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1.
The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity and thermotropic changes in membrane structure.
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Arrhenius plots of the non-latent UDP-glucuronyltransferase (p-nitrophenol acceptor) activity of guinea-pig microsomal membranes prepared with 154 mM-KCl were linear from 5 to 40 degrees C. Arrhenius plots for other microsomal preparations from guinea pig and rat liver that show various degrees of transferase latency, exhibited two linear regions intersecting at a sharp transition point near 20-25 degrees C. This discontinuity was abolished or greatly decreased when transferase latency was removed by treating the membranes with perturbants of phospholipid bilayer strucutre. The fluorescent probe N-phenyl-1-naphthyl-amine detected a thermotropic change in the fluidity of the phospholipid acyl chains of all the microsomal membrane preparations studied, at temperatures close to those of the Arrhenius-plot transitions. It is concluded that the thermotropic change in the structure of the membrane bilayer probably is a 'phase separation' or clustering of phospholipids, which affects a permeability barrier that restricts access of substrate to the transferase molecules. 相似文献
2.
The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity (reverse reaction).
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Arrhenius plots of the non-latent UDP-glucuronlytransferase reverse reaction (p-nitrophenyl glucuronide donor) activity of guinea-pig microsomal membranes prepared with 15 mM-KCl were linear from 5 to 40 degrees C. These plots for other preparations from guinea-pig and rat liver (i.e. preparations that show transferase latency) exhibited two linear regions intersecting at a transition point near 19--21 degrees C. This discontinuity was abolished when latency was removed by treating the membranes with perturbants of phospholipid-bilayer structure. Thus the temperature-depdendnces of the reverse reaction catalysed by the enzymes of these various preparations are similar to those of the corresponding forward reactions [Pechey, Graham & Wood (1978) Biochem. J. 175, 115--1124]. Perturbants activated the enzyme of KCl-prepared guinea-pig microsomal membranes only slightly and caused no significant alteration to Arrhenius plots of its forward or reverse reaction activities. These results support the 'compartmentation' theory of UDP-glucuronyltransferase lactency. 相似文献
3.
Properties of membrane-bound bilirubin UDP-glucuronyltransferase in rough and smooth endoplasmic reticulum and in the nuclear envelope from rat liver.
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We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations. 相似文献
4.
P. Steinberg R. Steinbrecher D. Schrenk P. Munzel M. Bruck H. Gschaidmaier F. Oesch K. W. Bock 《Cell biology and toxicology》1994,10(1):59-65
The activities of several different phase I and phase II drug-metabolizing enzymes were measured in freshly isolated oval cells from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6 weeks and alsoin vitro in the established oval cell line OC/CDE 6. No cytochrome P450 was spectrophotometrically measurable in both preparations and two cytochrome P450-dependent monoxygenase activities, aminopyrineN-demethylase and ethoxyresorufinO-deethylase, could not be detected in the oval cells of both sources. However, cytosolic glutathione transferase, microsomal expoxide hydrolase and UDP-glucuronosyltransferase activities were clearly measurable in oval cells. Similar enzyme activities were found in freshly isolated and cultured oval cells. The highest activities of these three enzymes were detected during the exponential growth phase of the cultured cells; thereafter the activities decreased until the cells reached confluency. Changes in phenol UDP-glucuronosyltransferase (UGT1A1) mRNA levels paralleled the variations in UDP-glucuronosyltransferase activity, i.e. they were high in exponentially growing oval cells and low in confluent cell cultures. Taking into account that oval cells are able to proliferate in the livers of rats continuously fed a choline-deficient/DL-ethionine-supplemented diet and that none of the analyzed drug metabolizing enzymes are involved in the activation or detoxication ofDL-ethionine, the described pattern might be part of a more general, nonspecific, protection mechanism enabling these cells to overcome the cytotoxic effects of a variety of carcinogens and to proliferate even in their presence. Furthermore, the expression of microsomal epoxide hydrolase, cytosolic glutathione transferase and UDP-glucuronosyltransferase appears to depend on the proliferative status of the cells.Abbreviations CDE
choline-deficient/DL-ethionine-supplemented diet
- GST
glutathione transferase
- mEH
microsomal epoxide hydrolase
- UGT
UDP-glucuronosyltransferase 相似文献
5.
