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1.
Summary. Hepatocytes were cultured for 3 days as spheroids (aggregates) or as monolayers in basal medium and in sulfur amino acid-supplemented
media. Cultured hepatocytes had low levels of cysteine dioxygenase (CDO) activity and normal levels of γ-glutamylcysteine
synthetase (GCS) and cysteinesulfinate decarboxylase (CSDC) activities compared to freshly isolated cells. CDO activity increased
and GCS activity decreased in a dose-response manner in cells cultured in either methionine- or cysteine-supplemented media.
CSDC activity was not significantly affected by methionine supplementation. Changes in CDO and GCS were associated with changes
in cysteine catabolism to taurine plus sulfate and in synthesis of glutathione, respectively. These responses are similar
to those observed in liver of intact rats fed diets supplemented with sulfur amino acids. A near-maximal response of CDO or
GCS activity was observed when the medium contained 1.0 mmol/L of methionine plus cyst(e)ine. Changes in CDO and GCS activities
did not appear to be mediated by changes in the intracellular glutathione concentration. Cultured hepatocytes offer a useful
model for further studies of cysteine metabolism and its regulation in response to sulfur amino acid availability.
Received June 2, 1999/Accepted September 16, 1999 相似文献
2.
Stipanuk MH 《Neurochemical research》2004,29(1):105-110
The first-pass metabolism of dietary sulfur amino acids by the liver and the robust upregulation of hepatic cysteine dioxygenase activity in response to an increase in dietary protein or sulfur amino acid level gives the liver a primary role in the removal of excess cysteine and in the synthesis of taurine. Hepatic taurine synthesis is largely restricted by the low availability of cysteinesulfinate as substrate for cysteinesulfinate decarboxylase, and taurine production is increased when cysteinesulfinate increases in response to an increase in the hepatic cysteine concentration and the associated increase in cysteine dioxygenase activity. The upregulation of cysteine dioxygenase in the presence of cysteine is a consequence of diminished ubiquitination of cysteine dioxygenase and a slower rate of degradation by the 26S proteasome. 相似文献
3.
Summary. Alterations in hepatic metabolism of S-amino acids were monitored over one week in male rats treated with a single dose of
ethanol (3 g/kg, ip). Methionine and S-adenosylhomocysteine concentrations were increased rapidly, but S-adenosylmethionine,
cysteine, and glutathione (GSH) decreased following ethanol administration. Activities of methionine adenosyltransferase,
cystathionine γ-lyase and cystathionine β-synthase were all inhibited. γ-Glutamylcysteine synthetase activity was increased from t = 8 hr, but GSH level did not return to control for 24 hr. Hepatic
hypotaurine and taurine levels were elevated immediately, but reduced below control in 18 hr. Changes in serum and urinary
taurine levels were consistent with results observed in liver. Cysteine dioxygenase activity was increased rapidly, but declined
from t = 24 hr. The results show that a single dose of ethanol induces profound changes in hepatic S-amino acid metabolism,
some of which persist for several days. Ethanol not only inhibits the cysteine synthesis but suppresses the cysteine availability
further by enhancing its irreversible catabolism to taurine, which would play a significant role in the depletion of hepatic
GSH.
Received April 26, 2002 Accepted June 12, 2002 Published online October 14, 2002
Authors' address: Young C. Kim, Ph.D., Professor of Toxicology, College of Pharmacy, Seoul National University, San 56-1 Shinrim-Dong, Kwanak-Ku,
Seoul, Korea, Fax: +82-2-872-1795, E-mail: youckim@snu.ac.kr
Abbreviations: CβS, cystathionine β-synthase; CDC, cysteine sulfinate decarboxylase; CDO, cysteine dioxygenase; CγL, cystathionine γ-lyase; GCS, γ-Glutamylcysteine synthetase; GSH, glutathione; MAT, methionine adenosyltransferase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine. 相似文献
4.
