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1.
Chymotrypsin inactivation of lysophosphatidic acid acyltransferase activity in detergent-disrupted rat liver microsomes, but not in intact microsomes, falsely indicated a lumenal location for the enzyme. Inhibition by several other proteases in the absence of detergent showed that lysophosphatidic acid acyltransferase activity is located on the cytoplasmic surface of microsomes. Chymotrypsin inactivation did not occur in vesicles disrupted by nitrogen cavitation unless deoxycholate was present, suggesting that deoxycholate exposes a cryptic chymotrypsin cleavage site. Criteria for localization of lumenal microsomal enzymes should include studies using several proteases and/or employ more than one method of microsomal disruption. 相似文献
2.
Acyl coenzyme A:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase (EC 2.3.1.23) is capable of forming lipid bilayer vesicles from its soluble substrates lysophosphatidylcholine (LPC) and oleoyl CoA. This suggested a purification method in which rat liver microsomes are first washed with deoxycholate to increase specific activity of the endogenous acyltransferase approximately fivefold, then solubilized by the detergent effect of excess LPC and oleoyl CoA in 1:1 stoichiometric ratios. As the LPC is converted to phosphatidylcholine by acyl group transfer, the detergent effect is lost and lipid vesicles containing the enzyme activity are produced. Other microsomal proteins are excluded from the vesicles. The vesicles may be separated by density gradient flotation and are found to contain acyltransferase with a specific activity of 9–10 µmol/mg/min. This reflects a purification of approximately 140-fold, about ten times greater than achieved in previous studies. 相似文献
3.
O Yukawa M Miyahara N Shiraishi T Nakazawa 《International journal of radiation biology and related studies in physics, chemistry, and medicine》1985,48(1):107-115
Radiation-induced damage to the reconstituted system of membrane-bound enzyme, D-beta-hydroxybutyrate dehydrogenase obtained from rat liver mitochondria, was investigated in relation to the lipid peroxidation of membranes. The activity of D-beta-hydroxybutyrate dehydrogenase in fresh mitochondria was very low in general and was not affected by irradiation because of little incorporation of substrates into mitochondria. However, the enzyme activity in one-day-aged mitochondria or submitochondrial particles was five times higher than that of fresh mitochondria and decreased with increasing radiation dose accompanying the increase in peroxidation of membrane lipids. The activity of D-beta-hydroxybutyrate dehydrogenase in the reconstituted system of the purified enzyme with irradiated liver microsomes or irradiated liposomes was decreased considerably in comparison with either unirradiated control or irradiated enzyme. Therefore, the radiation-induced decrease in the enzyme activity was thought to be caused mainly by peroxidation of membrane lipids and not to be due to direct damage by radiation to the enzyme molecule itself. Irradiation of microsomes, a component of the reconstituted system, caused decreases in phosphatidylcholine and phosphatidylethanolamine content and an increase in lysophosphatidylcholine content. In addition, arachidonic acid contents in phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were also markedly decreased with increasing radiation dose. These results are discussed in terms of a mechanism involving radiation-induced damage to membrane function and structures. 相似文献
4.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1996,1299(2):160-166
Fatty acid ethyl esters are a family of non-oxidative metabolites of ethanol present in many tissues after ethanol consumption. In this report we demonstrate the existence in human liver of an acyl-CoA: ethanol acyltransferase activity which may be responsible in part for the synthesis of these compounds in vivo. The effects of oleoyl-CoA and ethanol concentrations, presence or absence of bovine serum albumin and detergent, pH and enzyme concentration on this activity have been determined. Acyl-CoA: ethanol acyltransferase activity is localised in the membrane-bound fraction. Using inhibitors directed against related enzyme activities, it has been shown that the activity is not related to serine-dependent carboxylesterases or acyl-CoA: cholesterol acyltransferase, but that it may be associated with acyl-CoA hydrolase activity. We have also compared acyl-CoA: ethanol acyltransferase activity with fatty acid ethyl ester synthase activity in microsomes and cytosol from the same liver. Our data indicate that these activities are comparable in vitro (on a units/g liver basis), and suggest that both may be significant in vivo. 相似文献
5.