Rat liver nuclei have 2 to 12% of the corresponding microsomal aryl hydrocarbon hydroxylase, aminopyrine and benzphetamine N-demethylase, NADPH-cytochrome c reductase, and epoxide hydrase activities. Nuclear membranes were prepared from isolated liver nuclei by a sucrose density centrifugation technique. A 2.5- to 10.2-fold increase in the specific enzyme activities was observed in nuclear membrane as compared to intact nuclei. Several properties of the rat liver nuclear membrane and microsomal epoxide hydrase have been compared. Nuclear epoxide hydrase was similar to the corresponding microsomal enzyme in being induced by phenobarbital whereas 3-methylcholanthrene did not produce any effects. Nuclear membrane and microsomal epoxide hydrase were inhibited to a similar degree by 1,1,1-trichloropropene oxide, cyclohexene oxide, an trans-stilbene oxide. The apparent Km value of nuclear membrane epoxide hydrase was 20 μm for benzo(a)pyrene 4,5-oxide, which is 5.5-fold lower than the corresponding microsomal Km value (112 μm). Nuclear membranes were prepared from isolated nuclei of rat kidney, lung, spleen, and heart by the DNase digestion method. Epoxide hydrase activity in intact nuclei was in the following order: kidney > lung ? spleen, or heart. Increases of 2.2- and 2.5-fold in specific epoxide hydrase activity were observed in kidney and lung when nuclear membranes were compared to intact nuclei. DMSO, dimethylsulfoxide 相似文献
6.
Characterization of a thyroid-hormone-binding site on nuclear envelopes and nuclear matrices of the male-rat liver 总被引:1,自引:0,他引:1
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Nuclear envelopes and nuclear matrices were isolated from the male-rat liver. Incubation of 125I-labelled 3,3',5-tri-iodothyronine (T3) with the nuclear-envelope fraction resulted in specific binding of T3 to the membranes. Maximum specific binding occurred at 30 degrees C after 2h incubation. Storage for 1 week at -80 degrees C resulted in no loss of binding. Scatchard analysis revealed a class of binding sites with KD 86 nM. 3,3',5'-Tri-iodothyronine was as effective a competitor of [125I]T3 binding to nuclear envelopes as was L-T3 itself, and tri-iodothyroacetic acid was 70% as potent as T3. L- and D-thyronine did not compete for [125I]T3 binding. Incubation of nuclear envelopes with 0.6 M-NaCl before addition of T3 resulted in the complete loss of specific binding sites, whereas exposure of the membranes to 2.0 M-NaCl after incubation with T3 did not extract binding sites. Nuclear matrices, after incubation with [125I]T3 under the same conditions, were shown to possess a class of binding sites with a similar KD but with approx. 30% of the maximum binding capacity. Nuclear envelopes from hypothyroid animals may possess slightly lower numbers of binding sites compared with nuclear envelopes from the intact animal, whereas nuclear matrices from hypothyroid animals have the same number of binding sites as do nuclear envelopes from the intact animal. In conclusion, nuclear envelopes and nuclear matrices have a class of binding sites with relatively high affinity for T3. It is distinct from nuclear and cytosolic binding sites. 相似文献
7.