Translation of preprochymosin in vitro. Evidence for folding of prochymosin to the native conformation. 总被引:1,自引:0,他引:1
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The uptake and metabolism of 35S-labelled sulphur amino acids were compared in periportal (PP) and perivenous (PV) rat hepatocytes, isolated by digitonin/collagenase perfusion, to identify the factors underlying the previously observed [Kera, Penttilä & Lindros, Biochem. J. (1988) 254, 411-417] higher rate of GSH replenishment in PP cells. The buthionine sulphoximine-inhibitable synthesis of GSH was faster in PP than in PV hepatocytes with both cysteine (6.1 versus 5.0 mumol/h per g of cells) and methionine (4.5 versus 3.3 mumol/h per g) as well as with endogenous precursors and L-2-oxo-4-thiazolidinecarboxylate as substrates. However, the uptake of cysteine by PP cells was slower than by PV cells (8.6 versus 10.3 mumol/h per g of cells), whereas methionine was taken up at similar rates. The activity of gamma-glutamylcysteine synthetase (GCS) was slightly higher in digitonin lysates from the PP than from the PV zone. Production of sulphate, the major catabolite of [35S]cysteine sulphur, as well as incorporation of the label into protein occurred at similar rates in PP and PV cells. Taurine, on the other hand, was produced from [35S]cysteine much faster by PV than by PP cells (0.7 versus 0.1 mumol/h per g of cells). Accordingly, the taurine content of PV hepatocytes tended to be higher and to increase faster during incubation with methionine. These results imply that metabolism of taurine is highly zonated within the acinus. They also suggest that both the slightly lower GCS activity and the fast metabolism of cysteine to taurine limit the capacity of PV hepatocytes to synthesize GSH. 相似文献
5.
Summary. Glutathione (reduced form GSH and oxidized form GSSG) constitutes an important defense against oxidative stress in the brain,
and taurine is an inhibitory neuromodulator particularly in the developing brain. The effects of GSH and GSSG and glycylglycine,
γ-glutamylcysteine, cysteinylglycine, glycine and cysteine on the release of [3H]taurine evoked by K+-depolarization or the ionotropic glutamate receptor agonists glutamate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate
(AMPA) and N-methyl-D-aspartate (NMDA) were now studied in slices from the hippocampi from 7-day-old mouse pups in a perfusion system.
All stimulatory agents (50 mM K+, 1 mM glutamate, 0.1 mM kainate, 0.1 mM AMPA and 0.1 mM NMDA) evoked taurine release in a receptor-mediated manner. Both
GSH and GSSG significantly inhibited the release evoked by 50 mM K+. The release induced by AMPA and glutamate was also inhibited, while the kainate-evoked release was significantly activated
by both GSH and GSSG. The NMDA-evoked release proved the most sensitive to modulation: L-Cysteine and glycine enhanced the
release in a concentration-dependent manner, whereas GSH and GSSG were inhibitory at low (0.1 mM) but not at higher (1 or
10 mM) concentrations. The release evoked by 0.1 mM AMPA was inhibited by γ-glutamylcysteine and cysteinylglycine, whereas
glycylglycine had no effect. The 0.1 mM NMDA-evoked release was inhibited by glycylglycine and γ-glutamylcysteine. In turn,
cysteinylglycine inhibited the NMDA-evoked release at 0.1 mM, but was inactive at 1 mM. Glutathione exhibited both enhancing
and attenuating effects on taurine release, depending on the glutathione concentration and on the agonist used. Both glutathione
and taurine act as endogenous neuroprotective effectors during early postnatal life.
Authors’ address: Prof. Simo S. Oja, Brain Research Center, Medical School, FI-33014 University of Tampere, Finland 相似文献
6.
The effect of cold hardening on the accumulation of glutathione (GSH) and its precursors was studied in the shoots and roots
of wheat (Triticum aestivum L.) cv. Cheyenne (Ch, frost-tolerant) and cv. Chinese Spring (CS, moderately frost-sensitive), in a T. spelta L. accession (Tsp, frost-sensitive) and in chro- mosome substitution lines CS (Ch 5A) and CS (Tsp 5A). The fast induction
of total glutathione accumulation was detected during the first 3 d of hardening in the shoots, especially in the frost-tolerant
Ch and CS (Ch 5A). This observation was corroborated by the study of de novo GSH synthesis using [35S]sulfate. In Ch and CS (Ch 5A) the total cysteine, γ-glutamylcysteine (precursors of GSH), hydroxymethylglutathione and GSH
contents were greater during the 51-d treatment than in the sensitive genotypes. After 35 d hardening, when the maximum frost
tolerance was observed, greater ratios of reduced to oxidised hydroxymethylglutathione and glutathione were detected in Ch
and CS (Ch 5A) compared to the sensitive genotypes. A correspondingly greater glutathione reductase (EC 1.6.4.2) activity
was also found in Ch and CS (Ch 5A). It can be assumed that chromosome 5A of wheat has an influence on GSH accumulation and
on the ratio of reduced to oxidised glutathione as part of a complex regulatory function during hardening. Consequently, GSH
may contribute to the enhancement of frost tolerance in wheat.
