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The formation of bilirubin and p-nitrophenyl glucuronides by rat liver

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1. Glucuronide formation of bilirubin and p-nitrophenol in vitro with excess of UDP-glucuronic acid by UDP-glucuronyltransferase from livers of young and adult rabbits was studied. 2. The development of UDP-glucuronyltransferase for the two substrates followed a markedly different pattern during maturation of young rabbits, p-nitrophenol-conjugation ability being much higher at birth than that for bilirubin. 3. Mg(2+) increased bilirubin conjugation, but inhibited p-nitrophenyl glucuronide formation. 4. p-Nitrophenol acted as a potent non-competitive inhibitor for bilirubin conjugation but bilirubin did not affect p-nitrophenyl glucuronidation. 5. The enzyme for bilirubin conjugation was inactivated at pH9 during treatment with snake venom, whereas in the same preparation the activity of the corresponding enzyme for p-nitrophenol was enhanced. In addition, some solubilization of the latter enzyme could be achieved by this method. 6. The possibility of the existence of more than one enzyme system for the formation of O-glucuronides is discussed.

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... Because of its clinical implications the enzyme activity with bilirubin as substrate has attracted particular attention. Initial reports (Isselbacher, Chrabas & Quinn, 1962;Tomlinson & Yaffe, 1966) indicated that attempts to solubilize liver microsomes by a variety of procedures resulted in partial or complete loss of bilirubin glucuronyltransferase activity. Halac & Reff (1967) reported, however, that a relatively stable, solubilized enzyme activity was obtained by dialysis of rat liver microsomes against alkaline EDTA followed by treatment with deoxycholate. ...
... However, glucuronyltransferase activity was inhibited by the greater affinity of ATP for Mg2+ than Ca2+ concentrations of bilirubin above about 0.3mM. suggests that there is another factor too, possibly This has also been observed with rabbit liver the relative affinities of the enzyme for the two microsomes (Tomlinson & Yaffe, 1966). cations. ...
Article
1. It was confirmed that bilirubin glucuronyltransferase can be obtained in solubilized form from rat liver microsomes. 2. Michaelis-Menten kinetics were not followed by the enzyme with bilirubin as substrate when the bilirubin/albumin ratio was varied. High concentrations of bilirubin were inhibitory. 3. The K(m) for UDP-glucuronic acid at the optimum bilirubin concentration was 0.46mm. 4. Low concentrations of Ca(2+) were inhibitory in the absence of Mg(2+) but stimulatory in its presence; the converse applied for EDTA. 5. UDP-N-acetylglucosamine and UDP-glucose enhanced conjugation by untreated, but not by solubilized microsomes. 6. The apparent 9.5-fold increase in activity after solubilization was probably due to the absence of UDP-glucuronic acid pyrophosphatase activity in the solubilized preparation. 7. The activation of solubilized enzyme activity by ATP was considered to be a result of chelation of inhibitory metal ions. 8. The solubilized enzyme activity was inhibited by UMP and UDP. The effect of UMP was not competitive with respect to UDP-glucuronic acid. 9. A number of steroids inhibited the solubilized enzyme activity. The competitive effects of stilboestrol, oestrone sulphate and 3beta-hydroxyandrost-5-en-17-one, with respect to UDP-glucuronic acid, may be explained on an allosteric basis.
... This leads to the phenomenon of latency of UGT activity in preparations of ER (i.e., microsomes). The phenomenon of latency derives from experimental observations that in isolated microsomal preparations, UGTs generally show increased activities after the membranes are physically or chemically disrupted [20][21][22][23][24][25][26][27][28]. Other enzymes present in the lumen of the ER, such as glucose-6-phosphatase, also display latency [29]. ...
Article
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Uridine diphosphate-glucuronosyltransferases (UGTs) are phase 2 conjugation enzymes mainly located in the endoplasmic reticulum (ER) of the liver and many other tissues, and can be recovered in artificial ER membrane preparations (microsomes). They catalyze glucuronidation reactions in various aglycone substrates, contributing significantly to the body's chemical defense mechanism. There has been controversy over the last 50 years in the UGT field with respect to the explanation for the phenomenon of latency: full UGT activity revealed by chemical or physical disruption of the microsomal membrane. Because latency can lead to inaccurate measurements of UGT activity in vitro, and subsequent underprediction of drug clearance in vivo, it is important to understand the mechanisms behind this phenomenon. Three major hypotheses have been advanced to explain UGT latency: compartmentation, conformation, and adenine nucleotide inhibition. In this review, we discuss the evidence behind each hypothesis in depth, and suggest some additional studies that may reveal more information on this intriguing phenomenon.
... occurs at different rates. These rate differences are consistent with the existence of either separate transferase enzymes or a single enzyme with different substrate affinities [1,4,13,14,33]. Thus, the homozygous jaundiced Gunn rat conjugates p-nhrophenol normally but has only a partial capacity to conjugate o-aminophenol and is unable to conjugate bilirubin [6,28,34]. Consequently, measurements of UDP-glucuronyltransferase activity for bilirubin should employ bilirubin as the substrate in an assay system that is optimal for conjugation of that substrate and permits accurate determination of the conjugated products. ...
Article
Extract: Optimal conditions for the in vitro assay of bilirubin uridine diphospho- (UDP) glu-curonyltransferase activity in rat liver microsomes are described. Solvent partitioning was used to separate the conjugated from nonconjugated bilirubin, thus avoiding dependency on the rate of coupling with diazotized sulfanilic acid for the distinction between bilirubin and its conjugated form. The inclusion of uridine diphospho-N-ace-tylglucosamine (UDPNAG) in the reaction mixture permitted the rate of conjugation of bilirubin by fresh rat liver homogenates and microsomes to occur at greater saturation of the available enzyme with the substrates bilirubin and UDP-glucuronic acid. Liver microsomes, isolated in 0.15 M KC1, increased their activity for bilirubin conjugation and decreased their dependency on UDPNAG during the first 10 days of storage at — 15°. Chromatographic separation of the azo pigments of the conjugated bilirubin gave evidence to suggest that bilirubin monoglucuronide was the initial product and bilirubin diglucuronide appeared in increasing amounts in more prolonged incubations. These results suggested that bilirubin monoglucuronide can be intermediate to the formation of bilirubin diglucuronide. Bilirubin UDP-glucuronyltrans-ferase activity in hepatic microsomes of adult homozygous Gunn rats was not demonstrable. In microsomes of heterozygous Gunn rats and normal Wistar and Sprague-Dawley rats bilirubin UDP-glucuronyltransferase activity was found to be 31.0 and 58.0 μg bilirubin conjugated/mg microsomal N/30 min, respectively. Measurements in developing rats indicated that the maturation in enzyme activity occurred by at least two distinct means: increase of specific activity of the microsomes, and an increase in the content of microsomes per gram of liver (Table IV).
