Differential regulation by triiodothyronine of substrate-specific uridinediphosphoglucuronate glucuronosyl transferases in rat liver

Hepatic uridinediphosphoglucroonate glucuronosyl transferase (UDPglucuronyltransferase, EC 2.4.1.17) functionally heterogeneus; 4-nitrophenol and bilirubin are representative subtrates for two separated from of the enzyme. UDPglucuronyltransferase activity for bilirubin and 4-nitrophenol was separated from solubilized rat liver microsomes by DEAE-cellulose chromatography and corresponding enzymes were purified. A radioimmunoassay was developed using a rabbit antiserum against purified rat 4-nitrophenol-specific UDPglucuronyltransferase, which precipitated enzyme activities toward both 4-nitrophenol and bilirubin. After treatment with triiodothyronine(T₃) (0.55 mg/kg body weight), hepatic microsomal UDPglucuronyltransferase activity for 4-nitropheelos was increased 400% as compared to controls; the enzyme activity for bilirubin was decreased by 80%; the changes in the substrate-specific enzyme activities were reflected in the enzymatically active fractions separated after DEAE-cellulose chromatography. The changes in enzyme activities paralleled changes in the concentrations of the two corresponing UDP glucuronyltransferase proteins in the chromatographic fractions, as measured by radioimmunoassay. The results indicate that the opposite effects of T₃ on the two forms of UDPglucuronyltransferase activity is due to its differential effect on corresponding enzyme proteins.     

Activation of hepatic microsomal glucuronyltransferase from gunn rats by exposure to light

Exposure of hepatic microsomes from icteric and nonicteric rats to 250 foot candles of blue fluorescent light for 4 hours at 4–6°C significantly increased the activity of UDPglucuronyltransferase activity using p-nitrophenol as a substrate. The light exposure reduced serum bilirubin concentration from icteric rats or bilirubin of fortified human serum albumin by 70 per cent but there was no difference in light activation of hepatic microsomes from icteric or nonicteric rats. Light exposure also decreased the activation of UDPglucuronyltransferase produced by Triton X-100.     

The heterogeneity of uridine diphosphate glucuronyltransferase from rat liver

1. The glucuronide conjugation of p-nitrophenol, phenolphthalein, o-aminophenol and 4-methylumbelliferone by rat liver microsomes has been studied. The detergent Triton X-100 activated UDP-glucuronyltransferase activity towards all these substrates, therefore the optimum activating concentration was added in all experiments. 2. Mg²⁺ enhanced the conjugation of the substrates. 3. With phenolphthalein substrate inhibition occurred but this could be relieved by adding albumin, which binds excess of phenolphthalein. 4. Kinetic constants of the substrates and UDP-glucuronate have been determined. Mutual inhibition was found with the substrates p-nitrophenol, 4-methylumbelliferone and phenolphthalein. p-Nitrophenol conjugation was inhibited competitively by phenolphthalein and 4-methylumbelliferone. 5. o-Aminophenol did not inhibit the conjugation of the other three substrates because these are conjugated preferentially to o-aminophenol. 6. It is concluded that the four substrates are conjugated by one enzyme at the same active site.     

Enzymes of the glucuronic acid pathway in Wistar and Gunn rats and their heterozygotes

A significant difference in UDP glucuronyltransferase activity (with p-nitrophenol as an acceptor) was found in the liver and kidneys of homozygous Wistar and Gunn rats. There was also a significant difference in hepatic UDP glucuronyltransferase activity between homozygous Wistar and heterozygous Gunn rats when the enzyme preparations were first activated by adding surfactants to the reaction mixture. This determination of surfactant-activated UDP glucuronyltransferase can be used to distinguish Wistar rats from heterozygous Gunn rats. Other enzymes of the glucuronic acid pathway were also studied in the liver and kidneys of homozygous Wistar and Gunn rats, but no differences were found.This study has been supported by grants from the U.S. Public Health Service (AM-06018-09 and the National Research Council for Natural Sciences, Finland.     

Assay for the terminal enzyme of the stearoyl coenzyme A desaturase system using chick embryo liver microsomes.