Nuclear gangliosides were characterized using two distinct fractions of large (N1) and small (N2) nuclear populations from rat brain. The ganglioside concentration of N1 nuclei from adult rat brain was 0.92 microg sialic acid/mg protein, which was about 3.8 times higher than that of N2 nuclei. N1 and N2 nuclear gangliosides showed similar compositional profiles; they contained major gangliosides of GM1, GD1a, GD1b, and GT1b, with GM3 in lesser amounts. c-Series gangliosides such as GT3, GQ1c, and GP1c were also detected in both nuclear preparations. Nuclear localization of gangliosides was confirmed by immunofluorescence with anti-GM1 antibody, cholera toxin B subunit, and c-series ganglioside-specific monoclonal antibody A2B5. Developmental changes of nuclear gangliosides were examined using rats of different ages ranging from embryonic day 14 (E14) to postnatal 7 weeks. The concentration of N1 nuclear gangliosides changed only slightly during development and did not correlate with that of whole-brain gangliosides. The developmental pattern of ganglioside composition of N1 nuclei was also distinguished from that of microsomal membranes; the ganglioside changes in N1 nuclei included reduced expression of di- and polysialogangliosides at E16 and higher proportions of GM3 at early and late stages of the period. These findings suggest that gangliosides in nuclear membranes are developmentally regulated in a distinct manner in brain cells. 相似文献
8.
H Okuda N Tavoloni C L Kiang M J Jones P D Berk 《Biochemical and biophysical research communications》1983,117(2):406-412
The conjugates formed in vitro by bilirubin UDP-glucuronyl transferase were studied by examining reaction products as intact tetrapyrroles, rather than as dipyrrolic azoderivatives. Bile pigments were extracted from conventional microsomal enzyme reaction mixtures by affinity chromatography over albumin-agarose, eluted with 50% ethanol, and separated by a silica gel thin layer chromatographic system. In the presence of UDPGA, native and activated microsomal preparations all formed both bilirubin mono- and diglucuronides from unconjugated bilirubin, and bilirubin diglucuronide from bilirubin monoglucuronide. No significant non-enzymatic conversion of mono- to diglucuronide occurred without UDPGA, or in the presence of denatured enzyme. Hence, bilirubin diglucuronide is a major product of bilirubin-UDP-glucuronyl transferase. 相似文献
9.
Changes in the localization of terminal transferase during the cell cycle in random cultures of human pre-T leukemia line RPMI-8402 were examined by light and electron microscopy on immunoperoxidase-stained preparations. Paraformaldehyde-fixed and saponin-permeabilized human cells were used with a monoclonal anti-human terminal deoxynucleotidyl transferase (TdT) primary reagent to demonstrate changes in enzyme distribution occurring between interphase and mitosis. Nuclear localization is found uniformly during interphase. At metaphase, however, the majority of TdT staining appears randomly distributed in the cytoplasm and traces of TdT staining remain associated with mitotic chromatin. At later phases, when the daughter cells are forming, the enzyme again appears to be restricted to the new nuclear structure. 相似文献
10.
R Juvonen M Laitinen E Hietanen M Nieminen 《The International journal of biochemistry》1984,16(10):1083-1086
The UDP-glucuronosyltransferase activity towards 4-nitrophenol, 1-naphthol and phenolphthalein was measured from the hepatic microsomes of the reindeer (Rangifer tarandus tarandus) after summer, autumn and winter feeding periods. The microsomes were digested with trypsin or digitonin. The UDP-glucuronosyltransferase activity with 4-nitrophenol and 1-naphthol as aglycones was lower in reindeer on winter food than in ones on summer food after trypsin and digitonin digestion. The activity towards phenolphthalein was the same in each feeding period. The different seasonal feeding affects the structure of microsomal membranes and this is reflected as modifications of the UDP-glucuronosyltransferase towards different substrates. 相似文献
11.