Article
Full-text available
Treatment of bovine liver microsomes with a partially purified preparation of phospholipase A from Naja naja venom leads to activation of UDP-glucuronyltransferase with p-nitrophenol as glucuronyl acceptor. This stimulation of activity is due to a 6-fold increase in activity at Vmax. As activity at Vmax increases, there is a progressive decrease in binding affinity of the enzyme for both substrates, and although the enzyme remains stable at 23°, it becomes unstable at 37°. This unstable form of UDP-glucuronyltransferase decays to another stable form with a maximum activity 2.5-fold greater than that of untreated enzyme. As with phospholipase A, treatment with phospholipase C also activates UDP-glucuronyltransferase, but to a lesser extent. In addition to phospholipases other agents which can alter microsomal lipids also activate UDP-glucuronyltransferase. Triton X-100, sonication, and exposure to pH 9.8 increased activity at Vmax and had variable effects on the binding constants for UDP-glucuronic acid and p-nitrophenol. Maximal activation by these treatments was less than that obtained with phospholipase A; no two treatments had similar effects on all kinetic parameters of the enzyme. Nevertheless, additive effects could not be demonstrated. Although Triton stimulated glucuronidation of p-nitrophenol by the enzyme, it was without effect on the reverse reaction. This fact plus the other data indicate that the activation of UDP-glucuronyltransferase in these experiments cannot be attributed to compartmentation of the enzyme but is due to phospholipid-induced alterations of enzyme conformation.
Chapter
It was pointed out by With(122) in his exhaustive monograph on bile pigments that we are indebted to the work of Hans Fischer and his colleagues for much of our knowledge of the bile pigments. Their metabolism, principally that of bilirubin IXa, is of importance to the pediatrician because hyperbilirubinemia is singular among the common symptoms appearing in the newborn in that it in itself, regardless of cause, can produce severe and irrevocable damage to the developing CNS.
Chapter
This chapter discusses UDP-glucuronyltransferase (GT). Important biological compounds, such as bilirubin, steroid hormones, thyroid hormones, phenols produced by the intestinal flora, drugs, and other exogenous substances or their metabolic products, can be conjugated in the organism by GT to glucuronides and excreted in this form in the bile and urine. GT is not present in muscle, spleen, and leucocyte. To determine the activity of this enzyme in very small amounts of tissue, such as that obtained from needle biopsy material, only microanalytical, chemical, or radiochemical methods are suitable. UDPGA solution is stable for only a few days, even in the deep freeze. In duplicate experiments, the accuracy is better than 5%; no statistical evaluation is yet available. The GT activity in human liver is significantly decreased in Gilbert's disease, in cases of Crigler–Najjar disease, in viral hepatitis, and in drug-induced hepatitis, while the activity is only moderately decreased in cirrhosis and steatosis.
Chapter
Drug metabolism refers to any chemical change which a drug undergoes during its sojourn in the body. These chemical changes can involve oxidation, reduction, cleavage (most often hydrolysis) and conjugation (with glucuronic acid, etc.). These chemical changes can have quite different effects on the action of any given drug, and the point I wish to emphasize is that drug metabolism and detoxication are not synonymous. Drug metabolism can cause little or no change in drug action, increase in drug action, or decrease in drug action. Besides these quantitative considerations, metabolism can affect drug action qualitatively—making a stimulant of the central nervous system from an inactive parent molecule or a molecule having a CNS depressant action.
Chapter
Wie der Titel angibt, sollen diejenigen Hyperbilirubinämien behandelt werden, bei denen, auf Grund eines familiären Vorkommens und anderer Kriterien, eine vererbte Störung angenommen werden kann. Außerdem muß die Störung in der Leber lokalisiert sein. Eine Abgrenzung ist heute noch schwierig, weil es erst bei wenigen der in Frage kommenden Syndrome gelungen ist, die eigentliche Ursache mit Sicherheit aufzuklären, z. B. den Defekt des Enzyms Glucuronyltransferase beim Crigler-Najjar-Syndrom. Bei den meisten anderen hereditären hepatischen Ikterusformen ist man nicht in der Lage, etwas Sicheres über die Pathogenese, geschweige denn über die Ursache der Störung auszusagen. Dadurch ist es nicht möglich zu entscheiden, ob klinisch ähnliche, aber nicht ganz identische Krankheitsbilder durch einen einheitlichen Defekt bedingt sind, oder ob sie auf unter-schiedlichen pathogenetischen Mechanismen beruhen (z. B. Dubin-Johnson- und Rotor-Syndrom).
Chapter
Type I: A hereditary disorder of bilirubin metabolism in which bilirubin glucuronyl transferase activity is lacking abd the bile is free of bilirubin glucuronide. Severe unconjugated hyperbilirubinemia usually leads to kernicterus in infancy.
Article
The clearance of bilirubin in the newborn infant presents special problems. Bilirubin is produced more actively than at later ages; maternal inhibitors (presumably progestational hormones) may interfere with hepatic bilirubin metabolism, and deconjugation in the intestine may allow reabsorption of unconjugated pigment. In addition, deficiencies in hepatic uptake, conjugation and excretion may contribute to neonatal hyperbilirubinemia. Hemolytic conditions accelerate the accumulation of bilirubin after birth. Despite the frequent coincidence of these factors in the newborn period, severe hyperbilirubinemia is relatively rare in normal newborn infants. Hepatic processes which are normally inoperative or peripheral in adults may minimize the tendency to bilirubin retention in the immature organism. Alternate pathways of heme catabolism may produce pyrrolic compounds which are less toxic or easier to excrete than bilirubin. During the first few days after birth hepatic glucuronide stores may be conserved by conjugation of bilirubin to its monoglucuronide rather than diglucuronide derivative, or by formation of nonglucuronide conjugates. The development and regulation of bilirubin metabolism has been investigated in the newborn rat. The enzyme system responsible for bilirubin production is fully developed in utero, whereas conjugating activity is poorly developed. After birth heme catabolism is stimulated by heme and by hormones responsive to hypoglycemia, i.e., glucagon and epinephrine. Thus, hemolysis and starvation enhance the capacity for bilirubin production. In contrast, the postnatal development of conjugating activity is retarded by hemolysis and starvation. In addition to hemolysis, hypoglycemia is frequently present in erythroblastosis fetalis. Hypoglycemia may develop in infants of diabetic mothers and in babies whose feedings are delayed after birth. Under these circumstances bilirubin accumulates rapidly in newborn human infants, compared with adults. The metabolic relationships observed in newborn rats may clarify the factors responsible for severe neonatal hyperbilirubinemia in these situations.
Article
The study of glucuronidation reactions began 100 years ago with the isolation of a conjugate of o-nitrotoluene from the urine of dogs. Acid hydrolysis of this conjugate yielded an acidic sugar as one of the products. An identical sugar was obtained on acid hydrolysis of a conjugate of chloral hydrate exreted in human urine. The formula of the reducing sugar was given correctly as $$ {\left( {{\rm{CHOH}}} \right)_4}\left\{ {\begin{array}{*{20}{c}} {{\rm{CHO}}}\\ {{\rm{COOH}}} \end{array}} \right. $$by Schmiedeberg and Meyer in 1879 (Smith and Williams, 1970). A wide variety of organic compounds are now known to be conjugated with glucuronic acid. Predominant among these are aromatic molecules containing phenolic and carboxylic groups which form O-ether and O-ester glucuronides, respectively. Several aromatic thiols are glucuronidated, and N-glucuronides also have been reported (Dutton, 1966). The donor of the glucuronic acid moiety in these reactions was identified as UDP-glucuronic acid (Dutton and Storey, 1953; Dutton, 1966).
