Substrate specificity and properties of uridine diphosphate glucuronyltransferase purified to apparent homogeneity from phenobarbital-treated rat liver.

1. The purification to homogeneity of stable highly active preparations of UDP-glucuronyltransferase from liver of phenobarbital-treated rats is briefly described. 2. A single polypeptide was visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, of mol.wt.57000. 3. Antiserum raised against the pure enzyme produces a single sharp precipitin line after Ouchterlony double-diffusion analysis. 4. The pure UDP-glucuronyltransferase isolated from livers of untreated and phenobarbital-pretreated rats appears to be the same enzyme. 5. The Km (UDP-glucuronic acid) of the pure enzyme is 5.4 mM. 6. The activity of the pure enzyme towards 2-aminophenol can still be activated 2-3-fold by diethylnitrosamine. 7. UDP-glucose and UDP-galacturonic acid are not substrates for the purified enzyme. 8. The final preparation catalysed the glucuronidation of 4-nitrophenol, 1-naphthol, 2-aminophenol, morphine and 2-aminobenzoate. 9. Activities towards 4-nitrophenol, 1-naphthol and 2-aminophenol were all copurified. The proposed heterogeneity of UDP-glucuronyltransferase is discussed.     

Studies of the catalytic mechanism of microsomal UDP-glucuronyltransferase. Alpha-glucuronidase activity and its stimulation by phospholipids

The rate at which a specific, purified form of microsomal UDP-glucuronyltransferase (designated as the GT2P type of this enzyme) catalyzes the hydrolysis of UDP-glucuronic acid was measured with pure, delipidated enzyme and enzyme reconstituted with different lysophosphatidylcholines. This activity of the GT2P type of UDP-glucuronyltransferase is referred to as alpha-glucuronidase activity. For delipidated enzyme, the rate of hydrolysis of UDP-glucuronic acid catalyzed by GT2P extrapolated to infinite concentrations of UDP-glucuronic acid was 1 X 10(-9) mol/min/mg of protein. This compares with a rate of glucuronidation of p-nitrophenol of 96 X 10(-9) mol/min/mg of enzyme, for delipidated enzyme. Addition of oleoyl- or myristoyllysophosphatidylcholine to GT2P did not affect the alpha-glucuronidase activity significantly. This activity was stimulated, however, in the presence of compounds that bind at the aglycone site but that do not undergo glucuronidation. alpha-Glucuronidase activity extrapolated to infinite concentration of UDP-glucuronic acid was 4.0 X 10(-9) mol/min/mg for delipidated enzyme assayed in the presence of less than saturating concentrations of p-nitrophenyl phenyl ether. Moreover, when the aglycone site of GT2P was occupied by ethers, the alpha-glucuronidase activity of this enzyme was enhanced by addition of phospholipids to delipidated enzyme. The extent of activation of the alpha-glucuronidase activity of GT2P, when the aglycone site was occupied, depended on the acyl chain of the lipid added to delipidated enzyme. These data indicate that the GT2P form of UDP-glucuronyltransferase catalyzes the hydrolysis of UDP-glucuronic acid at a significant rate and that lysophosphatidylcholines can influence this rate.     

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction

After force-feeding a protein-free diet to male rats for 5-7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not significantly alter the ratio of these major membrane components. Protein deficiency profoundly altered the phospholipid composition of microsomal membranes. The most striking difference in microsomal phospholipid composition between control and protein-deficient rats was their content of lysophosphatides. Whereas microsomal membranes from protein-deficient rats contained significant proportions of lysophosphatidylcholine and lysophosphatidylethanolamine very little or no lysophosphatides were detected in control preparations. Pretreatment of microsomal fractions from normal rats with phospholipase A markedly increased their UDP-glucuronyltransferase activity as did their pretreatment with lysophosphatidylcholine. It is concluded that the quantities of lysophosphatides present in microsomal membranes from protein-deficient rats were sufficient to have caused the increased UDP-glucuronyltransferase activities of these preparations. Evidence is presented suggesting that these changes in microsomal phospholipid composition and UDP-glucuronyltransferase activity caused by protein deficiency reflect changes that occur in vivo. The possible physiological significance of these findings is discussed.     