Recovery of ribophorins and ribosomes in "inverted rough" vesicles derived from rat liver rough microsomes
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《The Journal of cell biology》1982,93(1):111-121
Treatment of rat liver rough microsomes (3.5 mg of protein/ml) with sublytical concentrations (0.08%) of the neutral detergent Triton X-100 caused a lateral displacement of bound ribosomes and the formation of ribosomal aggregates on the microsomal surface. At slightly higher detergent concentrations (0.12-0.16%) membrane areas bearing ribosomal aggregates invaginated into the microsomal lumen and separated from the rest of the membrane. Two distinct classes of vesicles could be isolated by density gradient centrifugation from microsomes treated with 0.16% Triton X-100: one with ribosomes bound to the inner membrane surfaces ("inverted rough" vesicles) and another with no ribosomes attached to the membranes. Analysis of the fractions showed that approximately 30% of the phospholipids and 20-30% of the total membrane protein were released from the membranes by this treatment. Labeling with avidin-ferritin conjugates demonstrated that concanavalin A binding sites, which in native rough microsomes are found in the luminal face of the membranes, were present on the outer surface of the inverted rough vesicles. Freeze-fracture electron microscopy showed that both fracture faces had similar concentrations of intramembrane particles. SDS PAGE analysis of the two vesicle subfractions demonstrated that, of all the integral microsomal membrane proteins, only ribophorins I and II were found exclusively in the inverted rough vesicles bearing ribosomes. These observations are consistent with the proposal that ribophorins are associated with the ribosomal binding sites characteristic of rough microsomal membranes. 相似文献
6.
The distribution of cyclic AMP-dependent protein kinase activity in porcine thyroid glands has been studied. Enzyme activity catalyzing phosphorylation of exogenous substrate (protamine) from ATP, and cyclic AMP binding were determined in parallel in subcellular fractions purified by differential centrifugation and flotation on sucrose density layers. Both activities were found in all the studied fractions; they were quantitatively the highest in the cytosol but particles showed the highest specific activities.Latent protein-kinase activity was unmasked by action of detergents on microsomes (× 5–10 fold) and solubilized (85 to 99 p. cent of the initial total activity). Cyclic AMP binding capacity was also recovered in detergent-treated microsomal extracts in spite of reduced cyclic AMP binding in the presence of detergent.Protein kinase activity and cyclic AMP-binding proteins were less represented in purified nuclei than in microsomes. Again both activities were unmasked by detergent.Preparations highly enriched in Golgi membranes were compared to rough microsomal preparations. Higher protein kinase activity was detected in rough microsomes as compared to Golgi membranes, whereas the reverse was true for cyclic AMP binding. Both activities were equalized after detergent treatment. Since unmasking of protein kinase activity was the highest in Golgi membranes, this fraction contains more enzyme activity and cyclic AMP binding capacity than rough microsomes.The localization of endogeneous protein substrates of cyclic AMP-dependent protein kinases was investigated using purified soluble protein kinase subcellular fractions. The better endogeneous substrates seemed to be localized in the rough microsomal and in the nuclear fractions. 相似文献
7.
Zohreh T. Farahbakhsh David W. Deamer Nancy M. Lee Horace H. Loh 《Journal of neurochemistry》1986,46(3):953-962
A new method using lysophosphatide and acyl-CoA as detergents has been used to solubilize the rat brain opiate receptor. After solubilization, lysophosphatide and acyl-CoA can be almost completely removed by an enzymatic reaction that uses an acyltransferase from rat liver microsomes and reconstitutes the solubilized receptor in membranous vesicles. Morphological studies performed with negative staining and freeze-fracture electron microscopy revealed that the general appearance and intramembrane particle distribution of fracture faces in the reconstituted membrane are similar to those of the native membrane; this indicates that hydrophobic protein components of the original membrane were incorporated during reconstitution. Reconstituted membrane, however, contained higher levels of phosphatidylcholine and lower levels of cholesterol. The activities of the membrane-bound enzymes Na+, K+-ATPase and Ca2+, Mg2+-ATPase in the reconstituted system were 24 and 3%, respectively, those of the native membrane. Although binding of opiate ligands to the reconstituted membrane was stereospecific and saturable, higher concentrations of some of the unlabeled ligands were required to inhibit binding of the radiolabeled ligands. These changes in receptor characteristics are likely due to changes in lipid composition, physical state, and/or distribution of the lipids in the reconstituted membrane bilayer. This conclusion is supported by an increase in the affinity of opiate ligands for reconstituted membrane after adjustment of the latter's lipid composition to match more closely that of the original membrane. This was accomplished by treatment with phospholipid exchange protein to remove the excess phosphatidylcholine and by incorporation of cholesterol into the reconstituted membrane. 相似文献
8.