A set of inhibitors for rat liver microsomal glutathione transferase have been characterized. These inhibitors (rose bengal, tributyltin acetate, S-hexylglutathione, indomethacin, cibacron blue and bromosulphophtalein) all have I50 values in the 1-100 microM range. Their effects on the unactivated enzyme were compared to those on the N-ethylmaleimide- and trypsin-activated microsomal glutathione transferase. It was found that the I50 values were decreased upon activation of the enzyme (5-20-fold), except for S-hexylglutathione, where a slight increase was noted. Thus, the activated microsomal glutathione transferase is generally more sensitive to the effect of inhibitors than the unactivated enzyme. It was also noted that inhibitor potency can vary dramatically depending on the substrate used. The I50 values for the N-ethylmaleimide- and trypsin-activated enzyme preparations are altered in a similar fashion compared to the unactivated enzyme. This finding indicates that these two alternative mechanisms of activation induce a similar type of change in the microsomal glutathione transferase. 相似文献
12.
A temporary but marked postnatal decline in UDP-glucuronosyltransferase activity occurs in homogenates and microsomes from rat liver. The profile of this trough and its time of occurrence (maximal over 13-16 days) are almost identical with the two substrates 2-aminophenol and 1-naphthol, whose rates of glucuronidation differ 10-fold. The trough is greatest with digitonin-activated preparations, least with fresh latent ('native') enzyme and intermediate when the native enzyme is treated with its specific activator UDP-N-acetylglucosamine (UDP-GlcNAc). Less detailed evidence supports similar conclusions with 4-nitrophenol as substrate. The trough is not due to the presence of an inhibitor of the transferase in rat liver at 15 days of age. Over the whole perinatal period, including the time of the trough, the enzyme in homogenates can be activated by UDP-GlcNAc; the microsomal enzyme is activated to a rather lesser degree perinatally, and evidence suggests this may be due to artefacts introduced during tissue fractionation. When the overall process of glucuronidation is studied in snips of intact liver offered high concentrations of the two different phenols, the trough is again evident over the same period as observed with broken cells, and of equal depth for both substrates. The infant rat is therefore probably less able to glucuronidate hepatically these phenols over the suckling or early weaning period than are the adult, late foetus or newborn, and may be especially incompetent at 13-16 days of age. 相似文献
13.
Glutathione dependent control of protein disulfide-sulfhydryl content by subcellular fractions of hepatic tissue 总被引:4,自引:0,他引:4
The disulfide-sulfhydryl (SS/SH) ratios of subcellular fractions of rat hepatic tissue were found to vary diurnally with the ratio lowest in the early morning and highest in the early evening. These changes were found in the nuclear, microsomal and cytosol fractions. The primary reaction is the reversible formation of mixed disulfides of glutathione with proteins. This formation is controlled by the activity of thiol transferase and the level of oxidized glutathione (GSSG) as substrate. Several enzymes including mitochondrial and microsomal oxidases, glutathione reductase and peroxidase and glucose-6-phosphate dehydrogenase were found to control the levels of GSSG. An NADPH-dependent microsomal oxidase system, inhibited by GSSG, was found to produce activated oxygen which served as substrate for flutathione peroxidase. Evidence is presented for the concept that the formation of mixed disulfides of proteins with glutathione is a mechanism for maintenance of a disulfide-sulfhydryl ratio such that the integrity of particulate membranes is maintaine during oxidative and reductive stresses on the hepatic cells. 相似文献
14.
Nuclear membranes and other subcellular fractions derived from bovine brain cortex were investigated for the existence of GTP-binding proteins. By using photolytic labeling with [alpha-32P]GTP a 29 kDa GTP-binding protein was shown to be present in nuclear membranes which was not present in the plasma membranes nor in microsomal or cytosolic fractions. Two-dimensional gel electrophoresis revealed that this protein is rather acidic with a pI lower than 4.5. Members of the heterotrimeric Gi/o family are not present in the nuclear envelope: a 39 kDa protein, ADP ribosylated by pertussis toxin, was shown to originate from plasma membrane contamination. 相似文献
15.