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Phospholipid depletion and re-activation of guinea-pig liver microsomal enzyme.

More than 80% of the phospholipid component of guinea-pig liver microsomal membranes (prepared with 154mM-KCl) was removed by treatment with phospholipase A followed by extraction of the lysophosphatides and fatty acids produced with albumin. Delipidation strongly inactivated the highly active UDP-glucuronyltransferase of these preparations and activity was restored by mixtures of phosphatidylcholine and lysophosphatidylchlone. However, small quantities of lysophosphatides were still associated with the delipidated fractions after extraction with albumin and might have influenced the inactivation and re-activation observed. To eliminate these uncertainties, microsomal proteins and phospholipids were separated by gel filtration on Sephadex G-150 in the presence of cholate. This technique also strongly inactivated the enzyme but did not generate membrane-active phospholipid degradation products. High transferase activity was again restored to the delipidated protein by choline glycerophosphatides. These results confirm the view that the fully active form of microsomal UDP-glucuronyltransferase is phospholipid-dependent.     

Development of uridine diphosphate-glucuronyltransferase activity in cultures of chick-embryo liver

1. The liver of the domestric fowl (Gallus gallus) remains capable of conjugating o-aminophenol with glucuronic acid after 8 days' culture. The pathway of o-aminophenyl glucuronide formation in cultured liver, as in fresh tissue, includes the enzyme UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activity in chick-embryo liver increases on culture from very low to adult values within 6-8 days. 3. The development of UDP-glucuronyltransferase activity in cultured chick-embryo liver requires certain serum factors in the medium. The requirements change with embryo age. Liver from embryos younger than 15 days develops enzyme activity equally well in media containing either foetal or adult serum; liver from embryos older than 16 days develops activity only with adult serum. The development of enzyme activity in liver from the older embryos appears to be stimulated by diffusible factors in adult serum and inhibited by diffusible factors in foetal serum. It is suggested that the stimulation and inhibition of enzyme formation by small, diffusible molecules may be part of the mechanism regulating UDP-glucuronyltransferase activity in vivo. 4. Liver from 19-day-old chick embryos cultured with foetal serum begins to develop UDP-glucuronyltransferase activity if transferred to an adult-serum medium. Its capacity to develop UDP-glucuronyltransferase activity in adult serum survives in a foetal-serum medium for at least 5 days, the longest period tested. 5. The activity of UDP-glucuronyltransferase reached in 19-day chick-embryo liver after 1 or 2 days with adult serum is maintained without further increase after transfer to a foetal-serum medium. After 3 days with adult serum UDP-glucuronyltransferase activity continues to increase when the tissue is transferred to a foetal-serum medium. Thus liver from 19-day-old embryos requires 3 days with adult serum before development of enzyme activity becomes independent of a continuous adult-serum environment.     

Altered sexual differentiation of hepatic uridine diphosphate glucuronyltransferase by neonatal hormone treatment in rats

The hepatic microsomal enzyme UDP-glucuronyltransferase undergoes a complex developmental pattern in which enzyme activity is first detectable on the 18th day of gestation in rats. Prepubertal activities are similar for males and females. However, postpubertal sexual differentiation of enzyme activity occurs in which male activities are twice those of females. Neonatal administration of testosterone propionate or diethylstilboestrol to intact animals resulted in lowered UDP-glucuronyltransferase activity in liver microsomal fractions of adult male rats, whereas no changes were observed in the adult females and prepubertal male and female animals. Neonatal administration of testosterone propionate and diethylstilboestrol adversely affected male reproductive-tract development as evidenced by decreased weights of testes, seminal vesicles and ventral prostate. Diethylstilboestrol also markedly decreased spermatogenesis. Hypophysectomy of adult male rats resulted in negative modulation of microsomal UDP-glucuronyltransferase and prevented the sexual differentiation of enzyme activity. In contrast hypophysectomy had no effect on female UDP-glucuronyltransferase activity. A pituitary transplant under the kidney capsule was not capable of reversing the enzyme effects of hypophysectomy, therefore suggesting that the male pituitary factor(s) responsible for positive modulation of UDP-glucuronyltransferase might be under hypothalamic control in the form of a releasing factor. Neonatal testosterone propionate and diethylstilboestrol administration apparently interfered with the normal sequence of postpubertal UDP-glucuronyltransferase sexual differentiation.     