Received: 24 March 1999 / Accepted: 19 July 1999 相似文献
7.
Yu. A. Zolotarev A. K. Dadayan E. V. Bocharov Yu. A. Borisov B. V. Vaskovsky E. M Dorokhova N. F. Myasoedov 《Amino acids》2003,24(3):325-333
Summary. The mechanism of the reaction of high temperature solid state catalytic isotope exchange (HSCIE) of hydrogen in peptides with spillover-tritium at 140–180°C was analyzed. This reaction was used for preparing [3H]enkephalins such as [3H]DALG with specific activity of 138 Ci/mmol and [3H]LENK with specific activity of 120 Ci/mmol at 180°C. The analogues of [3H]ACTG4–10 with specific activity of 80 Ci/mmol, [3H]zervamicin IIB with specific activity of 70 Ci/mmol and [3H]conotoxin G1 with specific activity 35 Ci/mmol were produced. The obtained preparations completely retained their biological
activity. [3H]Peptide analysis using 3H NMR spectroscopy on a Varian UNITY-600 spectrometer at 640 MHz was carried out. The reaction ability of amino fragments
in HSCIE was shown to depend both of their structures and on the availability and the mobility of the peptide chain. The reaction
of HSCIE with the β-galactosidase from Termoanaerobacter ethanolicus was studied. The selected HSCIE conditions allow to prepare [3H] β-galactosidase with specific activity of 1440 Ci/mmol and completely retained its the enzymatic activity.
Received November 30, 2001 Accepted January 31, 2002 Published online December 18, 2002
Acknowledgments The work was supported by the Russian Foundation for Basic Research, grant 01-04-48519a.
Authors' address: Dr. Yurii A. Zolotarev, Institute of Molecular Genetics, Russian Academy of Sciences, pl. Kurchatova 2, 123182, Moscow, Russia,
Fax: +7 (095) 196-0221, E-mail: zolya@img.ras.ru
Abbreviations: HSCIE, the reaction of high temperature solid state catalytic isotope exchange; HS, hydrogen spillover; 3H NMR, tritium nuclear magnetic spectroscopy; CtxG1, conotoxin G1; AchR, acetylcholine receptor; HF, Hartree-Fock ab initio quantum-chemical calculation method 相似文献
8.
λ-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization 总被引:1,自引:0,他引:1
λ-Glutamylcysteine synthetase activity (EC 6.3.2.2) was analysed in Sephacryl S-200 eluents of extracts from cell suspension
cultures ofNicotiana tabacum L. cv. Samsun by determination of λ-glutamylcysteine as its monobromobimane derivative. The enzyme has a relative molecular
mass (Mr) of 60000 and exhibits maximal activity at pH 8 (50% at pH 7.0 and pH9.0) and an absolute requirement for Mg2+. With 0.2mM Cd2+ or Zn2+, enzyme activity was reduced by 35% and 19%, respectively. Treatment with 5 mM dithioerythritol led to a heavy loss of activity
and to dissociation into subunits (Mr 34000). Buthionine sulfoximine andl-methionine-sulfoximine, known as potent inhibitors of λ-glutamylcysteine synthetase from mammalian cells, were found to be
effective inhibitors of the plant enzyme too. The apparent Km values forl-glutamate,l-cysteine, and α-aminobutyrate were, respectively, 10.4mM, 0.19 mM, and 6.36 mM. The enzyme was completely inhibited by glutathione
(Ki=0.42 mM). The data indicate that the rate of glutathione synthesis in vivo may be influenced substantially by the concentration
of cysteine and glutamate and may be further regulated by feedback inhibition of λ-glutamylcysteine synthetase by glutathione
itself. λ-Glutamylcysteine synthetase is, like glutathione synthetase, localized in chloroplasts as well as in the cytoplasm.
Chloroplasts fromPisum sativum L. isolated on a Percoll gradient contained about 72% of the λ-glutamylcysteine synthetase activity in leaf cells and 48%
of the total glutathione synthetase activity. In chloroplasts ofSpinacia oleracea L. about 61% of the total λ-glutamylcysteine synthetase activity of the cells were found and 58% of the total glutathione
synthetase activity. These results indicate that glutathione synthesis can take place in at least two compartments of the
plant cell.