Article
Activity of bilirubin glucuronyl transferase in mouse liver homogenates is low at birth, reaches a peak at 14 days of age, 2 to 3 times greater than adult values. Kinetic studies showed marked differences between microsomes derived from adult and 14 day old animals. Michaelis Menten constants [K(m)] were 5.9 x 10-5 M and 4 x 10-4 M respectively at these 2 ages. K(m) values increased from 5.9 x 10-5 M to 6.7 x 10-4 M when enzymes derived from adult animals were solubilized with deoxycholate. In contrast, solubilization did not modify the K(m) values in preparations from 14 day old mice. The newborn K(m) value for unsolubilized enzyme was 8.8 x 10-5 M and 6.7 x 10-4 M for the solubilized preparation. Pretreatment of pregnant animals with barbiturates to induce enzymic activity was necessary for the kinetic studies in the newborn. These findings indicate that different forms of the enzyme exist at the several stages of development studied. Alterations produced by solubilization procedures suggest a common structural form which undergoes alteration during maturation.
Article
Two adult siblings with congenital non-hæmolytic unconjugated hyperbilirubinæmia and glucuronyl-transferase deficiency were treated with sodium phenobarbitone. Bilirubin levels began to fall 12-36 hours after therapy was begun. The brother was treated for eleven days with concomitant, striking reduction in bilirubin level and with a return to former levels after therapy was discontinued. The sister was maintained on therapy with normal bilirubin levels for 5 months; bilirubin levels slowly rose to pretreatment levels during a 5-month tapering of phenobarbitone, but again fell when the dose of phenobarbitone was increased. The mechanism of this phenobarbitone effect upon bilirubin metabolism is unknown. The most intriguing hypothesis is that glucuronyl transferase is enhanced; a second possibility is augmentation of pre-existing alternative pathways of bilirubin metabolism. Changes in sulphobromophthalein-sodium metabolism were also noted during phenobarbitone therapy, possibly indicating a drug effect on the hepatic excretory transport system.
Article
Treatment of rats with phenobarbital stimulates the in vitro activities of hepatic side-chain hydroxylase and aromatic hydroxylase. This effect is paralleled by enhanced in vivo metabolism of toluene and benzene and results in increased tolerance of the rats to the narcotic action of toluene and the leukcopenic action of benzene. No comparable effect of the pretreatment is observed on the rates of oxidation of aromatic alcohol to the corresponding acid, phenolic sulfation, phenolic glucuronidation, or glycine conjugation of benzoic acid. Rapid disappearance of toluene from blood due to enhanced hepatic metabolism together with reduced sensitivity of central nervous system offer an explanation for the shortened sleeping time after toluene injection. The protective effect of phenobarbital against the leukopenic action of benezene is discussed in relation to the etiology of benzene intoxication.
1.1. The relationship between the different activating principles acting of glucuronyltransferase (UDP-glucuronate glucuronyltransferase EC 2.4.1.17) in vitro has been studied using five acceptor substrates (bilirium o-aminophenol, 4-methylumbelliferone, p-nitrophenol and phenolphthalein) in assay of enzyme activity2.2. Preincubation of mouse and rat-liver suspensions in vitro resulted in an increased activity of glucuronyltransferase which was highly variable depending on the acceptor substrate used. While 4-methylumbelliferone glucuronyltransferase exhibited an activity that was 45 times the initial one at maximum, the o-amninophenol enzyme was not activated by this procedure3.3. Detergents (Triton X-roo and digitonin) and UDP-N-acetylglucosamine were also found to activate glucuronyltransferase; only bilirubin glucuronyltransferase was not activated by UDP-N-acetylglucosamine4.4. Activation of glucuronyltransferase could not be increased by combining preincubation, detergents or UDP-N-acetylglucosamine, whereas activation of ratliver o-aminophenol gluconyltransferase by detergents and UDP-N-acetylglucosamine was strongly potentiated by diethylnitrosamine5.5. The slightly different kinetic of nonactivated and activated enzyme that were found at varying substrate (UDP-glucuronic acid, p-nitrophenol concentrations could not explain the activation observed6.6. The results are compatible with an activation by preincubation, detergents and UDP-N-acetylglucosamine due to exposition of active sites of glucuronyltransferases that have been nonfunctioning in unactivated tissue homogenates7.7. The degree of activation at constant detergent concentration was the same over a wide range of enzyme-protein concentrations8.8. The pH optimum for detergent-activated p-nitrophenol glucuronyltransferase was found at 6.2 6.6 while the activity towards bilirubin and o-aminophenol was maximum at pH 7.6. Detergent-activated 4-methylumbelliferone and phenolphthalein glucuronyltransferases, on the other hand, revealed about the same activity in the range between pH 6.2 and 7.6
Article
Flodgaard, H. J. & Brodersen, R. Bilirubin Glucuronide Formation in Developing Guinea Pig Liver. Scand. J. din. Lab. Invest. 19, 149–155, 1967. Formation of bilirubin diglucuronide by guinea pig liver tissue slices and microsomes was studied, using a radioisotope derivative method for the quantitative determinations, at various stages of perinatal development. O-aminophenol inhibits competitively the conjugation of bilirubin and vice versa. From the Km and Ki values it is concluded that bilirubin and OAP are conjugated by two different enzymes. The liver slices rapidly lose UDPGA to the medium, and the rate of bilirubin conjugation decreases. From the course of these processes an approximate value of the Michaelis constant for UDPGA was calculated. The possible role of the low UDPGA content of the liver of the foetus and newborn in the pathogenesis of neonatal jaundice is discussed.
Article
1.1. Solubilization of UDP glucuronosyltransferase (pi-nitrophenol) from rat liver was achieved by digesting microsomes first with trypsin and then with digitonin. Sixty to sixty-five per cent of the enzyme activity was recovered in the solubilized fraction obtained by centrifugation at 105,000 g for 60 minutes. The remaining activity was resedimented.2.2. The frozen solubilized preparation maintained 60–70 % of its activity after two weeks.3.3. The enzyme was further purified by the elution of the solubilized fraction from a column of Sepharose 6B. After gel filtration the increase in the specific activity was 40–fold in the best preparations. This increase in the specific activity was, however, partially due to the activation of the latent enzyme during trypsin-digitonin treatments.4.4. The partially-purified preparation was shown to contain other proteins and membrane phospholipids, which suggests that UDP glucuronosyltransferase may still be part of a complex structure derived from the microsomal membrane.