Reactivation of lipid-depleted Ca2+-ATPase by a nonionic detergent.

The Ca2+-ATPase of sarcoplasmic reticulum can be reversibly delipidated by precipitation with polyethyleneglycol in the presence of deoxycholate and glycerol to as low as 4 mol of phospholipid/mol of enzyme polypeptide and can then be reactivated to 90% of its original ATPase activity by the addition of phosphatidylcholine. Furthermore, the preparation exhibits nearly the same activity if the nonionic detergent dodecyl octaoxyethyleneglycol monoether is substituted for the added phospholipid. The delipidated ATPase is soluble in the detergent and retains activity for several days. This is the first report of the Ca2+-ATPase retaining high activity with less than about 30 mol of phospholipid bound per mol of polypeptide.     

Stimulation of defective Gunn-rat liver uridine diphosphate glucuronyltransferase activity in vitro by alkyl ketones.

Addition of alkyl ketone (10mM) to Gunn-rat liver homogenates increased UDP-glucuronyltransferase activity towards 2-aminophenol by 10--20 fold, up to enhanced values of enzyme activity observed with similarly treated Wistar-rat liver homogenates. Alkyl ketones also activate the defective enzyme purified from Gunn-rat liver. This genetic deficiency of UDP-glucuronyltransferase activity is no longer apparent when assayed in the presence of alkyl ketones.     

Studies on the purification of rat liver uridine diphosphate glucuronyltransferase.

1. A stable, more highly purified, preparation of UDP-glucuronyltransferase was obtained than previously reported. 2. Enzyme activity towards o-aminophenyl and p-nitrophenyl was increased 43- and 46-fold respectively. 3. The final preparation contains only three staining polypeptide bands visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The only known major accompanying protein appears to be epoxide hydratase. 5. The purified enzyme activity towards o-aminophenol can still be activated 3 fold by diethylnitrosamine. 6. On evidence from purification, o-aminophenol and p-nitrophenol appear to be glucuronidated by the same enzyme protein. The possible recognition of the UDP-glucuronyltransferase enzyme is discussed.     

Reversible ATP-induced inactivation of branched-chain 2-oxo acid dehydrogenase.

1. Antiserum was raised against purified Wistar-rat liver UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activities towards 4-nitrophenol, bilirubin, 1-naphthol and morphine were co-immunoprecipitated from solubilized Wistar-rat liver preparations. 3. UDP-glucuronyltransferase activities towards 1-naphthol, 2-aminophenol and 4-nitrophenol were precipitated from solubilized Gunn-rat liver preparations by this antiserum. 4. UDP-glucuronyltransferase activities towards 1-naphthol, 4-nitrophenol and bilirubin, from Wistar-rat liver, were slightly inhibited by antiserum, whereas 1-naphthol UDP-glucuronyltransferase activity from Gunn-rat livers was greatly inhibited. 5. Measurable Wistar-rat liver glucuronyltransferase activities in washed immunoprecipitates indicate that the enzyme(s) were not merely inhibited by antiserum. 6. Immunoglobulin G purified from this antiserum immunoprecipitated transferase activities towards 4-nitrophenol, bilirubin and 1-naphthol. 7. The washed immunoprecipitates from both rat strains, containing UDP-glucuronyltransferase activity, appear to be similar when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 8. Radial-immunodiffusion studies suggest that a smaller amount of UDP-glucuronyltransferase protein is present in Gunn-rat liver than in Wistar-rat liver. 9. The significance of these results in relation to the genetic deficiency in the Gunn rat is discussed.     

