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1.
NADH diferric transferrin reductase in liver plasma membrane   总被引:6,自引:0,他引:6  
Evidence is presented that rat liver plasma membranes contain a distinct NADH diferric transferrin reductase. Three different assay procedures for demonstration of the activity are described. The enzyme activity is highest in isolated plasma membrane, and activity in other internal membranes is one-eighth or less than in plasma membrane. The activity is inhibited by apotransferrin and antitransferrin antibodies. Trypsin treatment of the membranes leads to rapid loss of the transferrin reductase activity as compared with NADH ferricyanide reductase activity. Erythrocyte plasma membranes, which lack transferrin receptors, show no diferric transferrin reductase activity, although NADH ferricyanide reductase is present. The transferrin reductase is inhibited by agents that inhibit diferric transferrin reduction by intact cells and is activated by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate) detergent. Inhibitors of mitochondrial electron transport have no effect on the activity. We propose that the NADH diferric transferrin reductase in plasma membranes measures the activity of the enzyme that causes the reduction of diferric transferrin by intact cells. This transmembrane electron transport system requires the transferrin receptor for diferric transferrin reduction. Because the transmembrane electron transport has been shown to stimulate cell growth, the reduction of diferric transferrin at the cell surface may be an important function for diferric transferrin in stimulation of cell growth, in addition to its role in iron transport.  相似文献   

2.
Iron-limited cells of the green alga Chlorella kesslerii use a reductive mechanism to acquire Fe(III) from the extracellular environment, in which a plasma membrane ferric reductase reduces Fe(III)-chelates to Fe(II), which is subsequently taken up by the cell. Previous work has demonstrated that synthetic chelators both support ferric reductase activity (when supplied as Fe(III)-chelates) and inhibit ferric reductase. In the present set of experiments we extend these observations to naturally-occurring chelators and their analogues (desferrioxamine B mesylate, schizokinen, two forms of dihydroxybenzoic acid) and also two formulations of the commonly-used herbicide N-(phoshonomethyl)glycine (glyphosate). The ferric forms of the larger siderophores (desferrioxamine B mesylate, schizokinen) and Fe(III)-N-(phoshonomethyl)glycine (as the isopropylamine salt) all supported rapid rates of ferric reductase activity, while the iron-free forms inhibited reductase activity. The smaller siderophores/siderophore precursors, 2,3- and 3,4-dihydroxybenzoic acids, did not support high rates of reductase in the ferric form but did inhibit reductase activity in the iron-free form. Bioassays indicated that Fe(III)-chelates that supported high rates of ferric reductase activity also supported a large stimulation in the growth of iron-limited cells, and that an excess of iron-free chelator decreased the growth rate. With respect to N-(phosphonomethyl)glycine, there were differences between the pure compound (free acid form) and the most common commercial formulation (which also contains isopropylamine) in terms of supporting and inhibiting ferric reductase activity and growth. Overall, these results suggest that photosynthetic organisms that use a reductive strategy for iron acquisition both require, and are potentially simultaneously inhibited by, ferric chelators. Furthermore, these results also may provide an explanation for the frequently contradictory results of N-(phosphonomethyl)glycine application to crops: we suggest that low concentrations of this molecule likely solubilize Fe(III), making it available for plant growth, but that higher (but sub-lethal) concentrations decrease iron acquisition by inhibiting ferric reductase activity.  相似文献   

3.
R Horn 《Biophysical journal》1991,60(2):329-333
In perforated patch recording, the pore former nystatin is incorporated into a cell-attached patch, to increase its conductance. The possibility of lateral diffusion of nystatin through the membrane and under the glass-membrane seal was examined by reversing the nystatin gradient. Namely, a cell-attached patch on a cell was examined while placing nystatin into the bath. The reversal potential and current-voltage relationship of single Ca2+ activated K+ channels in the patch were readily changed by varying the K+ concentration in the bath, showing that nystatin was active in the cell membrane outside of the patch. However, the patch itself did not become leaky. The absence of a conductance induced in the patch by the nystatin in the rest of the plasma membrane of the cell suggests that the lateral diffusion of nystatin is inhibited by the glass-membrane seal.  相似文献   

