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1.
Human Cu,Zn-superoxide dismutase (hSOD1) has 4 cysteines per subunit. Cys57 and Cys148 are involved in an intrasubunit disulfide bond, while Cys6 and Cys111 are free. Cys6 is buried within the protein while Cys111 is on the surface, near the dimer interface. We examined by liquid chromatography-mass spectrometry the commercially purchased hSOD1 isolated from erythrocytes as well as hSOD1s isolated from human erythrocytes, brain, and hSOD1 expressed in Sf9, yeast, and E. coli. Our goal was to ascertain whether the Cys111 modification occurred naturally in vivo. Only the Sigma erythrocyte hSOD1 appeared to contain a trisulfide crosslink between the Cys111 residues. Thus it failed to react with N-ethylmaleimide, showed absorbtion at 325 nm that was eliminated by 2-mercaptoethanol, and had a mass 30 units more than expected for the native dimer. We examined the possibility that different purification methods might cause this modification in erythrocyte hSOD1. None of the procedures examined for hSOD1 purification produced such a trisulfide. In disagreement with Liu et al. [Biochemistry, 2000, 39, 8125-8132], complete derivitization of both Cys111s of hSOD1 from Sf9 cells with N-ethylmaleimide, 4-vinylpyridine, and by 5,5′-dithiobis(2-nitrobenzoic acid) were readily achieved; indicating that steric hindrance was not a problem. 相似文献
2.
Fernando R. Coelho Asif Iqbal Edlaine Linares Daniel F. Silva Filipe S. Lima Iolanda M. Cuccovia Ohara Augusto 《The Journal of biological chemistry》2014,289(44):30690-30701
The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1WT and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp32 residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp32 residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1WT and hSOD1G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp32 residue in the process. The results showed that Trp32 residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp32 residue (bovine SOD1 and hSOD1W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp32 residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1. 相似文献
3.
Human erythrocytes were exposed to oxidative stress by iodate and periodate. Oxidation causes a time- and concentration-dependent increase in membrane permeability for hydrophilic molecules and ions. The induced leak discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy (Ea = 1–4 kcal · mol?1). These results are reconcilable with the formation of aqeous pores. The pore size was approximated to be between 0.45 and 0.6 nm. This increase in permeability is reversible upon treatment with dithioerythritol. Blocking of membrane thiol groups with N-ethylmaleimide protects the membranes against leak formation. The oxidation causes dithioerythritol-reversible modification of membrane proteins as indicated by the gel electrophoretic behavior. These modifications can also be suppressed by blocking the membrane thiol groups with N-ethylmaleimide. About half of the membrane methionine is oxidized to acid hydrolysis-stable derivatives. A fast saturating increase in diene conjugation was observed in whole cells but not in isolated membranes, with only minor degradation of fatty acid chains. The oxidation of cell membrane lipids as well as oxidation of cell surface carbohydrates are not involved in leak formation. Taken together with earlier data (Deuticke, B., Poster, B., Lütkemeier, P., and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196–210), these findings indicate that formation of disulfide bonds by different oxidative mechanisms results in leaks with similar properties. 相似文献
4.
The transport of several neutral amino acids by human erythrocytes in vitro was studied. The measurements made included steady-state distributions, kinetics of initial rates of uptake, effects of monovalent cations and anions, general mutual inhibitory interactions, kinetics of inhibitions, effluxes, ability to produce accelerative exchange diffusion, and the inhibitory action of the thiol reagent N-ethylmaleimide. The results are interpreted as showing that the human erythrocyte membrane possesses several distinct transport systems for these amino acids, including one Na+-dependent system and one dependent on both Na+ and a suitable anion, that are qualitatively similar to those systems previously described in pigeon erythrocytes and mammalian reticulocytes. Quantitatively, however, the systems differ among the different kinds of red cell and a major difference lies in their abilities to produce accelerative exchange diffusion. 相似文献
5.