The NADH-dependent stearoyl CoA desaturase of hepatic microsomes (EC 1.14.99.5) is an enzyme system consisting of cytochrome b5 reductase (EC 1.6.2.2), cytochrome b5, and the terminal desaturase. We have developed a simple method for routine assay of the terminal enzyme based on complementation of the enzyme with chick embryo liver microsomes lacking desaturase activity. Desaturation of [1-14C]stearoyl CoA by the enzyme-microsome mixture is then assayed by thin-layer chromatography of the reaction products and determination of the amount of oleate formed. Microsomes from the livers of starved-refed rats were used as the source of the stearoyl CoA desaturase. The enzyme alone, solubilized and free from cytocrome b5 reductase and cytochrome b5, was unable to catalyze the desaturation of stearoyl CoA. However, after preincubation with chick embryo liver microsomes in the presence of 1% Triton X-100, the enzyme was active. The enzyme activity was linear with time and desaturase protein under the conditions described and depended on the concentrations of Triton X-100 present in the preincubation and the assay. The optimum concentrations of Triton X-100 were 1% for the preincubation and 0.1-0.15% in the assay. The desaturation activity was dependent on NADH and O2, and was inhibited 95% by 1 mM KCN. The use of chick embryo liver microsomes in this method eliminates the need to use purified cytochrome b5 reductase, cytochrome b5, and liposomes for routine assays and greatly reduces the complexities of timing and order of addition encountered in the existing assays.     

Properties of microsomal phospholipases in rat liver and hepatoma.

Phospholipase A1, A2 and lysophospholipase activities in microsomes of Novikoff hepatoma host rat liver and regenerating rat liver were compared using 1-[9', 10'-3H2]palmitoyl-2-[1'-14C] linoleoyl-sn-glycero-3-phosphoethanolamine, 1-[1' -3H-]hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine, and 1-[9', 10'-3H2]palmitoyl-sn-glycero-3-phosphoethanolamine as substrates. 1. Microsomes of all three tissues showed two pH dependent peaks of hydrolytic activity, one at pH 7.5 and another at pH 9.5. 2. Phospholipid hydrolytic activity in microsomes from host liver and regenerating liver require Ca2+ for hydrolysis at pH 9.5, but not at pH 7.5. Hepatoma microsomes require Ca2+ for activity at both pH values. 3. Phospholipase A1 activity, stimulated by addition of Triton X-100 to the incubation mixtures, was detected in both host liver and regenerating liver microsomes. There was no evidence of phospholipase A1 activity in hepatoma microsomes. 4. Phospholipase A2 was detected in microsomes of all three tissues using 1-[1'-3H] hexadecyl-2-acyl-sn-glycero-3-phosphoethanolamine as a substrate. The activity required calcium and was inhibited by Triton X-100. 5. Lysophospholipase activity was evident in the microsomes from all three tissues. The activity was inhibited by both Ca2+ and Triton X-100. 6. Differences were also detected between host liver and hepatoma microsomal phospholipid hydrolase activities with respect to the effect of increasing protein concentration, apparent Michaelis-Menten constants, and time course of the reaction.     

Long-chain acyl CoA synthetase in microsomes from rat brain gray matter and white matter

Long-chain acyl coenzyme A (CoA) synthetase in homogenates and microsomes from rat brain gray and white matter was studied. The formation of the thioesters of CoA was studied upon addition of [1-¹⁴C]-labeled fatty acids. The maximal activities were seen with linoleic acid, followed by arachidonic, palmitic, and docosahexaenoic acids in both gray and white matter homogenates and microsomes. The specific activities in microsomes were 3–5 times higher than in homogenates. The presence of Triton X-100 in the assay system enhanced the activity of long-chain acyl CoA synthetase in homogenates. The effect was more pronounced in palmitic and docosahexaenoic acid activation. The apparentK _m values andV _max values for palmitic and docosahexaenoic acids were much lower than for linoleic and arachidonic acids. The presence of Triton X-100 in the medium caused a definite decrease in the apparentK _m and Vmax values for all the fatty acid except palmitic acid in which case the reverse was true. There were no significant differences observed in the kinetic measurements between gray and white matter microsomes. These findings are similar to those resulting from the known interference of Triton X-100 in the measurement of kinetic variables of long-chain acyl CoA synthetase of liver microsomes. In this work, no correlation was observed between the fatty acid composition of gray and white matter and the capacity of these tissues for the activation of different fatty acids.     