Chapter
IntroductionBiological Oxidation–Reduction Processes and Oxidative PhosphorylationBiochemistry and Pharmacology of Naturally Occurring Compounds Containing the Nitro or Nitroso GroupBiochemistry and Pharmacology of Synthetic Compounds Containing the Nitro or Nitroso GroupThe Role of Nitro and Nitroso Compounds in the Formation of MethemoglobinAddendum and Final RemarksReferences
Article
1Initial velocity studies for the two-substrate system of bilirubin glucuronyl transferase were performed with Triton X-100 as an activator. These studies were performed by varying the concentration of bilirubin in the presence of different concentrations of uridine 5′-diphosphate glucuronic acid (UDP-GlcUA) and gave an intersecting pattern (characteristic of a sequential mechanism) when the data were graphed as double-reciprocal plots.2Competitive inhibition was observed for UDP-GlcUA and noncompetitive inhibition for bilirubin when UDP (product) or UMP (dead end) were used as inhibitors. Alternate product inhibition with phenolphthalein glucuronide produced noncompetitive inhibition for both UDP-GlcUA and bilirubin.3It was concluded that the reaction under study probably has an ordered mechanism although the iso-Theorell-Chance mechanism is still a possibility.4Kinetic constants (mean for 3 animals) for a sequential mechanism yielded 52.3 μM as the apparent Km for bilirubin, 2.3 mM as the apparent Km for UDP-GlcUA and 20.2 nmol bilirubin conjugated per mg of microsomal protein per 30 min as the V for this reaction.
Article
Glucuronidation of p-aminophenol (PAP), p-nitrophenol (PNP), and bilirubin has been studied in cultures of a clonal cell line with liver-like functions and homogenates from the same cells. The purpose was to study the effect of simultaneous addition of substrates to cultures of living cells and to homogenates with an excess of UDPGA, and to compare the glucuronidation rates in living cells and in homogenates from the same cells, fortified with UDPGA. The rates of glucuronidation of PAP, PNP, and bilirubin by cells in culture were about 50, 70, and 15 nmol/mg protein/hour respectively. In fortified cell homogenates the respective glucuronidation rates were about 500, 1800, and 15 nmol/mg protein/hour. PNP inhibited the glucuronidation of PAP and bilirubin both in cell cultures and cell homogenates. PAP inhibited the glucuronidation of bilirubin in cell cultures. Bilirubin did not inhibit the glucuronidation of the other substrates. PNP glucuronide did not inhibit the glucuronidation of bilirubin or PAP in cell cultures or homogenates.
Article
Neugeborene Kaninchen erhielten Phenobarbital oral in einer Konzentration von 5–7 mg/kg; durch mehrtgige Verabfolgung lie sich eine Aktivittssteigerung der Bilirubin-Glucuronyltransferase erreichen. Eine deutlichere Aktivittszunahme sahen wir nach zustzlicher Phenobarbitalgabe an die Muttertiere einige Tage vor der Geburt. Bei menschlichen Neugeborenen prften wir die Glucuronidierungsfhigkeit fr Paracetamol mit und ohne Phenobarbitalvorbehandlung. Nach mehrmaliger Gabe von 3–5 mg/kg Phenobarbital erfolgte ein Anstieg der Glucuronidierungsrate fr Paracetamol. Es wre vorstellbar, da sich bei menschlichen Neugeborenen auch die Bilirubin-Glucuronyltransferase, wie im Tierversuch, durch therapeutische Phenobarbitaldosen in ihrer Aktivitt vermehren lt.Newborn rabbits were given Phenobarbital in doses of 5–7 mg/kg. Application over several days induced activation of glucuronyl transferase. A further increase in activation could be achieved by treatment of the mother animals for several days before birth. In human newborns we tested the glucuronidation of Paracetamol with and without induction by Phenobarbital. After several doses of 3–5 mg/kg Phenobarbital an increased rate of glucuronidation of Paracetamol was seen. It may be assumed that the glucuronyl transferase for bilirubin can be induced by therapeutic doses of Phenobarbital in human newborn infants in a similar manner to that demonstrated in animal experiments.
The glucuronidation of bilirubin by UDPglucuronyltransferase (EC 2.4.1.17) was investigated using a kinetic assay. The role of albumin in the assay was studied at concentrations of bilirubin both above and below its limit of solubility. In assays saturated with respect to bilirubin, albumin was almost without affect on initial rates. At concentrations of bilirubin below its limit of solubility, albumin was inhibitory. Thus, the bilirubin-albumin complex is not a substrate for the enzyme. In assays containing saturating concentrations of bilirubin, the rate of glucuronidation was influenced by conditions which “salt in” bilirubin. Using a bisubstrate kinetic analysis, the dissociation constant for the enzyme bilirubin complex was determined to be 12–18 μM. The enzyme was shown to be stimulated by the allosteric effector UDP-N-acetylglucosamine which caused an increase in the apparent affinity of the enzyme for UPDglucuronic acid. The bilirubin conjugating form of UDPglucuronyl-transferase showed the same general behavior toward sulfhydryl reagents and perturbers of the lipid environment as previously studied forms. However, because of differences in the response of the rate of conjugation of bilirubin to metals, UDP-N-acetylglucosamine, and sulfhydryl reagents, it has been concluded that the bilirubin conjugating enzyme differs from the p-nitrophenol, o-aminophenol and o-aminobenzoate forms of UDPglucuronyltransferase.
1.1. Purification of UDP-glucuronyltransferase (UDP-glucuronate glucuronyltransferase, EC 2.4.1.17) from guinea pig liver, achieved by ultrasonication, sucrose gradient fractionation and Sephadex G-200 elution, increased the capacity to conjugate bilirubin in vitro 20-fold and 50-fold. The stability of enzymatic activity in this preparation was greater than previously described for this enzyme in preparations with a comparable increase in initial activity and was strikingly similar with both substrates. The purification procedure did not separate conjugating ability for any of the substrates tested.2.2. The inadequacy of gravitational criteria alone in indicating solubilization of a membrane-bound enzyme was demonstrated by electron microscopy. Methods used unsuccessfully in attempting true solubilization are listed.3.3. The purified enzyme preparation was shown to contain a number of proteins including microsomal enzymes and lipids. The possible effect of these on kinetic studies is critically discussed.4.4. Application of the purification procedure to livers of cat, Gunn rat and hypophysectomized or thyroidectomized rats also yielded a stable purified enzyme preparation, without separating conjugating ability for the substrates tested.5.5. The results of these investigations are discussed and their relationship to possible multiplicity of UDP-glucuronyltransferase considered.
1.1. The glucuronide conjugation of bilirubin by rat liver microsoma UDP-glucuronyltransferase (UDPglucuronate glucuronyltransferase (acceptor unspecific), EC 2.4.1.17) was studied; the enzyme preparation was activated by addition of Triton X-100.2.2. when bilirubin was dissolved in NaOH solution or solubilized in an albumin solution, equal initial velocities could be obtained at pH 7.3, but much higher concentrations of albumin-solubilized bilirubin were required for this.3.3. Bilirubin glucuronide conjugation could be inhibited by other substrates such as , 4-methylumbelliferone, phenolphthalein and .4.4. glucuronide conjugation was inhibited by very low concentrations of bilirubin (10 μM) only if bilirubin was added in the absence of albumin. With increasing concentrations of albumin this inhibition disappeared. Increasing concentrations of microsomal enzyme protein also reversed the inhibitory effect of bilirubin. The inhibition appeared to be competitive.5.5. Contrary to most earlier findings, the present results suggest that bilirubin and are conjugated at the same active site.