Purification and properties of 5-hydroxytryptamine UDP-glucuronyltransferase from rat liver microsomes

5-Hydroxytryptamine UDP-glucuronyltransferase was highly purified from untreated rat liver microsomes. The specific activity towards 5-hydroxytryptamine was increased 178-fold over the starting solubilized microsomes with a final yield of 3%. The final preparation contained two major and one minor Coomassie brilliant blue staining polypeptide bands visible after SDS-polyacrylamide gel electrophoresis. One of the major bands was identified as 3-methylcholanthrene-inducible UDP-glucuronyltransferase, so the other (molecular weight of 55,500) appeared to be 5-hydroxytryptamine UDP-glucuronyltransferase. Concanavalin A reacted with the 55,500-dalton polypeptide. Phospholipid was indispensable for the enzyme activity. The enzyme activity in the final preparation was activated by divalent cations. Simple Michaelis-Menten kinetics were followed with respect to 5-hydroxytryptamine, but deviations from this kinetics were observed with respect to UDP-glucuronic acid and Mg2+. As regards Mg2+ stimulation, further experiments indicated that the added Mg2+ was non-competitive with 5-hydroxytryptamine, but at low concentrations of Mg2+ it was competitive with UDP-glucuronic acid and at high concentrations of Mg2+ it was non-competitive with UDP-glucuronic acid. The final preparation showed high substrate specificity towards 5-hydroxytryptamine among endogenous substrates tested. From these results, it was concluded that the enzyme described here is a new form of UDP-glucuronyltransferase isozyme, and its activity showed a peculiar dependence on Mg2+.     

Bovine adrenal tyrosine hydroxylase: purification and properties.

Bovine adrenal tyrosine hydroxylase has been obtained in a form that is 85 to 90% pure. Sodium dodecyl sulfate-gel electrophoresis and density gradient centrifugation studies have established that the subunit molecular weight of the chymotrypsin-solubilized enzyme is 34,000. The presence of iron in the purified enzyme (0.50 to 0.75 mol of iron/mol of enzyme) has been established. Crude particulate tyrosine hydroxylase can be activated by the phospholipid, phosphatidyl-L-serine, or by exposure to enzymatic phosphorylating conditions. Both forms of activation lower the Km of the enzyme for its 2-amino-4-hydroxypteridine cofactor. By contrast, tyrosine hydroxylase that has been solubilized by chymotrypsin cannot be activated by either of these methods.     

Lipoprotein lipase hydrolysis of trioleoylglycerol in a phospholipid interface. Effect of cholesteryl oleate on catalysis

The effect of cholesteryl oleate on the lipoprotein lipase-catalyzed hydrolysis of trioleoylglycerol was determined in monolayers of egg phosphatidylcholine at a constant surface pressure of 24 mN m-1. The phospholipid monolayers contained 1.0 to 7.5 mol % trioleoylglycerol and various amounts (0 to 20 mol %) of cholesteryl oleate. The initial rates of trioleoylglycerol hydrolysis were determined with lipoprotein lipase purified from bovine milk. In phospholipid monolayers containing 5.0 or 7.5 mol % trioleoylglycerol, the further addition of cholesteryl oleate caused a decrease in lipoprotein lipase activity. In contrast, addition of cholesteryl oleate to phospholipid monolayers containing 1.0 or 2.5 mol % trioleoylglycerol enhanced enzyme activity; a 3-fold enhancement was observed with 5.0-7.5 mol % cholesteryl oleate. Based on force-area measurements, the cholesteryl ester-mediated decrease in lipoprotein lipase activity observed at high substrate concentrations may be explained by displacement of trioleoylglycerol from the interface, thereby reducing the interfacial trioleoylglycerol concentration available for enzyme catalysis. One explanation for the cholesteryl oleate-mediated enhancement of lipoprotein lipase activity at low trioleoylglycerol concentrations is that the additional spreading of cholesteryl oleate disrupts microemulsions of trioleoylglycerol, thereby increasing the effective monomer substrate concentration available for enzyme catalysis. Based on these monolayer studies with model systems, we suggest that the relative amount of cholesteryl esters in plasma triacylglycerol-rich lipoproteins plays a regulatory role in determining the rate at which triacylglycerols are cleared from the circulation.     