The intrinsically active, sn-glycerol-3-phosphate acyltransferase present in membranes prepared from both wild type Escherichia coli and from strains which overproduce the enzyme can be kinetically distinguished from a latent enzyme species which is unmasked by solubilization and reconstitution. Both membrane-associated and solubilized/reconstituted enzyme preparations exhibited cooperativity with respect to sn-glycerol-3-phosphate and palmitoyl-coenzyme A substrates; positive cooperativity in membranes toward palmitoyl-coenzyme A (napp = 4) and negative cooperativity toward sn-glycerol-3-phosphate (napp = 0.75) were significantly altered upon solubilization and reconstitution. Since the degree of alteration increased with the amount of sn-glycerol-3-P acyltransferase present in the membranes, a detergent-dissociable homooligomerization of the sn-glycerol-3-phosphate acyltransferase was considered as an underlying mechanism. This possibility was investigated by changing the protein-to-Triton X-100 ratio of homogeneous enzyme prior to reconstitution and then analyzing the subsequent migration of samples on a Sephacryl S-300 sizing column. The elution positions were consistent with monomeric and dimeric polypeptide bound to micelles of Triton X-100. Hill coefficients for monomeric, reconstituted enzyme preparations were comparable to those obtained for the active, membrane-associated sn-glycerol-3-phosphate acyltransferase. The reduced cooperativity of dimeric, reconstituted enzyme preparations correlated closely to the Hill coefficient values obtained for latent, solubilized/reconstituted sn-glycerol-3-phosphate acyltransferase from membranes of Escherichia coli which overproduce the enzyme. The physiological significance of these findings is discussed. 相似文献
9.
Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic detergent, Nonidet P-40. The preparation exhibits a single major band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a specific content of 11.5 nmol of P-450/mg of protein. The purified enzyme displays spectroscopic properties typical of the ferric and ferrous forms of cytochrome P-450. Full enzymatic activity can be reconstituted with rabbit liver microsomal cytochrome P-450 reductase and Nonidet P-40. Purified aromatase cytochrome P-450 displays catalytic characteristics similar to the enzyme in intact microsomes in the aromatization of androstenedione, 19-hydroxyandrostenedione and 19-oxoandrostenedione. Testosterone and 16 alpha-hydroxytestosterone are aromatized at maximal rates similar to androstenedione, and all substrates exhibit relative affinities corresponding to those observed in microsomes. We have raised rabbit antibodies to the purified enzyme which show considerable specificity and sensitivity on immunoblots. 相似文献
10.
EDTA/KCl- or pyrophosphate-treated rough microsomes of rat liver clearly showed the co-translational cleavage of pre-human placental lactogen and translocation of the product into membrane vesicles. The signal peptidase fraction was isolated by chromatography on Sephacryl S-300 of deoxycholate-treated membranes and reconstituted into liposomes by dialysis or by the Biobeads SM-2 method. Assay of the signal peptidase activity was performed with pre-human placental lactogen synthesized by the reticulocyte lysate system programmed with human placental lactogen mRNA. The signal peptidase reconstituted into liposomes showed stable activity over the temperature range of 0 to 45 degrees C; in contrast, the detergent-solubilized signal peptidase of dog pancreatic membranes was completely inactivated at the unusually low temperature of 37 degrees C. It was shown that this inactivation was due to the presence of detergent. Signal peptidase from rat liver was insensitive to a variety of protease inhibitors, like the enzyme from dog pancreas, but differed from the latter in being inhibited by chymostatin and TPCK. 相似文献
11.