Topology of glutathione-insulin transhydrogenase in rat liver microsomes

Glutathione-insulin transhydrogenase (EC 1.8.4.2) catalyzes the inactivation of insulin through scission of the disulfide bonds to form insulin A and B chains. In the liver, the transhydrogenase occurs primarily in the microsomal fraction where most of the enzyme is present in a latent (‘inactive’) state. We have isolated rat hepatic microsomes with latent transhydrogenase activity being an integral part of the vesicles. We have used these vesicles to study the topological location of glutathione-insulin transhydrogenase by investigating the effects of detergents (Triton X-100 and sodium deoxycholate), phospholipase A₂ and proteinases (trypsin and thermolysin) on the latent enzyme activity. Treatment of intact vesicles with variable concentrations of detergents and phospholipase A₂ resulted in the unmasking of latent transhydrogenase activity. The extent of unmasking of transhydrogenase activity is dependent upon the concentration of detergent or phospholipase used and is accompanied by a parallel release of the enzyme into the soluble fraction. Activation of the transhydrogenase by phospholipase A₂ is partially inhibited by bovine serum albumin and the extent of inhibition is inversely proportional to the phospholipase concentration. In intact vesicles, latent transhydrogenase activity is resistant to proteolytic inactivation by both trypsin and thermolysin, while in semipermeable and permeable vesicles these proteases inactivate 60 and 25% of the total transhydrogenase activity, respectively. Together these results indicate that in microsomes transhydrogenase is probably weakly bound to membrane phospholipid components and that most of the enzyme is present on the cisternal surface (i.e., the luminal surface of endoplasmic reticulum) of microsomes. Each detergent and phospholipase apparently unmasks glutathione-insulin transhydrogenase activity through disruption of the phospholipid-enzyme interaction followed by translocation of the enzyme to the soluble (cytoplasmic) fraction and not through increases in substrate availability.     

Dietary cholesterol caused modification in the structure and function of rat hepatic microsomes, studied by fluorescent probes

A 4% cholesterol diet fed to rats for four weeks was found to increase the phospholipid and cholesterol contents and the activities of drug metabolizing enzymes in rat liver microsomes.Microsomes from rats on a high cholesterol diet were able to enhance the fluorescence of membrane bound 1-anilinonaphthalene 8-sulphonate (1,8-ANS) and ethidium bromide more than microsomes from rats on a standard diet.In the case of 1,8-ANS, the enhanced fluorescence was found to be due to the increased affinity of the molecules for microsomes. In the case of ethidium bromide the fluorescence increased partly because of the larger amount of binding sites and partly because of the enhanced quantum yield of the molecules.P-nitrophenol was found to compete with 1,8-ANS for the same binding sites in microsomes. On the other hand, 1,8-ANS lowered the rate of drug metabolism when present in the incubation mixture.In vitro treatments of microsomes with trypsin, phospholipase A or digitonin altered the binding properties of 1,8-ANS and ethidium bromide to microsomes.It is concluded that the binding sites of 1,8-ANS in microsomes are important for the activity of drug-metabolizing enzymes. The mechanisms of dietary cholesterol in enhancing the drug metabolism and the role of microsomal phospholipids in regulating the activity of drug-metabolizing enzymes are discussed.     

Protein-lipid interactions in the sialic acid incorporating system of liver microsomes

The conditions for the incorporation of sialic acid (N-acetylneuraminic acid) from CMP-sialic acid into endogenous acceptors of rat liver microsomes has been studied. It is shown that the incorporating activity can be solubilized by extraction of the microsomes with a mild detergent, Triton X-100. The specific activity of the soluble system is about sixfold compared to the original microsomes. Removal of lipids from the system greatly reduces its ability to incorporate sialic acid. Recombination with phospholipids prepared from liver microsomes restores the activity. Other lipids are ineffective, and single phospholipid fractions are less effective than the phospholipid mixture. It is concluded that the system studied, comprising both sialyl transferase and sialyl acceptor-protein is a typical intrinsic membrane protein system, depedent on a hydrophobic environment for full activity.     