Article
An enzyme that conjugates the 16alpha-hydroxyl group of oestriol with glucuronic acid was found in the cytosol fraction of human liver. The enzymic activity could not be sedimented when the cytosol fraction was centrifuged at 158000g(av.) for 120min. The oestriol 16alpha-glucuronyltransferase was purified 100-fold by 0-30% saturation of the cytosol fraction with ammonium sulphate followed by filtration of the precipitate through Sephadex G-200. The activity was eluted at the void volume. The product of the reaction, oestriol 16alpha-monoglucuronide, was identified by paper chromatography and by crystallization of radioactive product to constant specific radioactivity. The optimum temperature was 37 degrees C, and the activation energy was calculated to be 11.1kcal/mol. The apparent Michaelis-Menten constants for oestriol and UDP-glucuronic acid were 13.3 and 100mum respectively. Cu(2+), Zn(2+) and Hg(2+) inhibited, whereas Mg(2+), Mn(2+) and Fe(2+) stimulated the enzyme. Substrate-specificity studies indicated that the amount of oestradiol-17beta, oestradiol-17alpha and oestrone conjugated was not more than about 5% of that found for oestriol. Oestriol 16alpha-monoglucuronide, a product of the reaction, did not inhibit the 16alpha-oestriol glucuronyltransferase; in contrast, UDP, another product of the reaction, inhibited the enzyme competitively with respect to UDP-glucuronic acid as the substrate, and non-competitively with respect to oestriol as the substrate. ATP and UDP-N-acetylglucosamine did not affect the oestriol 16alpha-glucuronyltransferase. 17-Epioestriol acted as a competitive inhibitor and 16-epioestriol as a non-competitive inhibitor of the glucuronidation of oestriol. 5alpha-Pregnane-3alpha,20alpha-diol also inhibited the enzyme non-competitively. It is most likely that the oestriol 16alpha-glucuronyltransferase described here is bound to the membranes of the endoplasmic reticulum.
Article
Salicylamide glucuronide (SAMG) in 0-6 and 6-12 hours-urine specimens was determined after oral administration of salicylamide in 7 normal volunteers (NV), in 51 cases of various liver diseases and hyperbilirubinemias, and in 19 cases after drug administration, to predict the in vivo drug metabolism in man and its change by drugs. Maximal glucuronide formation was obtained by 1.0 g of salicylamide administered to NV; thus, this dosage was used in the present study. SAMG as percent of total salicylamide, the percent of SAMG, from 0-6 hours-urine specimens was high and constant in NV (71.3 +/- 8.3 (Mean +/- S.D.)). 0-0.08% of the total salicylamide was confirmed as free salicylamide in 0-12 hours-urine specimens of NV. The percent of SAMG of 0-6 hours-urine specimens was 57.2 +/- 8.6 in acute hepatitis, 66.6 +/- 10.9 in chronic hepatitis, and 48.6 +/- 10.7 in liver cirrhosis (mean +/- S.D.). Free salicylamide increased slightly in liver diseases. Serum bilirubin levels tended to be inversely correlated with the percent of SAMG. In most cases of Gilbert's syndrome, the percent of SAMG remained at a normal level. The percent of SAMG in cases with unconjugated hyperbilirubinemias of other geneses were almost within normal limits. Bucolome and phenobarbital increased the percent of SAMG in patients with various liver diseases. After rifampicin or phenytoin administration, the percent of SAMG of the patients with lung tuberculosis or epilepsy did not surpass that of NV.
Article
Bilirubin UDP-glucuronyltransferase and UDP-xylosyltransferase activity could already be demonstrated in rat liver from day 19 of fetal life onwards (4 days before birth). Bilirubin-glucuronide was present in hepatocytes of 21-day-old fetal liver strongly suggesting that the enzyme detected in vitro was really active in vivo. This further supports the theory that secretion from the cell is also immature. The known deficiency of UDP-glucose dehydrogenase in fetal and neonatal rat liver could lead to decreased intracellular concentrations of UDP-glucuronic acid and possibly to increased concentrations of UDP-glucose. However, no glucosides were present in the fetal hepatocytes.
Article
[3H]epinephrine was injected into fetal rabbits through the uterine wall on day 26 of gestation in order to study placental transfer, and the uptake, retention and metabolism of the hormone in fetal tissues. Less than 0.1 per cent of the administered [3H] was found in maternal blood or within uninjected littermates. Accumulation of [3H]/g of tissue was greatest in the fetal kidney, liver and intestine, and less in the heart and lung. Levels of [3H] in the fetal serum, heart, lung and placenta were comparable in value at 5 and 30 min, but all fell by 60 min. In contrast, the fetal kidney and intestine show a 2- to 4-fold accumulation of [3H] in 30 min as compared to 5 min, while the fetal liver exhibits a 10-fold accumulation over 30 and 60 min. [3H] metabolites present in fetal serum at 30 min, expressed as a per cent of the total radioactivity, are 45 per cent for metanephrine and 27 per cent for O-methylated deaminated products, while 25 per cent is present as unmetabolized [3H]epinephrine. O-methylated metabolites are predominant in the liver, where 85 per cent of the radioactivity comprises metanephrine in conjugated and unconjugated forms. Administration of promethazine to the doe prior to injection of [3H]epinephrine into rabbit fetuses resulted in a 40–64 per cent decrease in accumulation of [3H] in the fetal brain, liver, lung, kidney, intestine and heart. The pattern of metabolites in the serum of promethazine-treated fetuses shows an increase in the per cent of [3H] in the O-methylated, deaminated fraction from 27 to 37 per cent. While the total per cent of O-methylated metabolite is not altered in the liver of treated fetuses, metanephrine is predominantly present in the conjugated form. The liver of the rabbit fetus is an important site for accumulation of catecholamine metabolites and is pharmacologically sensitive to promethazine administered to the mother.
Article
A rapid and sensitive method is described for assaying UDP-glucuronyl-transferase (EC 2.4.1.17) activity. The substrate is the relatively apolar phenolphthalein, labelled with 125I on one of its two phenolic rings leaving a free hydroxyl group for glucuronidation. Extraction with ethyl acetate leaves the glucuronide in the aqueous reaction medium which is then counted. Less than 10 mug microsomal protein is required and the glucuronidation of 10 ng 125I-labelled phenolphthalein (0.03 nmoles) is easily detected.
Article
Novobiocin inhibited bilirubin UDP-glucuronosyltransferase (EC 2.4.1.17) from rat liver both in vitro and in vivo, in a dose-dependent fashion. This inhibition was immediate, and was fully reversed when novobiocin was removed by dialysis or by ultracentrifugation through 0.6 M sucrose. The inhibition could not be explained by an alteration in the membrane conformation of this enzyme, since the same kinetic changes were observed in digitonin-activated and in non-activated microsomes. Novobiocin exerted a non-competitive inhibition of bilirubin UDP-glucuronosyltransferase with either bilirubin or UDP-glucuronic acid as the substrate. Kinetic studies demonstrated uncompetitive inhibition of novobiocin or bilirubin UDP-glucuronosyltransferase as a function of Mg2+ concentration, whether the assays were EDTA-free or not. Thus, similarities seem to exist between the known effect of novobiocin on membrane-bound enzymes of the bacterial wall and its inhibitory effect on bilirubin UDP-glucuronosyltransferase: both these enzymic systems require metal divalent cations for maximal activity. The uncompetitive inhibition pattern observed with novobiocin with regard to Mg2+ suggests that this antibiotic acts on bilirubin conjugation by affecting Mg2+-enzyme complexes.