4.
This paper critically examines the redox activity of K562 cells (chronic myelogenous leukemia cells) and normal peripheral blood lymphocytes (PBL). Ferricyanide reduction, diferric transferrin reduction, and ferric ion reduction were measured spectrophotometrically by following the time-dependent changes of absorbance difference characteristic for ferricyanide disappearance and for the formation of ferrous ion:chelator complexes. Bathophenanthroline disulfonate (BPS) and ferrozine (FZ) were used to detect the appearance of ferrous ions in the reaction mixtures when diferric transferrin or ferric reduction was studied. Special attention was devoted to the analysis of time-dependent absorbance changes in the presence and absence of cells under different assay conditions. It was observed and concluded that: (i) FZ was far less sensitive and more sluggish than BPS for detecting ferrous ions at concentrations commonly used for BPS; (ii) FZ, at concentrations of at least 10-times the commonly used BPS concentrations, seemed to verify the results obtained with BPS; (iii) ferricyanide reduction, diferric transferrin reduction and ferric ion reduction by both K562 cells and peripheral blood lymphocytes did not differ significantly; and (iv) earlier values published for the redox activities of different cells might be overestimated, partly because of the observation published in 1988 that diferric transferrin might have loosely bound extra iron which is easily reduced. It is suggested that the specific diferric transferrin reduction by cells might be considered as a consequence of (i) changing the steady-state equilibrium in the diferric transferrin-containing solution by addition of ferrous ion chelators which effectively raised the redox potential of the iron bound in holotransferrin, and (ii) changing the steady-state equilibrium by addition of cells which would introduce, via their large and mostly negatively charged plasma membrane surface, a new phase which would favor release and reduction of the iron in diferric transferrin by a ferric ion oxidoreductase. The reduction of ferricyanide is also much slower than activities reported for other cells which may indicate reduced plasma membrane redox activity in these cells.  相似文献   

5.
The transfer of iron from diferric transferrin to bathophenanthroline disulfonate was measured under varying conditions by spectrophotometry and EPR spectroscopy. Intact rat hepatocytes efficiently mediated the transfer of iron from human diferric transferrin to bathophenanthroline disulfonate. Isolated rat liver plasma membranes, in contrast, failed to facilitate the reaction at pH 7.4 in the presence of NADH, although the membranes were able to reduce ferricyanide and to oxidize NADH. Oxidation of NADH was stimulated by diferric transferrin. However, ferricyanide reductase and transferrin-stimulated NADH oxidase activities were apparently not linked to release of iron from transferrin. Our results, together with theoretical considerations, show that the ability (or inability) of intact cells or isolated plasma membranes to facilitate the transfer of iron from transferrin to strong diferric iron chelators does not allow interferences about the existence of an iron reduction step as part of the process of cellular uptake of iron from transferrin.  相似文献   

6.
Ubiquinol regeneration by plasma membrane ubiquinone reductase   总被引:1,自引:0,他引:1  
Summary Several enzyme systems have been proposed to play a role in the maintenance of ubiquinol in membranes other than the inner mitochondrial membrane. The aim of this study was to investigate the mechanisms involved in NADH-driven regeneration of antioxidant ubiquinol at the plasma membrane. Regeneration was measured by quantifying the oxidized and reduced forms of ubiquinone by electrochemical detection after separation by high-performance liquid chromatography. Plasma membrane incubation with NADH resulted in the consumption of endogenous ubiquinone, and a parallel increase in ubiquinol levels. The activity showed saturation kinetics with respect to the pyridine nucleotides and was moderately inhibited byp-hydroxymercuribenzoate. Only a slight inhibition was achieved with dicumarol at concentrations reported to fully inhibit DT-diaphorase. Salt-extracted membranes displayed full activity of endogenous ubiquinol regeneration, supporting the participation of an integral membrane protein. In liposomes-reconstituted systems, the purified cytochromeb 5 reductase catalyzed the reduction of the natural ubiquinone homologue coenzyme Q10 at rates accounting for the activities observed in whole plasma membranes, and decreased the levels of lipid peroxidation. Our data demonstrate the role of the cytochromeb 5 reductase in the regeneration of endogenous ubiquinol.Abbreviations AAPH 2,2-azobis-(2-amidinopropane) hydrochloride - CoQ coenzyme Q, ubiquinone - CoQH2 reduced coenzyme Q, ubiquinol - pHMB p-hydroxymercuribenzoate  相似文献   