Presence of UTP:D-GLUCOSE-1-phosphate uridylyltransferase in liver microsomes of the eel (Conger vulgaris)]

An activity UTP : D-glucose-1-phosphate uridylyltransferase is located in the microsomal membranes of conger liver. The properties of this enzyme are studied and compared to the soluble activity. The microsomal activity is partially liberated from the membrane by freezing and thawing and by the means of a neutral detergent, Triton X-100. The enzyme is latent in the membranes and totally inhibited by phospholipase A2. This microsomal enzyme could be the last of a membranous biosynthetic pathway for UDP-glucose, as conger liver microsomes contain also a membranous glucokinase and a membranous phosphoglucomutase.     

A simple procedure for the solubilization of NADH-cytochrome b5 reductase

NADH-cytochrome b₅ reductase has been solubilized by extraction of rabbit liver microsomes with 1 potassium phosphate buffer (pH 7.4), and has been purified to comparable purity with the Triton X-100-solubilized enzyme. Gel electrophoresis indicated an apparent molecular weight of 33,000 for both phosphate buffer-extracted and Triton X-100-solubilized enzymes. Phosphate buffer extraction provides a simple mild procedure for the extraction of NADH-cytochrome b₅ reductase that avoids detergents or proteolytic agents.     

Effects of membrane perturbants on UDP-glucuronosyltransferase activity in rat-liver microsomes. Circular dichroism studies.

Rat-liver microsomes were treated with two non-ionic detergents, Triton X-100 and Lubrol WX, with phospholipase A2, or with aqueous acetone solution. The activity of the membrane-bound UDP-glucoronosyltransferase (UDPGT, EC 2.4.1.17) was measured after the treatment with these perturbants. At the same time, modifications of the secondary structure of the microsomal proteins were followed and studied by circular dichroism (CD) spectroscopy. The detergents greatly activated UDPGT, maximally at a 1 mM concentration of either detergent. The maximally activating Triton X-100 treatment did not greatly change the ellipticity of the microsomes at 222 nm ((theta)222), whereas that with Lubrol WX affected the secondary structure of the membrane proteins more strongly. UDPGT activation also occurred in phospholipase A2-treated microsomes. Maximal activation was obtained after 1--5 min of incubation and was stable throughout the experiment. Phospholipase A2 at the ratio of microsomal protein to phospholipase 250 : 1 (w/w) slightly increased (theta)222 after 10 min of incubation and did not change it further even after 30 min of incubation. Treatment of liver microsomes with a 10 : 90 (v/v) aqueous acetone solution removed 90% of the total membrane phospholipids, particularly phosphatidylcholine and phosphatidylethanolamine. The UDPGT activity was decreased in lipid-depleted microsomes, and the enzyme was not reactivated when phosphatidylcholine-lysophosphatidylcholine liposomes were added at a low temperature. An even greater decrease was obtained when the lipid binding was carried out at 37 degree C. Lipid-depleted microsomes had a high (theta)222 associated with a red-shift of 2 nm, indicating partial aggregation of membrane proteins and an increase in the alpha-helical content of the protein after acetone extraction. However, this particular protein structure was partially reversible, since a binding of phospholipids to lipid-depleted microsomes gave a (theta)222 close to that found in control microsomes. The UDPGT activity was not dependent on the secondary structure of the membrane proteins.     

Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes.

NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.     

Phospholipase A2 activity towards phosphatidylcholine in mixed micelles: surface dilution kinetics and the effect of thermotropic phase transitions

Phospholipase A₂ will act on dipalmitoyl phosphatidylcholine as substrate when the phospholipid is part of a mixed micelle with Triton X-100 at a molar ratio of Triton to phospholipid of 2:1 or greater. Kinetic studies at high molar ratios of Triton X-100 to phospholipid are reported and show that the binding of phospholipase A₂ to substrate depends on the total concentration of Triton X-100 and phospholipid, but that the rate of enzymatic catalysis decreases proportionally to the Triton X-100 concentration. These results are interpreted in terms of a model involving surface dilution kinetics. The relationship of this model to that of competitive inhibition is discussed. In addition, the activity of phospholipase A₂ towards dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine at different temperatures is reported, and the results show a direct effect of the thermotropic phase transition of dipalmitoyl phosphatidylcholine on enzymatic activity.     

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B₁ and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.     