Article
The addition of digitonin or other detergents to liver microsomes from control and phenobarbital-treated rats increased the rate of glucuronidation of bilirubin, testosterone, estradiol, estrone and estriol. The extent of activation by digitonin as well as the concentration of detergent causing maximal activation was substrate dependent. Treatment of rats with sodium phenobarbital (75 mg/kg/day, i.p.) for 3 days significantly increased the glucuronidation of testosterone and estradiol per mg of protein but had no effect on estrone glucuronidation. Administration of 0.1% sodium phenobarbital in the drinking water for 20 days increased bilirubin, testosterone and estrone glucuronidation per mg of protein, but had little or no effect on estradiol and estriol glucuronidation. When UDP-glucuronyl transferase activity was calculated on a per g liver (wet wt) basis, however, an increase in the glucuronidation of estradiol and estriol was seen; this increase could be accounted for by a large stimulatory effect of phenobarbital on the synthesis of total microsomal protein. Liver microsomes from homozygous Gunn rats, which are deficient in the ability to conjugate bilirubin, did conjugate testosterone, estradiol. estrone and estriol, but some substrates were conjugated more rapidly than others. The addition ofdigitonin to liver microsomes from the homozygous Gunn rat increased the glucuronidation of testosterone but not that of estrone. Microsomal steroid glucuronidation in this animal was stimulated to varying degrees by the administration of sodium phenobarbital. The data suggest the presence of several glucuronyl transferases in liver microsomes.
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How closely the isolated liver of the rat would simulate the in vivo function of the organ in terms of the metabolic pattern of the compounds such as bromosulphophthalein, p-nitrophenol, hexobarbital, and indocyanine green was investigated. In order to produce tissue with the stimulated function, the animal was pretreated with phenobarbital and, for the reverse purpose, with ethionine. Some of the indices of the function employed herein, such as the appearance pattern of the compound, the rate of biochemical transformation or the biliary excretion, showed that the perfused liver would generally well reflect the in vivo situation. The method with isolated and perfused liver could exclude the participation of other organs and also the influence of the factors unavoidable in an in vivo experiment. Thus, it is suggested that the isolated perfused liver is useful for studying directly the functional level of the organ as drug metabolizing tissue.
Article
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The temperature dependence of two kinetic properties of the GT2P isoform of microsomal UDP-glucuronosyltransferase was studied for enzyme in intact microsomes and for pure enzyme reconstituted into different types of lipid bilayers. The properties studied were the non-Michaelis-Menten kinetics of the enzyme and activity at Vmax(app). For enzyme in intact microsomes, the pattern of non-Michaelis-Menten kinetics was seen at all temperatures in the range tested (23 to 48 degrees C), and the slopes of the Hill plots of the data were constant across this range of temperatures. Although non-Michaelis-Menten kinetics were present for pure enzyme in bilayers of 1,2-dimyristoylphosphatidylcholine or 1,2-dipalmitoylphosphatidylcholine only in the gel phase (Hockman, Y., Kelley, M., and Zakim, D. (1983) J. Biol. Chem. 258, 6509-6519), it was not possible to reconstitute this pattern of kinetics for enzyme at T greater than 40 degrees C. For example, GT2P displayed Michaelis-Menten kinetics in bilayers of 1,2-distearoylphosphatidylcholine at 44 degrees C. For enzyme in microsomes, activities at Vmax(app) increased with increasing temperature in the range 23 to 48 degrees C, with a discontinuity in the slope of the Arrhenius plot at 34 degrees C. This thermotropic property also could not be reconstituted with pure GT2P. Instead, activities at Vmax(app) for GT2P reconstituted in 1,2-dioleoylphosphatidylcholine, 1,2-distearoylphosphatidylcholine, or 1,2-stearoyl oleoylphosphatidylcholine increased in the range 23 to 37 degrees C, but then decreased at T greater than 37 degrees C. The fall in activity at T greater than 37 degrees C was reversible, indicating that GT2P undergoes a reversible change at 37 degrees C to a less active form of the enzyme. The differences between pure, reconstituted GT2P and microsomal GT2P indicate that the thermotropic properties of the microsomal enzyme are not properties of the enzyme per se but depend on interactions between it and other components in the microsome. The data suggest, therefore, that the properties of GT2P in microsomes results in part from an organization of components in the microsomal membrane.
Article
Divalent metal ions have two important effects on the properties of UDP-glucuronyltransferases. They alter the properties of the active sites of these enzymes and modify the effect of UDP-N-acetylglucosamine on their activities. The metal ion responsible for effects on the active site is different from the metal ion which modifies the properties of the allosteric site (UDP-N-acetylglucosamine site); that is, there are at least two metal binding sites in some forms of UDP-glucuronyltransferase. The effects of metal ions also differ, according to the compound used as glucuronyl acceptor. By studying the effects of metal ions on the rates of synthesis of p-nitrophenyl, o-aminobenzoyl, and o-aminophenyl glucuronides, three types of UDP-glucuronyltransferase reactions can be distinguished. In the type I reaction, for which p-nitrophenol serves as the prototype aglycone, divalent metal jons increase activity at Vmax, and are essential for stimulation by UDP-N-acetylglucosamine. In contrast to the type I reaction, divalent metal ions are not essential for stimulation of UDP-glucuronyltransferase by UDP-N-acetylglucosamine with o-aminophenol as aglycone (type Ia reaction). Divalent metal ions, however, increase activity at Vmax for the type Ia reaction. In the type II reaction, for which o-aminobenzoate is the prototype aglycone, metal ions decrease the concentrations of UDP-glucuronic acid needed for half-maximal rates of glucuronidation and are essential for stimulation by UDP-N-acetylglucosamine. Metal ions do not increase activity at Vmax. for the type II reaction. Interactions between metal ions and UDP-N-acetylglucosamine seem to be essential components of the regulatory apparatus of UDP-glucuronyltransferase catalyzed reactions since these interactions ensure rapid rates of glucuronidation under conditions in vivo.
Article
This chapter describes the methodology and properties of uridine diphosphate (UDP)-glucuronyltransferase and of the related UDP-glycosyltransferase activities. It also delineates the applications to human disease. Bilirubin derives largely from senescent erythrocyte hemoglobin. In most normal adult animals, conjugation of bilirubin with various sugars represents the major mechanism for its transformation. The reaction products are eliminated from the organism by excretion into the bile. Glucuronyl transfer is of predominant importance in the biotransformation of bilirubin in man and rat. Glucuronyl transference to bilirubin and to many other acceptor substrates (G5, L14, M14, P4, W4), with the exception of phenolphthalein (G5), is higher in the rough endoplasmic reticulum than it is in the smooth endoplasmic reticulum. The main field of application of the in vitro assays of bilirubin UDP glycosyltransferase activities is the evaluation of stimulation or inhibition—by drugs or endogenous substances—of the enzyme activities or of their maturation. Development of unconjugated hyper-bilirubinemia in some neonates is related to breast feeding.