7.
Members of the Rab family of small GTPases play important roles in membrane trafficking along the exocytic and endocytic pathways. The Rab11 subfamily consists of two highly conserved members, Rab11a and Rab11b. Rab11a has been localized both to the pericentriolar recycling endosome and to the trans-Golgi network and functions in recycling of transferrin. However, the localization and function of Rab11b are completely unknown. In this study green fluorescent protein (GFP)-tagged Rab11b was used to determine its subcellular localization. GFP-Rab11b colocalized with internalized transferrin, and using different mutants of Rab11b, the role of this protein in transferrin uptake and recycling was examined. Two of these mutants, Rab11b-Q/L (constitutively active) and Rab11b-S/N (constitutively inactive), strongly inhibited the recycling of transferrin. Interestingly, both of them had no effect on transferrin uptake. In contrast, the C-terminally altered mutant Rab11b-DeltaC, which cannot be prenylated and therefore cannot interact with membranes, did not interfere with wild-type Rab11b function. From these data we concluded that functional Rab11b is essential for the transport of internalized transferrin from the recycling compartment to the plasma membrane.  相似文献   

8.
Proton release from HeLa cells is stimulated by external oxidants for the transplasmalemma electron transport enzymes. These oxidants, such as ferricyanide and diferric transferrin, also stimulate cell growth. We now present evidence that proton release associated with the reduction of ferricyanide and diferric transferrin is through the Na+/H+ antiport. The stoichiometry of H+/e- release with diferric transferrin is over 50 to 1, which is greater than expected for oxidation of a protonated transmembrane electron carrier. Diferric transferrin induced proton release depends on external sodium and is inhibited by amiloride. Proton release is also inhibited when diferric transferrin reduction is inhibited by apotransferrin. A tightly coupled association between the redox system and the antiport is shown by sodium dependence and amiloride inhibition of diferric transferrin reduction. The results indicate a new role for ferric transferrin in growth stimulation by activation of the sodium-proton antiport.  相似文献   

9.
Incubation of adriamycin resistant Chinese hamster lung cells with low levels of N-ethylmaleimide (NEM) results in a major increase in the cellular accumulation of drug. When resistant cells are prelabeled with [32Pi] and thereafter treated with NEM there also occurs a selective superphosphorylation of an 180K plasma membrane glycoprotein (P-180). This phosphorylation reaction occurs at both serine and threonine residues. In similar experiments with drug sensitive cells only minor levels of this protein can be detected. Detailed studies have established that in cells which have reverted to drug sensitivity there is a parallel loss in the presence of phosphorylated P-180. Also in cells which have undergone partial reversion to drug sensitivity there is a correlation between levels of superphosphorylated P-180 and adriamycin resistance. These results provide evidence that adriamycin resistance is dependent on the presence of P-180. The results also suggest that the biological activity of this protein is highly regulated by phosphorylation and that in the superphosphorylated state P-180 is inactive and under these conditions the resistant cell is converted to a drug sensitive phenotype.  相似文献   

10.
The thioredoxin system facilitates proliferative processes in cells and is upregulated in many cancers. The activities of both thioredoxin (Trx) and its reductase (TrxR) are mediated by oxidation/reduction reactions among cysteine residues. A common target in preclinical anticancer research, TrxR is reported here to be significantly inhibited by the anticancer agent laromustine. This agent, which has been in clinical trials for acute myelogenous leukemia and glioblastoma multiforme, is understood to be cytotoxic principally via interstrand DNA crosslinking that originates from a 2-chloroethylating species generated upon activation in situ. The spontaneous decomposition of laromustine also yields methyl isocyanate, which readily carbamoylates thiols and primary amines. Purified rat liver TrxR was inhibited by laromustine with a clinically relevant IC50 value of 4.65 μM. A derivative of laromustine that lacks carbamoylating activity did not appreciably inhibit TrxR while another derivative, lacking only the 2-chloroethylating activity, retained its inhibitory potency. Furthermore, in assays measuring TrxR activity in murine cell lysates, a similar pattern of inhibition among these compounds was observed. These data contrast with previous studies demonstrating that glutathione reductase, another enzyme that relies on cysteine-mediated redox chemistry, was not inhibited by methylcarbamoylating agents when measured in cell lysates. Mass spectrometry of laromustine-treated enzyme revealed significant carbamoylation of TrxR, albeit not on known catalytically active residues. However, there was no evidence of 2-chloroethylation anywhere on the protein. The inhibition of TrxR is likely to contribute to the cytotoxic, anticancer mechanism of action for laromustine.  相似文献   