Cardiolipin is a major membrane polyglycerophospholipid that is required for the reconstituted activity of a number of key mitochondrial enzymes involved in energy metabolism. Cardiolipin is subjected to remodeling subsequent to its de novo biosynthesis to attain appropriate acyl composition for its biological functions. Yet, the enzyme(s) involved in the remodeling process have not been identified. We report here the identification and characterization of a murine gene that encodes an acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1). Expression of the ALCAT1 cDNA in either insect or mammalian cells led to a significant increase in acyl-CoA:monolysocardiolipin acyltransferase and acyl-CoA: dilysocardiolipin acyltransferase activities that exhibited a dependence upon ALCAT1 enzyme levels. The recombinant ALCAT1 enzyme recognizes both monolysocardiolipin and dilysocardiolipin as substrates with a preference for linoleoyl-CoA and oleoyl-CoA as acyl donors. In contrast, no significant increases in acyltransferase activities by the recombinant ALCAT1 were detected against either glycerol-3-phosphate or a variety of other lysophospholipids as substrates, including lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine. Immunocytohistochemical analysis showed that the ALCAT1 enzyme is localized in the endoplasmic reticulum, which is supported by a significant ALCAT activity in isolated liver and heart microsomes. Northern blot analysis indicates that the mouse ALCAT1 is widely distributed, with the highest expression in heart and liver. In support of a role for ALCAT1 in maintaining heart function, the ALCAT1 gene is conserved among different species of vertebrates, but not in non-atrium organisms. ALCAT1 represents the first identified cardiolipin-remodeling enzyme from any living organism; its identification implies a novel role for the endoplasmic reticulum in cardiolipin metabolism. 相似文献
12.
Microsomal T system: a stereological analysis of purified microsomes derived from normal and dystrophic skeletal muscle 总被引:3,自引:1,他引:2
Heterogeneous populations of microsomes obtained from normal and dystrophic chicken pectoralis muscle were separated into two subfractions by an iterative loading technique. The buoyant density of the sarcoplasmic reticulum (SR) microsomes was increased after loading them with calcium oxalate. Several incubations in the transport medium were necessary to load all of the SR. The fraction that did not form a pellet contained microsomes which displayed freeze-fracture faces that had a low density of particles. A stereological analysis was used on membrane fracture faces of intact muscle to generate reference particle density distributions, which were compared with the distributions measured on the microsomal fracture faces. The concave microsomal fracture faces of purified microsomes which did not load calcium oxalate had particle distributions nearly identical to the distributions of intact P-face T tubules. The morphological data suggest that this subfraction is microsomal T system. Biochemical measurements show negligible amounts of specific Na+, K+-ATPase activity, suggesting that there was little contamination from the surface membrane in this subfraction. Furthermore, an active Ca2+-ATPase is demonstrated in both normal and dystrophic T-tubular membranes. 相似文献
13.
The enzyme acyl-CoA:1-acyl-sn-glycero-3-phosphoinositol acyltransferase (LPI acyltransferase, EC 2.3.1.23) was purified approximately 11,000-fold to near homogeneity from bovine heart muscle microsomes. The purification was effected by extraction with the detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate, followed by chromatography on Cibacron blue agarose, DEAE-cellulose, and Matrex gel green A. The isolated enzyme was a single protein of 58,000 Da as measured by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. This purification procedure also allows isolation of the related enzyme lysophosphatidylcholine (LPC) acyltransferase, which was separated from LPI acyltransferase at the final chromatographic step. The purified LPI acyltransferase exhibits an absolute specificity for LPI as the acyl acceptor. Broader specificity was found for acyl-CoA derivatives as substrates, although the preferred substrates are long-chain, unsaturated derivatives: measured reactivities were in the order arachidonoyl-CoA greater than oleoyl-CoA greater than eicosadienoyl-CoA greater than linoleoyl-CoA. Little activity was found with palmitoyl-CoA or stearoyl-CoA as potential substrates. These properties are consistent with a role of the enzyme in controlling the acyl group composition of phosphoinositides. Comparison of LPC acyltransferase and LPI acyltransferase shows that these two enzymes have distinct kinetic and physical properties and are affected differently by local anesthetics, which are potent inhibitors. 相似文献
14.
Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney 总被引:1,自引:0,他引:1
M D Maines 《Archives of biochemistry and biophysics》1982,216(1):17-26
The acylation of sn-glycerol 3-phosphate with palmityl-CoA was compared in mitochondria and microsomes isolated from rat liver. Polymyxin B, an antibiotic known to alter bacterial membrane structure, stimulated the mitochondrial glycerophosphate acyltransferase but inhibited the microsomal enzyme. When mitochondrial and microsomal fractions were incubated at 4–6 °C for up to 4 h, the mitochondrial enzyme remained virtually unchanged while the microsomal enzyme lost about one-half of its activity. Incubations at higher temperatures also revealed that the mitochondrial enzyme was comparatively more stable under the conditions employed. The mitochondrial acyltransferase showed no sensitivity to bromelain, papain, Pronase, and trypsin, all of which strongly inhibited the microsomal enzyme. The differential sensitivity to trypsin was observed in mitochondria and microsomes isolated from other rat organs. However, the liver mitochondrial glycerophosphate acyltransferase was inhibited by trypsin in the presence of either 0.05% deoxycholate or 0.1% Triton X-100. The trypsin sensitivity of the mitochondrial glycerophosphate acyltransferase in the presence of detergent was not due to the presence, in the mitochondrial fraction, of a trypsin inhibitor which became inactivated by Triton X-100 or deoxycholate. The results suggest that the catalytic site of mitochondrial glycerophosphate acyltransferase is not exposed to the cytosolic side and it is located in the inner aspect of the outer membrane. 相似文献
15.
Comparative studies on mannosylphosphoryl dolichol and glucosylphosphoryl dolichol synthases 总被引:1,自引:0,他引:1
Factors affecting the synthesis of mannosylphosphoryl dolichol and glucosylphosphoryl dolichol hen oviduct microsomes were compared in order to gain insight into the properties of their respective synthases. A stabilized form of mannosylphosphoryl dolichol synthase, but not glucosylphosphoryl dolichol synthase, was released from microsomes by freezing the membranes after exposure to the detergent CHAPSO. The activation energy for mannosylphosphoryl dolichol synthesis in membranes was 9.4 glucosylphosphoryl dolichol synthesis in membranes had a similar activation energy, 8.1 kcal/mol, but below 18 degrees C the value was 16.7 kcal/mol. Tryptic digestion of sealed microsomes preferentially inactivated mannosylphosphoryl dolichol synthase; however, both synthases were equally inactivated in detergent-permeabilized microsomes. Periodate-oxidized UDP-Glc was used to probe the topological orientation of glucosylphosphoryl dolichol synthase in rat liver microsomes. Sealed microsomes treated with oxidized UDP-Glc were inactive in synthesis of glucosylphosphoryl dolichol. However, when these treated microsomes were permeabilized, glucosylphosphoryl dolichol synthase activity was readily detected. From these studies we conclude that although mannosyl- and glucosylphosphoryl dolichol synthases catalyze chemically similar reactions in the endoplasmic reticulum, they differ in several respects. These differences were interpreted in terms of a topological model in which the active sites of the two enzymes reside on opposite faces of the endoplasmic reticulum, with that of the glucosyl lipid synthase facing the lumen and that of the mannosyl lipid synthase facing the cytosol. 相似文献
16.
The thermal stability of glucose-6-phosphatase in rat liver microsomes was examined in untreated and cholate-treated microsomes. Activity of the enzyme was measured with both glucose-6-P and mannose-6-P as substrates. Heat treatment did not cause glucose-6-phosphatase activity to decline to zero with a single rate constant in untreated microsomes. Instead, heat treatment produced an enzyme with a small residual activity that was stable. The residual level of activity was not stimulated by addition of detergent. In untreated microsomes the energies of activation for the processes of decay were different for glucose-6-phosphatase and mannose-6-phosphatase activities, suggesting that the rate-limiting steps for the hydrolysis of these compounds were different. Treatment of microsomes with detergent increased the rate constants for the thermal decay of glucose-6-phosphatase by about 150 times, and, in contrast to untreated microsomes, glucose-6-phosphatase and mannose-6-phosphatase decayed to zero with a single rate constant in cholate-treated microsomes. Also, rate constants for thermal inactivation of glucose-6-phosphatase and mannose-6-phosphatase were the same in cholate-treated microsomes. Removal of cholate increased the stability of glucose-6-phosphatase but did not regenerate the form of the enzyme present in untreated microsomes. The data for the stability of glucose-6-phosphatase under different conditions provide evidence that the enzyme can exist in at least five different stable states that are enzymatically active. 相似文献
17.