FAD-dependent malate dehydrogenase, a phospholipid-requiring enzyme from Mycobacterium sp. strain Takeo. Purification and some properties

FAD-dependent malate dehydrogenase, a phospholipid-requiring enzyme, was homogeneously purified from the particulate fraction of Mycobacterium sp. strain Takeo. The isolated enzyme contains no FAD and few phospholipid, and has a specific activity of 300-360 units/mg of protein. In the assay system without addition of phospholipid (cardiolipin), the enzyme activity was only about 3% of maximum activity. The molecular weight was estimated to be 51 000-55 000 by four methods. Titration by p-chloromercuribenzoate revealed the presence of one cysteine residue/mol of enzyme. The isoelectric point was found to be pH 6.9 by isoelectric focusing. From circular dichroism spectral data, the enzyme protein was found to contain alpha-helix structure of 24%.     

Purification of rat-liver microsomal UDP-glucuronyltransferase. Separation of two enzyme forms inducible by 3-methylcholanthrene or phenobarbital.

Glucuronidation reactions catalysed by rat liver microsomal UDP-glucuronyltransferase are differentially inducible by 3-methylcholanthrene and phenobarbital. To elucidate the molecular basis of this functional heterogeneity the enzyme was purified from livers of rats pretreated with the inducing agents. Using cholate solubilization, chromatography on Bio-Gel A-1.5m and on DEAE-cellulose in the presence of the nonionic detergent Brij 58, two enzyme forms could be separated. Both forms were subsequently purified to apparent homogeneity by affinity chromatography on UDP-hexanolamine Sepharose 4B, 3-Methylcholanthrene-inducible enzyme activity towards 1-naphthol, 4-nitrophenol, 3-hydroxybenzo(a)pyrene and N-hydroxy-2-naphthylamine copurified with one enzyme form (enzyme 1). In contrast phenobarbital-inducible enzyme activity towards morphine, chloramphenicol and 4-hydroxybiphenyl was associated with the other enzyme fraction (enzyme 2). Sodium dodecylsulfate/polyacrylamide gels showed similar molecular weights of 54000 for enzyme 1 and 56000 for enzyme 2. The results suggest the presence of at least two forms of UDP-glucuronyltransferase in rat liver. Factors affecting enzyme activity in purified and membrane-bound states are discussed.     

Reconstitution of purified Wistar rat liver bilirubin UDP-glucuronyltransferase into Gunn-rat liver microsomes.

1. Reconstitution of purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver into Gunn-rat liver microsomes provides a better environment than phosphatidylcholine liposomes, such that the final specific activity of the Wistar-rat liver enzyme was increased up to 85 units/mg of protein. 2. Gunn- and Wistar-rat liver microsomes were equally effective for reconstitution of the purified enzyme. 3. The transferase activity does not appear to be fully expressed in the more rigid environment of foetal Wistar-rat liver microsomes. 4. These reconstitution experiments reveal a final specific activity for the purified bilirubin UDP-glucuronyltransferase consistent with the capacity of the whole rat liver to glucuronidate bilirubin and indicate that the absence of this enzyme activity in Gunn-rat liver microsomes is not due to an abnormal microenvironment.     

Stereospecificity and requirements for activity of the respiratory NADH dehydrogenase of Escherichia coli