Dedicated to Professor A. Prison on the occasion of his 80th birthday 相似文献
9.
Summary. The effect of dietary sulfur amino acids on the taurine content of rat blood and tissues was investigated. Three types of
diet were prepared for this study: a low-taurine diet (LTD), normal taurine diet (NTD; LTD + 0.5% Met), and high-taurine diet
(HTD; LTD + 0.5% Met + 3% taurine). These diets had no differing effect on the growth of the rats. The concentration of taurine
in the blood from the HTD- and NTD-fed rats was respectively 1,200% and 200% more than that from LTD-. In such rat tissues
as the liver, the taurine content was significantly affected by dietary sulfur amino acids, resulting in a higher content
with HTD and lower content with LTD. However, little or no effect on taurine content was apparent in the heart or eye. The
activity for taurine uptake by the small intestine was not affected by dietary sulfur amino acids. The expression level of
taurine transporter mRNA was altered only in the kidney under these dietary conditions: a higher expression level with LTD
and lower expression level with HTD.
Received January 8, 2002 Accepted January 18, 2002 Published online August 20, 2002
Authors' address: Dr. Hideo Satsu, Laboratory of Food Chemistry, Department of Applied Biological Chemistry, Graduate School of Agricultural
and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, Fax: +81-3-5841-8026 E-mail: asatsu@mail.ecc.u-tokyo.ac.jp
Abbreviations: HTD, high-taurine diet; NTD, normal taurine diet; LTD, low-taurine diet; TAUT, taurine transporter; CSA, cysteine sulfinate;
CDO, cysteine dioxygenase; CSAD, cysteine sulfinate decarboxylase; PBS, phosphate-buffered saline; DIDS, 4,4′-diisothiocyanostilbene-2′,2′-disulfonic
acid 相似文献
10.
M. Wimmer 《Histochemistry and cell biology》1989,92(4):331-336
Summary Phosphoenolpyruvate carboxykinase activity in rat liver was shown to be heterotopically distributed within the acinus under varying feeding conditions. Highest values of PEPCK activity were found in the periportal zone of the acinus from where it decreased continuously towards the perivenous zone. 84 h of starvation resulted in an increase of activity, which was most prominent in the perivenous zone, but nevertheless resulted in a steeper gradient. Refeeding of starved rats with a high carbohydrate diet for 6 nights led to a decrease in PEPCK activity which was most prominent in the periportal zone, but almost negligible in the perivenous zone, resulting in a further change in the activity gradient.Sex-dependent differences for total PEPCK activity were found i) in controls, where the activity was lower in females, ii) after starvation, where the induction was much higher in females, and iii) after refeeding of starved rats, where the activity in females remained higher compared to that of the controls. Differences in the intra-acinar localization of the activity in dependence of the sex were registrated in the control group and in starved rats. Livers from female rats contained a higher periportal/perivenous ratio compared to males. In starved and starved and refed animals the periportal/perivenous ratios were almost the same in both sexes. 相似文献
11.
By the use of a newly developed technique of ultrathin-layer electrophoresis, class I and class II alcohol dehydrogenase
activity could be demonstrated in microdissected samples of the periportal, intermediate, and perivenous zones of the liver
acinus in men and women. It could be demonstrated that both classes exhibit low activity in the periportal zone. From there,
a rising gradient in the direction of the perivenous end was apparent. This increase, however, was found to be significant
only in women. The analysis of class I alcohol dehydrogenase isoenzymes showed that the expression of α-, β-, and γ-containing
isoforms did not differ in relation to the intraacinar position. The constant proportions of the isoenzymes to the maxima
and minima of the total alcohol dehydrogenase activity support the view that the adult liver-specific isoenzyme pattern is
determined during postnatal development.
Accepted: 1 February 1999 相似文献
12.
Sex differences of the influence of T3 on the topical distribution of phosphoenolpyruvate carboxykinase activity in the liver acinus 总被引:1,自引:0,他引:1
M. Wimmer 《Histochemistry and cell biology》1989,92(2):109-113
Summary Phosphoenolpyruvate carboxykinase (PEPCK) activity was investigated in livers of triiodothyronine (T3) treated male and female rats with special regard to its intraacinar localization. In untreated controls of both, male and female rats, the activity was heterotopically distributed within the acinus with highest values in the periportal zone, and with lowest values in the perivenous zone. This periportal to perivenous activity gradient revealed to be under the influence of T3. Application of T3 resulted in a relative increase of PEPCK activity which was much greater in the livers of females than in males. The extent of T3-induced augmentation of PEPCK activity was dependent on the intraacinar position. In both sexes greatest relative activation was found in the perivenous zone. In female animals, the perivenous activity of T3 treated livers was comparable to that observed in the periportal zone of controls. 相似文献
13.