Tekle M Bentinger M Nordman T Appelkvist EL Chojnacki T Olsson JM 《Biochemical and biophysical research communications》2002,291(5):1128-1133
The possibility that ubiquinone biosynthesis is present in rat liver peroxisomes was investigated. The specific activity of trans-prenyltransferase was 30% that of microsomes, with a pH optimum of around 8. trans-Geranyl pyrophosphate was required as a substrate and maximum activity was achieved with Mn(2+). Several detergents specifically inactivated the peroxisomal enzyme. The peroxisomal transferase is present in the luminal soluble contents, in contrast to the microsomal enzyme which is a membrane component. The treatment of rats with a number of drugs has demonstrated that the activities in the two organelles are subjected to separate regulation. Nonaprenyl-4-hydroxybenzoate transferase has about the same specific activity in peroxisomes as in microsomes and like the transferase activity, its regulation differs from the microsomal enzyme. The results demonstrate that peroxisomes are involved in ubiquinone biosynthesis, and at least two enzymes of the biosynthetic sequence are present in this organelle. 相似文献
16.
P J Lad J W Brown W T Shier 《Biochemical and biophysical research communications》1978,85(4):1472-1479
Of seven marine sponges tested only two, and , yielded preparations that activated rat heart microsomal guanylate cyclase and exhibited direct hemolytic activity. These two preparations also inhibited basal and fluoride-activated adenylate cyclase in rat heart microsomes and glucagon-stimulated adenylate cyclase in rat liver plasma membranes. Hemolytic activity co-purified with nucleotide cyclase-modulating activity during a standard lipid fractionation procedure. This fraction was cytotoxic to 3T3-4a Swiss mouse fibroblasts. 相似文献
17.
The major polypeptides of the nuclear pore complex 总被引:24,自引:0,他引:24
Nuclear envelopes of maturing oocytes of various amphibia contain an unusually high number of pore complexes in very close packing. Consequently, nuclear envelopes, which can be manually isolated in great purity, provide a remarkable enrichment of nuclear pore complex material, relative to membranous and other interporous structures. When the polypeptides of nuclear envelopes isolated from oocytes of Xenopus laevis and Triturus alpestris are examined by gel electrophoresis, visualized either by staining with Coomassie blue or by radiofluorography after in vitro reaction with [3H]dansyl chloride, a characteristic pattern is obtained (10 major and 15 minor bands). This polypeptide pattern is radically different from that of the nuclear contents isolated from the same cell. Extraction of the nuclear envelope with high salt concentrations and moderately active detergents such as Triton X-100 results in the removal of membrane material but leaves most of the non-membranous structure of the pore complexes. The dry weight of the pore complex (about 0.2 femtograms) remains essentially unchanged during such extractions as measured by quantitative electron microscopy. The extracted preparations which are highly enriched in nuclear pore complex material contain only two major polypeptide components with apparent molecular weights of 150 000 and 73 000. Components of such an electrophoretic mobility are not present as major bands, if at all, in nuclear contents extracted in the same way. It is concluded that these two polypeptides are the major constituent protein(s) of the oocyte nuclear pore complex and are specific for this structure. When nuclear envelopes are isolated from rat liver and extracted with high salt buffers and Triton X-100 similar bands are predominant, but two additional major components of molecular weights of 78 000 and 66 000 are also recognized. When the rat liver nuclear membranes are further subfractionated material enriched in the 66 000 molecular weight component can be separated from the membrane material, indicating that this is relatively loosely associated material, probably a part of the nuclear matrix. The results suggest that the nuclear pore complex is not only a characteristic ubiquitous structure but also contains similar, if not identical, skeletal proteins that are remarkably resistant to drastic changes of ionic strength as well as to treatments with detergents and thiol reagents. 相似文献
18.
