 mM) does not support any detectable NADPH oxidation under the reaction conditions. The most likely explanation for the results is that the uncyclized aldehyde form of D-glucuronate is the product of the oxygenase reaction, and that it diffuses into solution and is captured by the reductase before it cyclizes to the more stable but less reactive hemiacetal form.
Three-dimensional X-ray diffraction data were used to determine the crystal structure of alpha-D-glucuronate CaBr times 3H20, a model system for investigating the factors involved in the binding of calcium ions to D-glucuronate residues of oligo-and poly-saccharides. Crystals of the salt are monoclinic, space group P21, having a = 6.410 (1), b = 10.784 (2), c = 8.879 (1) A, betta = 92.07 (1)degrees, and Z = 2. Instensity data for 1082 reflections were measured with an automated diffractometer. A trial structure, obtained by the heavy-atom method, was refined by least squares to R = 0.025. The absolute configuration was confirmed by anomalous-dispersion effects. An outstanding feature of the crystal packing is the interaction of D-glucuronate anions with calcium ions. The calcium ion is coordinated to three symmetry-related D-glucuronate anions and to two water molecules. The D-glucuronate anion binds calcium cations through three chelation sites: one that involves a carboxyl-oxygen atom combined with O-5; one that includes the second carboxyl-oxygen atom acting in concert with O-4, and one composed of the O-1-O-2 pair of hydroxyl groups.
Studies on the activity of the glucuronic acid pathway in alloxan diabetic rabbits were carried out. Amount of D-glucaric acid, L-ascorbic acid, and D-glucuronic acid in urine increased in the case of the alloxan diabetic rabbits. The transformation from D-glucuronolactone to D-glucaric acid was higher than normal in the diabetic animals. The expired 14-CO2 decreased and urinary excretion of labeled L-gulonic acid increased after administration of 6-14-C-glucuronolactone in the diabetic rabbits. L-Gulonic acid dehydrogenase, lactonase II, and beta-glucuronidase activities were reduced, and UDPGA-pyrophosphatase, D-glucuronic acid-1-phosphatase, and UDPGA-transferase activities increased in the diabetic rabbit liver. From these results, it may be concluded that an increase of endogenous D-glucuronic acid in the diabetic states could be attributed to a metabolid defect in the step of L-gulonic acid dehydrogenation and to the enhancement of UDPGA-pyrophosphatase and D-glucuronic acid-1-phosphate phosphatase activities.
1. The relative rates of hydrolysis of synthetically prepared beta-d-glucuronic esters [aglycone: benzoic acid, veratroic (3,4-dimethoxybenzoic) acid, indol-3-ylacetic acid and ethylbutyric acid], and beta-d-glucuronic ethers (aglycone: phenolphthalein, p-nitrophenol, 3,4-dimethoxyphenol, 3,4-dimethoxybenzyl alcohol) by commercial preparations of beta-glucuronidase from bovine liver and Escherichia coli were investigated. The rates of hydrolysis of all compounds tested were followed by measuring the formation of glucuronic acid under conditions which do not affect the glycosidic ester bond. 2. The pH profiles of the substrates in reaction with the enzyme from both sources were determined, and substrate-saturation curves at the optimal pH for each substrate were constructed; double-reciprocal plots of activity against concentration were linear. 3. Comparison of kinetic data indicates that neither the type of sugar-aglycone linkage, nor the aglycone structure alone can explain the observed K(m) and V(max.) values. 4. alpha-d-Glucuronic esters of benzoic and veratroic acid resisted hydrolysis by beta-glucuronidase from both sources.
Eleven tetrasaccharides were isolated from the repeating disaccharide region of porcine intestinal heparin after strong digestion with Flavobacterium heparinase. Their structures were determined by composition analysis, enzymatic analysis, and 1H NMR spectroscopy. Nine of them have the common tetrasaccharide backbone, delta HexA alpha 1-4GlcN alpha 1-4IdoA alpha 1-4GlcN, where delta HexA and IdoA represent 4,5-unsaturated hexuronic acid and L-iduronic acid, respectively, and their structural variations are based upon the positions of sulfate groups. The nine compounds include one hexasulfated, three pentasulfated and five tetrasulfated compounds, and four of them have not been isolated previously as discrete structures. The other two of the 11 tetrasaccharides have the following hitherto unreported structures with novel glucuronate 2-O-sulfate at the internal position: delta HexA(2-sulfate) alpha 1- 4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N-sulfate) and delta HexA(2-sulfate) alpha 1-4GlcN(N,6-disulfate) alpha 1-4GlcA(2-sulfate) beta 1-4GlcN(N,6-disulfate). Thus, 2-O-sulfated glucuronate in the highly sulfated tetrasaccharide structures typical of heparin has been demonstrated. The former and the latter tetrasaccharides account for 0.31 and 0.32% (w/w) of the starting heparin, respectively. Their yield, however, is an underestimation, since these tetrasaccharide structures in longer sequences will be degraded by heparinase. Although the latter tetrasaccharide described above was unexpectedly cleaved by heparinase into two disaccharide units, the former was not degraded by the enzyme most likely due to the lack of the 6-O-sulfate group on the GlcN residue at the reducing terminus. The results indicate its capability of catalyzing both anti and syn elimination, a property shared by heparitinases I and II and chondroitinase ABC. Both tetrasaccharides were degraded into disaccharides by heparitinase II. Therefore, it is necessary to reevaluate the disaccharide composition of heparin/heparan sulfate or oligosaccharide structures, which were previously determined after heparinase or heparitinase II digestion. It is no longer possible to conclude that the 2-O-sulfated unsaturated uronic acid residues obtained from heparin/heparan sulfate by lyase digestions are always derived from iduronate 2-O-sulfate residues in the original polymer. It is quite possible that the novel glucuronate 2-O-sulfate structure in the highly sulfated region of heparin is involved in some of the biological activities of heparin.
A new active site-directed photoaffinity analogue, [beta-32P]5-azido-UDP-glucuronic acid (UDP-GlcA), was enzymatically synthesized from [beta-32P]5-N3UDP-Glc using UDP-glucose dehydrogenase. The product was characterized by its mobility on ion exchange and two thin-layer chromatographic systems, by its UV absorbance at 288 nm, and the loss of this absorbance after UV irradiation of the compound. Photoincorporation of [beta-32P]5-N3UDP-GlcA into bovine liver UDP-Glc dehydrogenase (EC 1.1.1.22) was saturable with an apparent Kd of 12.5 microM, and was inhibited by the known active-site effectors UDP-GlcA, UDP-Glc, and UDP-xylose. When human liver microsomes with known UDP-glucuronosyltransferase (EC 2.4.1.17) activities were photolabeled with [beta-32P]5-N3UDP-GlcA, major photolabeled bands of 35-37 and 50-54 kDa were detected. When rat liver microsomes from phenobarbital-injected rats were photolabeled with [beta-32P]5-N3UDP-GlcA, there was a marked increase in photoincorporation of a 51-kDa protein as compared with control animals. Evidence is presented which suggests that the photolabeled 51-54-kDa proteins in the liver microsomes from both tissues are UDP-glucuronosyltransferase and that [beta-32P]5-N3UDP-GlcA represents a new alternative approach in the study of UDP-glucuronosyltransferase and other UDP-GlcA-utilizing enzymes.
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