Cysteine, gamma-Glutamylcysteine, and Glutathione Levels in Maize Seedlings : Distribution and Translocation in Normal and Cadmium-Exposed Plants
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The levels of cysteine (Cys), γ-glutamylcysteine (γEC), and glutathione (GSH) were measured in the endosperms, scutella, roots, and shoots of maize (Zea mays L.) seedlings. GSH was the major thiol in roots, shoots, and scutella, Cys predominated in endosperms. The endosperm, scutellum, and functional phloem translocation were required for maintenance of GSH pools in roots and shoots of 6-day-old seedlings. Exposure of roots to 3 micromolar Cd, besides causing a decline in GSH, caused an accumulation of γEC, as if the activity of GSH synthetase was reduced in vivo. [35S]Cys injected into endosperms of seedlings was partly metabolized to [35S]sulfate. The scutella absorbed both [35S]sulfate and [35S]Cys and transformed 68 to 87% of the radioactivity into [35S]GSH. [35S]GSH was translocated to roots and shoots in proportion to the tissue fresh weight. Taken together, the data supported the hypothesis that Cys from the endosperm is absorbed by the scutellum and used to synthesize GSH for transfer through the phloem to the root and shoot. The estimated flux of GSH to the roots was 35 to 60 nanomoles per gram per hour, which totally accounted for the small gain in GSH in roots between days 6 and 7. For Cd-treated roots the GSH influx was similar, yet the GSH pool did not recover to control levels within 24 hours. The estimated flux of GSH to the entire shoot was like that to the roots; however, it was low (11-13 nanomoles per gram per hour) to the first leaf and high (76-135 nanomoles per gram per hour) to the second and younger leaves. 相似文献
14.
Distribution of cyclic AMP phosphodiesterase in microdissected periportal and perivenous rat liver tissue with different dietary states 总被引:1,自引:0,他引:1
Summary Cyclic AMP phosphodiesterase was measured in liver homogenates and microdissected periportal and perivenous liver tissue from rats in different dietary states under different conditions of substrate saturation and effector stimulation. A radiochemical microtest, more sensitive by 2–3 orders of magnitude than the usual assay, was established for the determination of the activity in liver samples corresponding to 200–800 ng dry weight. At saturating cyclic AMP concentrations (46 M) phosphodiesterase was homogeneously distributed within the liver acinus of fed rats. Starvation for 48 h led to a decrease in the overall activity and to a heterogenous distribution with slightly higher activities in the perivenous zone. At physiological cyclic AMP concentrations (1.8 M) phosphodiesterase showed a flat zonal gradient in livers of fed rats with higher levels in the periportal zone; after 48 h starvation it was homogeneously distributed. In the presence of cyclic GMP (2 M) the basal activity at physiological substrate concentrations was stimulated to a greater extent in the perivenous zone. This led to a homogeneous activity distribution in the fed state and to a heterogenous pattern with a slight perivenous maximum in the fasted state. Thus there was no or only a small zonal heterogeneity of signal transmitting enzymes such as cyclic AMP phosphodiesterase and glucagon-stimulated adenylate cyclase (Zierz and Jungermann 1984). This similar signal transducing capacity in the periportal and the perivenous area will contribute to maintain the zonation of signal input due to the hormone concentration gradients across the liver acinus. 相似文献
15.
Maize (Zea mays L.) cell cultures incorporated radioactivity from [14C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5–20 min, the
CoA pool had lost its 14C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a
pulse–chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age.
In young (1–3 d) cultures, polysaccharide-bound [14C]feruloyl- and [14C]diferuloyl residues were both detectable within 1 min of [14C]cinnamate feeding. Thus, feruloyl residues were dimerised <1 min after their attachment to polysaccharides. For at least
the first 2.3 h after [14C]cinnamate feeding, polysaccharide-bound [14C]diferuloyl residues remained almost constant at ≈7% of the total polysaccharide-bound [14C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides <1 min after the biosynthesis of the latter,
and >10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H2O2 (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In
older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [14C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [14C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H2O2 induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H2O2-limited. In both 2- and 4-d-old cultures, polysaccharide-bound 14C-trimers and larger coupling products exceeded [14C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually
quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides
in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely
to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response
of cell walls to an oxidative burst are discussed.