NUCLEAR MEMBRANES FROM MAMMALIAN LIVER : I. Isolation Procedure and General Characterization 总被引:19,自引:14,他引:5
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Werner W. Franke Barbara Deumling Baerbel Ermen Ernst-Dieter Jarasch Hans Kleinig 《The Journal of cell biology》1970,46(2):379-395
Nuclear membranes were isolated from rat and pig liver by sonication of highly purified nuclear fractions and subsequent removal of adhering nucleoproteins in a high salt medium. The fractions were examined in the electron microscope by both negative staining and thin sectioning techniques and were found to consist of nuclear envelope fragments of widely varying sizes. Nuclear pore complex constituents still could frequently be recognized. The chemical composition of the nuclear membrane fractions was determined and compared with those of microsomal fractions prepared in parallel. For total nuclei as well as for nuclear membranes and microsomes, various enzyme activities were studied. The results indicate that a similarity exists between both fractions of cytomembranes, nuclear envelope, and endoplasmic reticulum, with respect to their RNA:protein ratio and their content of polar and nonpolar lipids. Both membranous fractions had many proteins in common including some membrane-bound enzymes. Activities in Mg-ATPase and the two examined cytochrome reductases were of the same order of magnitude. The content of cytochrome b5 as well as of P-450 was markedly lower in the nuclear membranes. The nuclear membranes were found to have a higher buoyant density and to be richer in protein. The glucose-6-phosphatase and Na-K-ATPase activities in the nuclear membrane fraction were very low. In the gel electrophoresis, in addition to many common protein bands, some characteristic ones for either microsomal or nuclear membranous material were detected. Significant small amounts of DNA and RNA were found to remain closely associated with the nuclear envelope fragments. Our findings indicate that nuclear and endoplasmic reticulum membranes which are known to be in morphological continuity have, besides a far-reaching similarity, some characteristic differences. 相似文献
19.
J.P. Blanchet 《Experimental cell research》1974,84(1-2)
Nuclear membranes and plasma membranes of chicken erythrocytes were tested for some enzyme activities and polypeptide content. Both membranes show ATPase and NADH cytochrome c reductase activities, which were higher for nuclear membrane preparations; besides the nuclear membrane ATPase is not stimulated by Na + K + ions. SDS solubilization of these membranes followed by separation using SDS polyacrylamide gel electrophoresis yields about 25 bands. Nuclear membranes and plasma membranes possess specific bands; some differences were seen when adult and embryonic membranes were compared. 相似文献
20.
Specific binding of androgen and androgen-receptor complex by microsomes from rat ventral prostate 总被引:1,自引:0,他引:1
Jaime Steinsapir Thomas G. Muldoon 《The Journal of steroid biochemistry and molecular biology》1990,37(5):697-705
Microsomes from rat ventral prostate show the presence of a high affinity-low capacity population of androgen-binding sites with affinity for ionic exchange resin similar to that of cytosol androgen receptor (AR), as manifested by similar results obtained with hydroxylapatite. The affinity for mibolerone was similar for both forms (Ka = 0.5-2.9 x 10(10) M-1). The membrane-bound form can be extracted in hypotonic buffer, with retention of binding properties. Isotonic sucrose allowed higher degree of extractability of the microsomal AR than 10% (v/v) glycerol. The presence of hormone lends stability to the microsomal AR, while high salt or nonionic detergents have a deleterious effect on their longevity. The microsomal receptor form is not sensitive to serine-proteases as opposed to the cytosol AR. After exhaustive extraction of binding sites, microsomes are capable of accepting cytosol mibolerone-receptor complexes to a level corresponding to the concentration of depleted binding sites; microsomes from non-target tissue do not manifest such capability. Microsomal AR complexes do not bind DNA and they are not activated after heat treatment. Mixed preparations of extracted microsomal complexes with cytosol complexes showed heat-induced increased ability to bind DNA to the same level of diluted cytosol complex alone, indicating the absence of a microsomal inhibitor of DNA binding. The results indicate the co-existence of a non-DNA binding form of the AR in the microsomal membranes with the classical DNA binding form of the AR present in the cytosol of ventral prostate homogenates. 相似文献