Izabela J. Kozka Michael K. Gould 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,797(2):212-220
These experiments examined the effects of N-ethylmaleimide on insullin- and oxidant-stimulated sugar transport in soleus muscle in terms of the Thiol-Redox model for insulin-stimulated adipocyte sugar transport (Czech, M.P. (1976) J. Cell. Physiol. 89, 661–668). Brief exposure (1 min) to N-ethylmaleimide (0.3?10 nM) inhibited the stimulatory effect of insulin (0.1 U/ml) on D-[U-14C]xylose uptake by rat soleus muscle. N-Ethylmaleimide also inhibited the stimulatory effects of H2O2 (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin was paralleled by the inhibition of 125I-labelled insulin binding by the muscle. N-ethylmaleimide lowered muscle ATP; however, its effects on sugar transport and 125I-labelled insulin binding could be dissociated from its effect on ATP. Exposing muscles to insulin prior to N-ethylmaleimide did not abolish the inhibitory effect of sulphydryl blockae on insulin-stimulated sugar transport, but did reduce the effect of the inhibitor by 20–30%. Conversely, when muscles were first allowed to bind 125I-labelled insulin and then exposed to the inhibitor, there was no effect of N-ethylmaleimide on pre-bound insulin. Exposure to diamide or vitamin K-5 before N-ethylmaleimide (1 mM) attenuated the inhibitory effet of sulphydryl blockade but no protective effect was observed with H2O2. None of the oxidants protected against the inhibitory effect of 3 nM N-ethylmaleimide. It is concluded that there are two N-ethylmaleimide-sensitive sites involved in the activation of muscle sugar transport at the post-receptor level. One of these would appear to be similar to the Thiol-Redox site described in the adipocyte; the other site appears to be an essential sulphydryl group whose function does not involve oxidation to a disulphide. 相似文献
6.
In order to elucidate the molecular basis of membrane shear elasticity, the effect of membrane protein modification by SH-reagents on the deformability of human erythrocytes was studied. Deformability was quantified by measuring the elongation of erythrocytes subjected to viscometric flow in a transparent cone-plate viscometer. Impermeable SH-reagents proved to have no mechanical effect. Many, but not all, permeable SH-reagents markedly decreased the elongation. Among these, bifunctional SH-reagents (e.g. diamide, tetrathionate and N,N′-p-phenylenedimaleimide) able to cross-link membrane SH-groups were more effective than monofunctional SH-reagents (e.g. N-ethylmaleimide and ethacrynic acid). The bifunctional SH-reagents produced a 50% decrease of elongation after modification of less than 5% of the membrane SH-groups. In contrast, for a comparable effect, more than 20% of the SH-groups had to be modified by the monofunctional reagents. The effect of SH-oxidizing agents was fully reversible after treatment with disulfide-reducing agents. All bifunctional SH-reagents induced a dimerization of a small fraction of spectrin. Analysis of the distribution of the diamide-induced disulfide bonds among the various membrane protein fractions showed that this agent preferentially acts on the spectrin polypeptides.The results provide direct experimental evidence that the native arrangement of spectrin is essential for the shear resistance of the erythrocyte membrane and that introduction of small numbers of intermolecular cross-links as well as modification within the molecule lead to a rapid loss of this function. 相似文献
7.