11.
Plasma-membrane-bound nitrate reductase (PM-NR) is located in roots and leaves of tobacco (Nicotiana tabacum L. cv. Samsun) and reduces nitrate with NADH as electron donor. When plasma membranes were prepared under specific protecting conditions, a PM-NR of roots was detected that accepts electrons from succinate to reduce nitrate. Comparison between the succinate dehydrogenase of mitochondria and the succinate-oxidising PM-NR of roots indicated that they are two different enzymes. Partial purification of the nitrate reductase forms by anion-exchange chromatography indicated that succinate and NADH supply electrons to the same plasma-membrane-bound protein. Received: 27 March 1997 / Accepted: 9 April 1997  相似文献   

12.
Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.Abbreviations L1 first lower phase - NR nitrate reductase - NRA nitrate-reductase activity - PM plasma membrane - T:p Triton X-100 (octylphenoxy polyethoxyethanol) to protein ratio - U2 second upper phase  相似文献   

13.
Chloroquine is a weak base which has been shown to inhibit lysosomal acidification. Chloroquine inhibits iron uptake in reticulocytes at a concentration of 0.5 mM. It is also effective in the control of malaria and other parasitic diseases. We now report that chloroquine inhibits NADH diferric transferrin reductase as well as the proton release stimulated by diferric transferrin from liver and HeLa cells. Ammonium chloride which also inhibits endosome acidification does not significantly inhibit the NADH diferric transferrin reduction. NADH diferric transferrin reductase of isolated rat liver plasma membrane is inhibited by chloroquine at concentrations similar to those required for inhibition of diferric transferrin reduction by whole cells. Ferricyanide reduction by whole cells is also inhibited by chloroquine. These observations provide an alternative mechanism for chloroquine control of acidification of endosomes and suggests a new approach to control of protozoal parasites through inhibition of a transmembrane oxidoreductase which controls transmembrane proton movement.  相似文献   

14.
The mitochondrial inner membrane anion channel (IMAC) is a channel, identified by flux studies in intact mitochondria, which has a broad anion selectivity and is maintained closed or inactive by matrix Mg2+ and H+. We now present evidence that this channel, like many other chloride/anion channels, is reversibly blocked/inhibited by stilbene-2,2-disulfonates. Inhibition of malonate transport approaches 100% with IC50 values of 26, 44, and 88 M for DIDS, H2-DIDS, and SITS respectively and Hill coefficients 1. In contrast, inhibition of Cl transport is incomplete, reaching a maximum of about 30% at pH 7.4 and 65% at pH 8.4 with an IC50 which is severalfold higher than that for malonate. The IC50 for malonate transport is decreased about 50% by pretreatment of the mitochondria withN-ethylmaleimide. Raising the assay pH from 7.4 to 8.4 increases the IC50 by about 50%, but under conditions where only the matrix pH is made alkaline the IC50 is decreased slightly. These properties and competition studies suggest that DIDS inhibits by binding to the same site as Cibacron blue 3GA. In contrast, DIDS does not appear to compete with the fluorescein derivative Erythrosin B for inhibition. These findings not only provide further evidence that IMAC may be more closely related to other Cl channels than previously thought, but also suggest that other Cl channels may be sensitive to some of the many regulators of IMAC which have been identified.  相似文献   

15.
Plasma membrane vesicles from adult rat brain synaptosomes (PMV) have an ascorbate-dependent NADH oxidase activity of 35-40 nmol/min/(mg protein) at saturation by NADH. NADPH is a much less efficient substrate of this oxidase activity, with a Vmax 10-fold lower than that measured for NADH. Ascorbate-dependent NADH oxidase activity accounts for more than 90% of the total NADH oxidase activity of PMV and, in the absence of NADH and in the presence of 1 mm ascorbate, PMV produce ascorbate free radical (AFR) at a rate of 4.0 +/- 0.5 nmol AFR/min/(mg protein). NADH-dependent *O2- production by PMV occurs with a rate of 35 +/- 3 nmol/min/(mg protein), and is a coreaction product of the NADH oxidase activity, because: (i) it is inhibited by more than 90% by addition of ascorbate oxidase, (ii) it is inhibited by 1 micro g/mL wheat germ agglutinin (a potent inhibitor of the plasma membrane AFR reductase activity), and (iii) the KM(NADH) of the plasma membrane NADH oxidase activity and of NADH-dependent *O2- production are identical. Treatment of PMV with repetitive micromolar ONOO- pulses produced almost complete inhibition of the ascorbate-dependent NADH oxidase and *O2- production, and at 50% inhibition addition of coenzyme Q10 almost completely reverts this inhibition. Cytochrome c stimulated 2.5-fold the plasma membrane NADH oxidase, and pretreatment of PMV with repetitive 10 microm ONOO- pulses lowers the K0.5 for cytochrome c stimulation from 6 +/- 1 (control) to 1.5 +/- 0.5 microm. Thus, the ascorbate-dependent plasma membrane NADH oxidase activity can act as a source of neuronal.O2-, which is up-regulated by cytosolic cytochrome c and down-regulated under chronic oxidative stress conditions producing ONOO-.  相似文献   