Article
The hepatic enzyme, bilirubin UDP glucuronyltransferase (UDPglucuronate glucuronyltransferase, EC 2.4.1.17) has been solubilized in stable form by treating rat liver microsomes with 10% (by weight) dissolved digitonin at pH 7.8. All enzyme activity was associated with the rough membranes of the endoplasmic reticulum as shown by ultracentrifugation of selected density gradients in combination with electron microscopy. Proof of solubilization was determined by analytic ultracentrifugation, gel filtration, and electron microscopy which indicated that the enzyme was an homogeneous protein with a calculated molecular weight in the range of 150 000. Studies to characterize enzyme kinetics and assay requirements were performed and differ from those reported for liver hemogenates.
Article
In rat liver microsomal preparations, bilirubin markedly stimulated the glucuronidation of morphine and p-nitrophenol catalyzed by UDPglucuronyltransferase (UDPGT, EC 2.4.1.17). The activation was not due to contamination of bilirubin with bile acids. At equimolar concentrations, the activating effect of bilirubin was greater than that produced by deoxycholate, a detergent well known as an activator of UDPGT. Other results suggest that bilirubin activation of UDPGT is similar to that produced by detergents. In experiments, the rate of urinary excretion of morphine glucuronide in rats treated with bilirubin was twice that of control animals. These results suggest that bilirubin may be a physiologic activator of UDPGT activity.
Article
1.1. UDP-glucuronyltransferase activity towards oestriol has been demonstrated in liver preparations from foetal and adult human and mouse, and from chick embryo and chick. It was also found in foetal human kidney and gut, but appeared absent from placenta.2.2. In human foetal liver and kidney levels of UDP-glucuronyltransferase activity towards oestriol were as low as those towards o-aminophenol, and developed with age.3.3. No UDP-glucosyltransferase activity towards oestriol was detected in human foetal preparations.4.4. UDP-glucuronyltransferase activity towards oestriol develops precociously in cultures of chick embryo liver; in cultures of human foetal liver the transferase activity towards oestriol declines whilst increasing towards o-aminophenol.5.5. Pre-treatment with phenobarbital increased UDP-glucuronyltransferase activity towards oestriol in chick embryo liver, both in ovo and in culture. Pre-treatment with phenobarbital increased this activity in maternal mouse liver but not in foetal liver, in utero or in culture, nor in cultured human foetal liver.6.6. UDP-glucuronyltransferase activities towards oestriol and towards o-amino phenol differ markedly in development, in culture and in response to phenobarbital pre-treatment and activation procedures; heterogeneity of the enzyme is probably responsible.
Article
The microsomal glucuronyltransferase catalyzing formation of p-nitrophenyl glucuronide in rat, guinea pig, and mouse livers, and transplantable rat hepatomas, was activated spontaneously during storage at 0 ° and by low concentrations (less than 0.05%) of deoxycholate and Triton X-100 added in vitro. Activity in liver microsomes was increased 5- to 10-fold, in hepatoma microsomes 20-fold, and in homogenates 2-to 5-fold. Higher concentrations of detergent were inhibitory to activity in fresh preparations, and preparations which had been fully activated spontaneously were inhibited by all concentrations of detergents. The activation was irreversible and not accompanied by solubilization of the enzyme. The Michaelis constants (for uridine diphosphate glucuronic acid) for detergent- and spontaneously activated liver and hepatoma microsomes appeared to be the same. Attempts to determine the mechanism of activation by kinetic analysis were unsuccessful, but the data were compatible with an increase in the relative site number per milligram microsomal protein during activation.
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After the intraportal injection of retinol-6,7-¹⁴C to rats, the 0-ether derivative of retinol, retinyl β-glucosiduronate, appears in the bile. Both retinoyl β-glucuronide and retinyl β-glucosiduronate are also synthesized in vitro when washed rat liver microsomes are incubated with uridine diphosphoglucuronic acid (UDPGA) and either retinoic acid or retinol, respectively. The synthesis of retinoyl β-glucuronide was also demonstrated in microsomes of the kidney and in particulate fractions of the intestinal mucosa. The glucuronides were characterized by their UV absorption spectra, by their quenching of UV light or fluorescence under it, by their thinlayer chromatographic behavior in two solvent systems, and by the identification of products released during their hydrolysis by β-glucuronidase. With retinoic acid as the substrate, the UDP glucuronyl transferase of rat liver microsomes had a pH optimum of 7.0, a temperature optimum of 38°C, and a marked dependence on the concentrations of both retinoic acid and UDPGA, but was unaffected by a number of possible inhibitors, protective agents, and competitive substrates. The conversion of retinal to retinoic acid and the synthesis of retinoyl β-glucuronide from retinoic acid could not be detected in whole homogenates, cell fractions, or outer segments of the bovine retina.
Article
The influence of phenobarbital pretreatment on the glucuronidation of and bilirubin has been compared in liver microsomes from Wistar rat, homozygous and heterozygous Gunn rat and cat. Both for Wistar rat and cat it appeared that phenobarbital has a different effect on and bilirubin glucuronidation. No bilirubin glucuronidating activity could be detected in homozygous Gunn rats both before and after exposure to phenobarbital. The glucuronidating capacity of Gunn rats, however, was strongly enhanced after phenobarbital treatment. These results are arguments in favour of the involvement of different enzymes in the glucuronidation of and bilirubin.
Article
1. The bilirubin UDP-glucuronyltransferase assay described by Van Roy & Heirwegh (1968) has been improved. 2. Extraction of final azo-derivatives is rendered more simple and efficient by thorough emulsification and by cooling. 3. Pretreatment of homogenates and cell fractions with digitonin increases the sensitivity of the assays and gives less variable results than those with untreated preparations. The activation procedure is flexible. 4. Blank values (obtained from incubation mixtures from which activating bivalent metal ion and UDP-glucuronic acid were omitted) are low. No endogenous conjugate formation could be detected except with untreated, fresh liver homogenates. Control incubation mixtures containing the latter preparations are preferably kept at 0 degrees C. 5. With activated microsomal preparations, rates of breakdown of UDP-glucuronic acid (as monitored by release of P(i)) were low. Little if any increase in enzyme activity was found when UDP-N-acetylglucosamine was included in the incubation mixtures. 6. Slight deviation from Michaelis-Menten kinetics with respect to bilirubin observed at low substrate concentrations is probably related to the use of binding protein in the assay mixtures. Michaelis-Menten kinetics were followed with respect to UDP-glucuronic acid. Part of the enzyme in microsomal preparations from rat liver functioned independently of added bivalent metal ions. Mn(2+) was slightly more, and Ca(2+) somewhat less, stimulatory than Mg(2+). The Mg(2+)-dependent fraction showed Michaelis-Menten kinetics with respect to the added Mg(2+). 7. The enzyme activities found were higher than values reported in the literature for untreated or purified preparations from rat liver. They were above reported values of the maximal biliary excretion rate of bilirubin.