Received: 19 January 2000 / Accepted: 13 April 2000 相似文献
16.
The biosynthesis of galactan was investigated using microsomal membranes isolated from suspension-cultured cells of potato
(Solanum tuberosum L. var. AZY). Incubation of the microsomal membranes in the presence of UDP-[14C]galactose resulted in a radioactive product insoluble in 70% methanol. The product released only [14C]galactose upon acid hydrolysis. Treatment of the product with Aspergillus niger endo-1,4-β-galactanase released 65–70% of the radioactivity to a 70%-methanol-soluble fraction. To a minor extent, [14C]galactose was also incorporated into proteins, however these galactoproteins were not a substrate for Aspergillus niger endo-1,4-β-galactanase. Thus, the majority of the 14C-labelled product was 1,4-β-galactan. Compounds released by the endo-1,4-β-galactanase treatment were mainly [14C]galactose and [14C]galactobiose, indicating that the synthesized 1,4-β-galactan was longer than a trimer. In vitro synthesis of 1,4-β-galactan
was most active with 6-d-old cells, which are in the middle of the linear growth phase. The optimal synthesis occurred at
pH 6.0 in the presence of 7.5 mM Mn2+. Aspergillus aculeatus rhamnogalacturonase A digested at least 50% of the labelled product to smaller fragments of approx. 14 kDa, suggesting that
the synthesized [14C]galactan was attached to the endogenous rhamnogalacturonan I. When rhamnogalacturonase A digests of the labelled product
were subsequently treated with endo-1,4-β-galactanase, radioactivity was not only found as [14C]galactose or [14C]galactobiose but also as larger fragments. The larger fragments were likely the [14C]galactose or [14C]galactobiose still attached to the rhamnogalacturonan backbone since treatment with β-galactosidase together with endo-1,4-β-galactanase
digested all radioactivity to the fraction eluting as [14C]galactose. The data indicate that the majority of the [14C]galactan was attached directly to the rhamnose residues in rhamnogalacturonan I. Thus, isolated microsomal membranes contain
enzyme activities to both initiate and elongate 1,4-β-galactan sidechains in the endogenous pectic rhamnogalacturonan I.
Received: 24 June 1999 / Accepted: 30 August 1999 相似文献
17.
Gribar JJ Ramachandra M Hrycyna CA Dey S Ambudkar SV 《The Journal of membrane biology》2000,173(3):203-214
P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as an ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine residues 91, 94, and
99 located within the first extracellular loop. We report here the biochemical characterization of glycosylation-deficient
(Gly−) P-gp using a vaccinia virus based transient expression system. The staining of HeLa cells expressing Gly− P-gp (91, 94, and 99N→Q), with P-gp specific monoclonal antibodies, MRK-16, UIC2 and 4E3 revealed a 40 to 50% lower cell-surface
expression of mutant P-gp compared to the wild-type protein. The transport function of Gly− P-gp, assessed using a variety of fluorescent compounds indicated that the substrate specificity of the pump was not affected
by the lack of glycosylation. Additional mutants, Gly− D (91, 94, 99N→D) and Gly−Δ (91, 94, 99 N deleted) were generated to verify that the reduced cell surface expression, as well as total expression, were
not a result of the glutamine substitutions. Gly− D and Gly−Δ Pgps were also expressed to the same level as the Gly− mutant protein. 35S-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of 35S-methionine/cysteine in full length Gly− P-gp compared to wild-type protein, but the half-life (∼3 hr) of mutant P-gp was essentially unaltered. Since treatment with
proteasome inhibitors (MG-132, lactacystin) increased only the intracellular level of nascent, mutant P-gp, the decreased
incorporation of 35S-methionine/cysteine in Gly− P-gp appears to be due to degradation of improperly folded mutant protein by the proteasome and endoplasmic reticulum-associated
proteases. These results demonstrate that the unglycosylated protein, although expressed at lower levels at the cell surface,
is functional and suitable for structural studies.
Received: 28 July 1999/Revised: 20 October 1999 相似文献
18.