16.
Doxorubicin (adriamycin) is cytotoxic to cells, but the biochemical basis for this effect is unknown, although intercalation with DNA has been proposed. This study suggests that the cytotoxicity of this drug may be due to inhibition of the plasma membrane redox system, which is involved in the control of cellular growth. Concentrations between 10–6–10–7 M adriamycin inhibit plasma membrane redox reactions >50%. AD32, a form of adriamycin which does not intercalate with DNA, but is cytotoxic, also inhibits the plasma membrane redox system. Thus, the cytotoxic effects of adriamycin, which limit its use as a drug, may be based on the inhibition of a transplasma membrane dehydrogenase involved in a plasma membrane redox system.  相似文献   

17.
G Warren  J Davoust    A Cockcroft 《The EMBO journal》1984,3(10):2217-2225
There is a marked reduction in the number of surface transferrin receptors as A431 cells enter mitosis which persists until telophase when receptors reappear to a level that exceeds the original interphase value. This is most simply explained by assuming that recycling of receptors back to the cell surface is inhibited as cells enter mitosis but that internalisation continues for a short while, causing surface receptor depletion. In telophase recycling would resume before internalisation giving a temporary excess of surface transferrin receptors.  相似文献   

18.
Retinoic acid inhibits the reduction of diferric transferrin through the transplasma membrane electron transport system on fetal rat liver cells infected with a temperature-sensitive SV40 virus when the cells are in the nontransformed state cultured at 40°C. When the cells are in the transformed state (grown at the permissive 33°C temperature), retinoic acid does not inhibit the diferric transferrin reduction. Inhibition of activity of nontransformed cells is specific for retinoic acid with only slight inhibition by retinol and retinyl acetate at higher concentrations. Isolated rat liver plasma membrane NADH diferric transferrin reductase is also inhibited by retinoic acid. The effect of transformation with SV40 virus to decrease susceptibility to retinoic acid inhibition stands in contrast to much greater adriamycin inhibition of diferric transferrin reduction in the transformed cells than in nontransformed cells.  相似文献   

19.
TRPM1 is the founding member of the melastatin subgroup of transient receptor potential (TRP) proteins, but it has not yet been firmly established that TRPM1 proteins form ion channels. Consequently, the biophysical and pharmacological properties of these proteins are largely unknown. Here we show that heterologous expression of TRPM1 proteins induces ionic conductances that can be activated by extracellular steroid application. However the current amplitudes observed were too small to enable a reliable biophysical characterization. We overcame this limitation by modifying TRPM1 channels in several independent ways that increased the similarity to the closely related TRPM3 channels. The resulting constructs produced considerably larger currents after overexpression. We also demonstrate that unmodified TRPM1 and TRPM3 proteins form functional heteromultimeric channels. With these approaches, we measured the divalent permeability profile and found that channels containing the pore of TRPM1 are inhibited by extracellular zinc ions at physiological concentrations, in contrast to channels containing only the pore of TRPM3. Applying these findings to pancreatic β cells, we found that TRPM1 proteins do not play a major role in steroid-activated currents of these cells. The inhibition of TRPM1 by zinc ions is primarily due to a short stretch of seven amino acids present only in the pore region of TRPM1 but not of TRPM3. Combined, our data demonstrate that TRPM1 proteins are bona fide ion-conducting plasma membrane channels. Their distinct biophysical properties allow a reliable identification of endogenous TRPM1-mediated currents.  相似文献   

20.
Studies reproted here indicate that a major cell surface glycoprotein of the rabbit bone marrow erythroid cell may be involved in binding transferrin. The glycoprotein has an apparent molecular weight of 18,000. It is suggested that bone marrow erythroid cells may provide an invaluable source of the red cell membrane transferrin receptor for future studies.  相似文献   

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