Article
This chapter discusses the physiology and metabolism of vitamin A. In modern biology, vitamin A possesses a feminine-like charm. It is unique structurally, is essential nutritionally, is elegant in its photoreceptor role, and yet is tantalizingly mysterious in its other biological involvements. The demonstration that β-carotene is cleaved into two molecules of retinal by a cytoplasmic enzyme of the intestine and liver lays to rest the old and frustrating controversy concerning the mechanism and mode of provitamin A transformation. The rapid hydrolysis and reesterification of vitamin A ester has been demonstrated quantitatively in which the specificity of the esterification reaction for long-chain saturated fatty acid has been reaffirmed. Retinol is mainly transported as an ester in the chylomicra of the lymph, but a significant, minor portion is apparently oxidized and transported via the portal blood. The puzzling rapid disappearance of vitamin A acid after its injection into animals has been resolved by the demonstration that retinoyl β-glucuronide is rapidly formed and excreted in the bile. As retinyl β-glucuronide, the O-ether derivative of retinol, is formed after the administration of retinol, retinoic acid is not an obligatory intermediate in retinol metabolism.
Article
The capacity of liver fractions to conjugate bilirubin and p-nitrophenol with glucuronic acid is increased several fold by dialysis against alkaline EDTA. UDPglucuronyltransferase (UDP-glucuronate glucuronyltransferase (acceptor-unspecific), EC 2.4.1.17), with activity towards bilirubin and p-nitrophenol, has been solubilized by treating microsomes activated with EDTA and deoxycholate.Kinetic data obtained with activated microsomes, and fractionation and inactivation experiments performed with deoxycholate-solubilized enzyme have yielded evidence in favor of the existence of at least two UDP-glucuronyltransferases.Microsomal pellets were examined by electron microscopy before EDTA, after EDTA, and after EDTA and deoxycholate. The most apparent change that is concomitant with the activation by EDTA is the transformation of rough membranes into smooth membranes.
Article
Treatment of lyophilized human liver microsomes with deoxycholate led to “solubilization” of oestriol 16α-glucuronyltransferase. The enzymic activity was stable in the presence of dithio-threitol and EDTA. A five fold enrichment of enzyme activity was obtained after filtration of the “solubilized” enzyme through Sepharose 4B. Phospholipase C reduced the enzyme activity by 70%, lysolecithin restored the activity to 75%. Phosphatidylcholine and sphingomyelin were found to be the major phospholipids in the partially purified oestriol 16α-glucuronyltransferase. From the ratio of void volume to elution volume, the apparent molecular weight of the active fraction was found to be approximately two million. These results suggest that the oestriol 16α-glucuronyltransferase “solubilized” from human liver microsomes may be a lipid-protein complex.
Article
CONGENITAL, non-obstructive, non-hæmolytic jaundice is a syndrome characterized by marked elevation of `indirect-reacting' (free) bilirubin in the blood1. Recent studies have shown that bilirubin forms a polar glucuronide which is readily excreted in the bile1. The synthesis of bilirubin glucuronide has been found to be catalysed by an enzyme system in the microsomes of the liver that transfers glucuronic acid from uridine diphosphate glucuronic acid to a Suitable aglycone2. We wish to describe experiments showing that the accumulation of free bilirubin in this syndrome is the result of a defect in the glucuronide-forming mechanism.
Article
Hepatic injury was produced in rats by a protein-deficient diet and by acute and chronic CCI 4 poisoning. Glucuronyl transferase activity in liver homogenates was measured and compared with that in a control group of animals. The homogenates were incubated in a suitable medium with uridine diphosphoglucuronic acid and with both bilirubin and phenolphthalein as acceptors of glucuronide. The glucuronide conjugation of bilirubin was significantly decreased by both nutritional cirrhosis and by acute and chronic CCI 4 toxicity. The conjugation of phenolphthalein was not affected by nutritional liver injury, but was markedly diminished by CCI 4 damage. The recovery after acute CCI 4 poisoning was more rapid for bilirubin conjugation than for phenolphthalein conjugation. The differences between bilirubin and phenolphthalein conjugation suggest that in the presence of liver damage the rate of bilirubin glucuronide synthesis should not be inferred from studies using other glucuronide acceptors, particularly phenolphthalein.
Article
1. Glucuronide synthesis from uridine diphosphate glucuronate and o-aminophenol or p-nitrophenol in the presence of uridine diphosphate transglucuronylase of mouse-liver homogenates has been studied with respect to inhibition by compounds known to be conjugated under the experimental conditions, and also by thiophenol. 2. Raising the o-aminophenol concentration decreased the inhibition of o-aminophenyl glucuronide synthesis by the alternative glucuronyl acceptors phenol, menthol and benzoic acid, but was without effect on that caused by p-nitrophenol and thiophenol. 3. Raising the p-nitrophenol concentration decreased or abolished the inhibition of p-nitrophenyl glucuronide synthesis due to phenol, menthol, benzoic acid, anthranilic acid, o-aminophenol and thiophenol. 4. The o-aminophenol system was much more readily inhibited by all compounds than the p-nitrophenol system. 5. In tris buffer, pH7.4, over 30% activation of the o-aminophenol system was achieved by 2mm-Mg(2+), but 10mm-Mg(2+) was inhibitory. The p-nitrophenol system showed only inhibition from 2mm-Mg(2+) upwards. 6. The results are discussed as suggesting that there are at least two uridine diphosphate transglucuronylases.
Article
1. A study of the catalysis of the formation of the glucuronides of o-aminophenol and p-nitrophenol by the uridine diphosphate transglucuronylase of homogenates of female mouse liver has been made, with reference to the effect of reagents reacting with thiol groups. 2. The synthesis of both glucuronides was completely inhibited by organic mercurials and N-ethylmaleimide. The inhibition was only partial with arsenite and the arsenoxides, iodoacetamide and o-iodosobenzoate. 3. The o-aminophenol system was much more sensitive than that for p-nitrophenol to all the thiol reagents, except N-ethylmaleimide, which was equally active in both systems. 4. At very low concentrations of the organic mercurials, the o-aminophenol system was activated. 5. With o-aminophenyl glucuronide formation, complete protection was given by glutathione and cysteine against the organic mercurials, N-ethylmaleimide and iodoacetamide, and partial protection against the arsenicals. Reversal was complete against the mercurials, and very limited against the arsenicals and iodoacetamide. The effects of N-ethylmaleimide and o-iodosobenzoate were irreversible. Results with p-nitrophenol were very similar. 6. Uridine diphosphate transglucuronylase was partially protected against p-chloromercuribenzoate and lewisite oxide by uridine diphosphate glucuronate, but not by o-aminophenol. 7. Glutathione did not prevent the decline in the rate of conjugation of o-aminophenol when homogenates were aged by incubation at 30 degrees . Cysteine was unable to prevent or reverse inactivation by ultrasonic radiation.
Article
Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical pro- cedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10). Although the reagent would seem to be recommended by its great sen- sitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of react- ants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances. Procedures are described for measuring pro- tein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein.
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