A microsomal preparation from suspension-cultured potato stem cells (Solanum tuberosum L. cv. AZY) was incubated with [14C]acetyl-CoA resulting in a precipitable radiolabeled product. Analysis of the product revealed that it consisted mostly of
acetylated proteins and cell wall polysaccharides, including xyloglucan, homogalacturonan and rhamnogalacturonan I. Thus,
acetyl-CoA is a donor-substrate for the O-acetylation of wall polysaccharides. A rhamnogalacturonan acetylesterase was used to develop an assay to measure and characterize
rhamnogalacturonan O-acetyl transferase activity in the microsomal preparation. Using this assay, it was shown that the transferase activity was
highest during the linear growth phase of the cells, had a pH-optimum at pH 7.0, a temperature optimum at 30 °C, an apparent
K
m of 35 μM and an apparent V
max of 0.9 pkat per mg protein. Further analysis of the radiolabeled acetylated product revealed that it had a molecular mass
>500 kDa.
Received: 3 July 1999; Accepted: 27 September 1999 相似文献
19.
Avilés C Torres-Márquez ME Mendoza-Cózatl D Moreno-Sánchez R 《Archives of microbiology》2005,184(2):83-92
To determine the onset of the Cd2+-hyperaccumulating phenotype in Euglena gracilis, induced by Hg2+ pretreatment (Avilés et al. in Arch Microbiol 180:1–10, 2003), the changes in cellular growth, Cd2+ uptake, and intracellular contents of sulfide, cysteine, γ-glutamylcysteine, glutathione and phytochelatins during the progress
of the culture were analyzed. In cells exposed to 0.2 mM CdCl2, the Cd2+-hyperaccumulating phenotype was apparent only after 48 h of culture, as indicated by the significant increase in cell growth
and higher internal contents of sulfide and thiol-compounds, along with a higher γ-glutamylcysteine synthetase activity. However,
the stiochiometry of thiol-compounds/Cd2+ accumulated was similar for both control and Hg2+-pretreated cells. Moreover, the value for this ratio was 2.1 or lower after 48-h culture, which does not suffice to fully
inactivate Cd2+. It is concluded that, although the glutathione and phytochelatin synthesis pathway is involved in the development of the
Cd2+-hyperaccumulating phenotype in E. gracilis, apparently other pathways and sub-cellular mechanisms are also involved. These may be an increase in other Cd2+ chelating molecules such as di- and tricarboxylic acids, phosphate and polyphosphates, as well as Cd2+ compartmentation into organelles.
César Avilés: In memoriam. 相似文献
20.
Summary. Resveratrol (3,4′,5-trihydroxy-trans-stilbene) is a naturally occurring phytoalexin and polyphenol existing in grapes and various other plants, and one of the
best known ‘nutriceuticals’. It shows a multiplicity of beneficial biological effects, particularly, by attenuating atherogenic,
inflammatory, and carcinogenic processes. However, despite convincing evidence from experimental and clinical studies, data
concerning the role of resveratrol and other members of the large polyphenols family for human health is still a matter of
debate. One reason for this is the lack of suitable sensitive and specific methods, which would allow direct assessment of
biodistribution, biokinetics, and the metabolic fate of these compounds in vivo. The unique features of positron emission tomography (PET) as a non-invasive in vivo imaging methodology in combination with suitable PET radiotracers have great promise to assess quantitative information on
physiological effects of polyphenols in vivo. Herein we describe the radiosynthesis of an 18F-labelled resveratrol derivative, 3,5-dihydroxy-4′-[18F]fluoro-trans-stilbene ([18F]-1), using the Horner-Wadsworth-Emmons reaction as a novel radiolabelling technique in PET radiochemistry for subsequent functional
imaging of polyphenol metabolism in vivo. In a typical “three-step/one-pot” reaction, 18F-labelled resveratrol derivative [18F]-1 could be synthesized within 120–130 min including HPLC separation at a specific radioactivity of about 90 GBq/μmol. The radiochemical
yield was about 9% (decay-corrected) related to [18F]fluoride and the radiochemical purity exceeded 97%. First radiopharmacological evaluation included measurement of biodistribution
ex vivo and positron emission tomography (PET) studies in vivo after intravenous application of [18F]-1 in male Wistar rats using a dedicated small animal PET camera with very high spatial resolution. Concordantly with data on
bioavailability and metabolism of native resveratrol from the literature, these investigations revealed an extensive uptake
and metabolism in the liver and kidney, respectively, of [18F]-1. This study represents the first investigation of polyphenols in vivo by means of PET. 相似文献