The specificity of the 3beta-hydroxysteroid dehydrogenase activity of bovine ovaries toward dehydroepiandrosterone and pregnenolone: evidence for multiple enzymes.

The 3beta-hydroxysteroid dehydrogenase activity in whole bovine ovaries was systematically studied using dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one) and pregnenolone (3 beta-hydroxy-5-pregnen-20-one) as substrates, in order to determine whether, in this tissue, the same or different 3beta-hydroxysteroid dehydrogenases metabolize these steroids. The majority of the activity, with both substrates was found in the microsomes. Detergent extraction of the microsomes indicated that more than one enzyme was present in this fraction. A number of experiments on the Triton X-100 extract of the microsomes (the stability of the activity, its nucleotide specificity and kinetic analyses) were most simply explained by a single enzyme metabolizing both steroids. However, the stereospecificity of hydride-ion transfer from pregnenolone to NAD+ (B transfer) was different than that from dehydroepiandrosterone to NAD+ (A and B transfer). Thus, as no single enzyme is known to catalyze the transfer of hydride-ion to both sides of NAD+, it is proposed that there are at least two 3beta-hydroxysteroid dehydrogenases in the Triton X-100 extract.     

Rat liver microsomal 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase: solubilization, separation and partial purification

Rat liver microsomes contain 3 alpha-hydroxysteroid dehydrogenase (HSD) (EC 1.1.1.50) and dihydrodiol dehydrogenase (DHD) (EC 1.3.1.20) activities. The two enzyme activities were solubilized by 10% Triton X-100 or 0.4% sodium deoxycholate. Unlike the cytosolic enzyme (Penning & Talalay (1983) Proc. Natl. Acad. Sci. U.S.A., 80, 4505), the microsomal HSD and DHD activities were not inhibited by indomethacin. Chromatography of the microsomal Triton X-100 extract on Affigel Blue and then on Phenyl-Sepharose gave an HSD preparation containing no detectable (less than 3 - 5%) DHD activity, whereas chromatography of the deoxycholate extract on Phenyl-Sepharose provided a DHD preparation that lacked measurable HSD activity. These results are in sharp contrast to the cytosolic enzyme where both HSD and DHD activities could be copurified to homogeneity (Penning et al. (1984) Biochem. J. 222, 601).     

Purification and partial characterization of squalene epoxidase from rat liver microsomes

Squalene epoxidase (EC 1.14.99.7, squalene 2,3-monooxygenase (epoxidizing) was purified to an apparent homogeneity from rat liver microsomes. The purification was carried out by solubilization of microsomes by Triton X-100, fractionation with ion exchangers, hydroxyapatite, Cibacron Blue Sepharose 4B, and chromatofocusing column chromatography. A total purification of 143-fold over the first DEAE-cellulose fraction was achieved. The purified enzyme gave a single major band on SDS-polyacrylamide gel electrophoresis and the Mr was estimated to be 51 000 as a single polypeptide chain. The enzyme showed no distinct absorption spectrum in the visible regions. The squalene epoxidase activity was reconstituted with the purified enzyme, NADPH-cytochrome P-450 reductase (EC 1.6.2.4), FAD, NADPH and molecular oxygen in the presence of Triton X-100. The apparent Michaelis constants for squalene and FAD were 13 microM and 5 microM, respectively. The Vmax was about 186 nmol per mg protein per 30 min for 2,3-oxidosqualene. The enzyme activity was not inhibited by potent inhibitors of cytochrome P-450. It is suggested that squalene epoxidase is distinct from cytochrome P-450 isozymes.     

Purification of squalene epoxidase from rat liver microsomes

Squalene epoxidase was purified from rat liver microsomes by DEAE-cellulose, alumina C_ν gel, hydroxylapatite, CM-Sephadex C-50 and Cibacron Blue Sepharose 4B in the presence of Triton X-100. The specific activity was increased 50 fold with a yield of about 10%. On SDS-polyacrylamide gel electrophoresis, the preparation gave one major band and one minor band with apparent molecular weights of 47,000 and 27,000 daltons, respectively. The protein of 47,000 was the most probable candidate for squalene epoxidase. Squalene epoxidase activity could be reconstituted in the squalene epoxidase preparation with the addition of NADPH-cytochrome P-450 reductase, FAD, and Triton X-100.