The respiratory NADH dehydrogenase of Escherichia coli has been further amplified in vivo by genetic methods. The enzyme, a single polypeptide of Mr 47 200 of known amino acid sequence [Young, I. G., Rogers, B. L., Campbell, H. D., Jaworowski, A., & Shaw, D. C. (1981) Eur. J. Biochem. 116, 165-170], constitutes 10-15% of the total protein in the amplified membranes. In situ in the membrane, the enzyme contains 1 mol of FAD/mol of subunit and has a specific NADH:ubiquinone-1 oxidoreductase activity of approximately 1100-1200 units mg-1 at 30 degrees C, pH 7.5. The purified enzyme contains phospholipid, which remains closely associated with it during gel filtration on Sephacryl S-300 in the presence of 0.1% (w/v) cholate at low ionic strength. Under these conditions the enzyme is extensively aggregated (apparent Mr greater than 10(6]. This procedure yielded enzyme with a specific activity of 980 units mg-1, similar to the value observed in the membrane. This preparation contained less than 0.1 mol of Fe/mol of enzyme, confirming that Fe is not involved in reduction of ubiquinone 1 catalyzed by the enzyme. Neutron activation analysis of purified enzyme has demonstrated the absence of 35 trace elements including Se, Zn, Mn, Co, W, Cu, and Fe. The enzyme polypeptide, prepared completely free of phospholipid, FAD, and ubiquinone by gel filtration in the presence of sodium dodecyl sulfate, has been reactivated. The results show that the only components necessary for catalysis of ubiquinone-1 reduction by NADH in this system are the enzyme polypeptide, FAD, and phospholipid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of sorghum grain tannins and dietary protein on the activity of liver UDP-glucuronyltransferase

The activity of liver microsomal UDP-glucuronyltransferase (EC 2.4.1.17), an enzyme known to detoxify phenolic compounds, was measured in chicks and rats fed high- (HTS) and low-tannin sorghums (LTS). In an initial investigation, activity was significantly elevated in chicks fed HTS-soybean meal diets over those fed the LTS control diet. Other studies were designed to differentiate between the effects due to tannin and those resulting from a protein deficiency which had previously been reported to increase the activity of this enzyme. In general, only a relatively small part of the increased activity observed by feeding HTS to chicks could be attributed to a tannin-induced protein deficiency. The same phenomenon of elevated activity produced by feeding HTS to chicks was not observed in the rat. These results would suggest that sorghum tannins, or their breakdown products, are absorbed and activating UDP-glucuronyltransferase in the chick, but not the rat.     

Kinetic properties of microsomal UDP-glucuronyltransferase. Regulation by metal ions

Treatment of microsomes with EDTA abolishes the stimulation of glucuronidation produced by UDP-N-acetylglucosamine. Addition of divalent metal ions to EDTA-treated microsomes restores the sensitivity of UDP-glucuronyltransferase to UDP-N-acetylglucosamine. Regulation of the activity of this enzyme by UDP-N-acetylglucosamine depends, therefore, on the presence of divalent metal ions. In addition, divalent metals increase the rate of glucuronidation of p-nitrophenol at V_max. The data indicate that metals are essential for the efficient function of UDP-glucuronyltransferase.     

Thermal stability of membrane-reconstituted yeast cytochrome c oxidase

The thermal dependence of the structural stability of membrane-reconstituted yeast cytochrome c oxidase has been studied by using different techniques including high-sensitivity differential scanning calorimetry, differential detergent solubility thermal gel analysis, and enzyme activity measurements. For these studies, the enzyme has been reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) vesicles using detergent dialysis. The phospholipid moiety affects the stability of the enzyme as judged by the dependence of the denaturation temperature on the lipid composition of the bilayer. The enzyme is more stable when reconstituted with the 18-carbon, unsaturated phospholipid (DEPC) than with the 14-carbon saturated phospholipid (DMPC). In addition, the shapes of the calorimetric transition profiles are different in the two lipid systems, indicating that not all of the subunits are affected equally by the lipid moiety. The overall enthalpy change for the enzyme denaturation is essentially the same for the two lipid reconstitutions (405 kcal/mol of protein for the DMPC and 425 kcal/mol for the DEPC-reconstituted enzyme). In both systems, the van't Hoff to calorimetric enthalpy ratios are less than 0.2, indicating that the unfolding of the enzyme cannot be represented as a two-state process. Differential detergent solubility experiments have allowed us to determine individual subunit thermal denaturation profiles. These experiments indicate that the major contributors to the main transition peak observed calorimetrically are subunits I and II and that the transition temperature of subunit III is the most affected by the phospholipid moiety. Experiments performed at different scanning rates indicate that the thermal denaturation of the enzyme is a kinetically controlled process characterized by activation energies on the order of 40 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)