A I Archakov I P Kanaeva S Z Kha?tina G I Bachmanova V I Solnyshkin 《Biokhimii?a (Moscow, Russia)》1979,44(3):490-497
A method for membrane reconstitution from cholate-solubilized microsomal proteins and lipids by a removal of the detergent on a column with charcoal has been developed. A comparative study showed that the membranes reconstituted by a dialysis or absorption do not differ from each other in terms of membrane proteins incorporation into lipid vesicles and cytochrome P-450 reconversion into cytochrome P-450. A possibility of biomembrane reconstitution from membrane proteins and lipids solubilized by a non-ionic detergent Triton X-100 was shown. A removal of the detergent results in a formation of membranes, which are chemically close to the original ones but ultrastructurally very different from the latter. On the other hand, absorption or dialysis of cholate-solubilized proteins and lipids results in reconstituted membranes with asymmetrically arranged intramembrane particles located on the hydrophobic surfaces of the membrane halves. The number and size of these particles are similar to those of the original microsomal membranes. 相似文献
18.
G Berthillier R Got 《Comptes rendus des séances de la Société de biologie et de ses filiales》1979,173(5):927-931
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. 相似文献
19.
Paul R.Ortiz De Montellano Bruce A. Mico James M. Mathews Kent L. Kunze Gerald T. Miwa Anthony Y.H. Lu 《Archives of biochemistry and biophysics》1981,210(2):717-728
The autocatalytic destruction of cytochrome P-450 by the following six substrates has been investigated in vivo and in vitro with microsomal and purified, reconstituted rat liver enzymes: 2-isopropyl-4-pentenamide (AIA), 1-ethinylcyclopentanol, 17α-propadienyl-19-nortestosterone, fluroxene, 5,6-dichloro-1,2,3-benzothiadiazole (DCBT), and 1-aminobenzotriazole (ABT). Administration of the first three substrates to rats pretreated with either phenobarbital (Pb) or 3-methylcholanthrene (3-MC), or their incubation with hepatic microsomes from such rats, produced a larger decrease in cytochrome P-450 levels in the membranes from Pb- than 3-MC-treated rats. Comparable losses, however, were observed in microsomes from rats pretreated with both Pb and 3-MC when the last three agents were used. Similar experiments were carried out using the major cytochrome P-450 isozymes purified from liver microsomes of Pb- or 3-MC-treated rats. The Pb isozyme was inactivated during catalytic turnover of all six substrates while only three substrates (DCBT, ABT, and fluroxene) were found to inactivate the 3-MC isozyme. Oxygen consumption studies with purified enzymes have shown that AIA is not a measurable substrate for the 3-MC isozyme, a fact which explains its failure to inactivate this isozyme. Similar studies with the Pb isozyme establish that one enzyme molecule is inactivated for approximately every 230–320 AIA molecules processed by the enzyme. 相似文献
20.
Conflicting data have been published regarding the effects of phenobarbital treatment on bilirubin UDP-glucuronyltransferase activity in native liver microsomes. Recent evidence suggests that the bilirubin UDP-glycosyltransferase system faces the interior of microsomal vesicles, and that expression of its activities in sealed microsomes may be rate-limited by transport of UDP sugars across the membrane. These observations raise the possibility that the reported variability in the effects of phenobarbital may reflect differences in integrity of the membrane in microsomal preparations. We examined the effect of phenobarbital on bilirubin UDP-glucosyltransferase and the UDP-glucuronyltransferase activities towards bilirubin, 4-nitrophenol, and 1-naphthol using native rat liver microsomes with verified vesicle integrity. Phenobarbital-induced microsomes in which the membrane permeability barrier was eliminated by pretreatment with detergent displayed markedly higher UDP-glycosyltransferase activities towards all tested substrates compared with activities in similarly disrupted microsomes from untreated rats. In contrast, none of the transferase activities tested were significantly enhanced by phenobarbital treatment when the enzymic activities were assayed in sealed microsomes. Addition to the enzyme assay mixture of UDPGlcNAc, a presumed physiological activator of the UDP-glucuronyltransferases, failed to expose the enhanced UDP-glucuronyltransferase concentration in phenobarbital-induced sealed microsomes. Our findings are consistent with the idea that transport of UDP sugar across the membrane may be rate-limiting for expression of UDP-glycosyltransferase activities in sealed microsomes. Quantitative assessment of membrane integrity is an essential prerequisite in experiments designed to study the regulation of the microsomal UDP-glycosyltransferase system. 相似文献