The effects of N-ethylmaleimide and p-hydroxymercuribenzoate on the ATPase activity of glycerinated Tetrahymena cilia, of 30 S dynein extracted from the cilia, and on the residual ATPase remaining after extraction were studied and correlated with the effects of these reagents on the pellet height response of these cilia. Simultaneous addition of N-ethylmaleimide and ATP to cilia caused a slight inhibition of ATPase activity. Preincubation of the cilia with low N-ethylmaleimide in the absence of ATP, however, enhanced the ATPase activity; the enhancement decreased with increasing time of preincubation. Preincubation of cilia with high N-ethylmaleimide caused increasing inhibition. p-Hydroxymercuribenzoate was more potent than N-ethylmaleimide, usually causing only an inhibition which increased if the cilia were preincubated with p-hydroxymercuribenzoate in the absence of ATP.The pellet height response of these cilia, which serves as a convenient assay of some events related to ciliary motility, was inhibited about 50% by high concentrations of N-ethylmaleimide in the presence of ATP. Preincubation of the cilia with low concentrations of N-ethylmaleimide led to complete loss of the pellet height response. p-Hydroxymercuribenzoate was a more potent inhibitor of the pellet height response than N-ethylmaleimide; complete inhibition was attained even in the presence of ATP, while preincubation with a low concentration of p-hydroxymercuri-benzoate caused a very rapid loss of pellet height response.The ATPase activity of the crude dynein obtained by extraction of cilia and removal of the axonemes was approximately doubled by preincubation with N-ethylmaleimide. 30 S Dynein, obtained from the crude dynein by sedimentation on a sucrose density gradient, was slightly inhibited by N-ethylmaleimide; p-hydroxy-mercuribenzoate was more potent. The residual ATPase activity remaining on the axonemes after two extractions was also only inhibited by N-ethylmaleimide and by p-hydroxymercuribenzoate.These results demonstrated that SH groups influence both the ATPase activity of dynein and the pellet height response of glycerinated cilia. The possible significance of the similarity in enhancing effect of N-ethylmaleimide on cilia ATPase and on myosin ATPase was discussed. 相似文献
8.
Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Lii-Nao countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect of the various reagents indicates that the sensitive SH-groups of the countertransport system are not externally exposed. N-Ethylmaleimide was used to probe for changes elicited by substrate cations in Lii-Nao countertransport. In Na- and Li-free medium, inhibition of Lii-Nao countertransport by N-ethylmaleimide of 35% was reached within 2 s. In Na or Li medium, maximal inhibition was twice as great, but was attained much more slowly, within 10 min. Kinetic data and Hill plot analysis indicate the involvement of two classes of SH-groups: one expressed in the various media with and without substrate cations, and an additional one, which becomes specifically available to N-ethylmaleimide in the presence of external Na or Li. The affinity of Na to the site promoting inhibition by N-ethylmaleimide (apparent Km 12 mM) is higher than the affinity of Na to its external countertransport site (apparent Km 25 mM), as reported by Sarakadi, B., Alifimoff, J.K., Gunn, R.B. and Tosteson, D.C. (1978) J. Gen. Physiol. 72, 249–265). Reactivity of was not modified by the media tested. It is concluded that external Na and Li cause a conformational change in the protein(s) of the countertransport system in human erythrocytes. 相似文献
9.
The less reactive SH groups of soybean β-amylase, SH4, SH5, and SH6, were modified with p-chloromercuribenzoic acid or N-ethylmaleimide, after the reactive SH groups, SHI, SH2, and SH3, were blocked with 5,5′-dithiobis-(2-nitrobenzoic acid) and cyanide. The enzyme activity decreased, accompanied by the modification of SH4. α-Cyclodextrin protected SH4 from the modification more effectively than maltose. The SH4-modified enzyme still bound to glucose, maltose, and α-cyclodextrin. SH4 was concerned with neither the catalysis nor substrate binding but its large substituent affected the substrate binding site. The sequencing of the 5-(iodoacetoamidoethyl)-aminonaphthalene-1-sulfonate-labeled peptides showed that SH4, SH5, and SH6 are Cys343, Cys82, and Cys208, respectively. Comparison of the primary structure of β-amylases also showed that the sequence around SH4 (Cys343), as well as SH2 (Cys95), is strongly conserved between higher plant and bacterial β-amylases. These results agree with the structure model deduced from X-ray crystallography of soybean β-amylase. 相似文献
10.
Myosin reacted with N-ethylmaleimide in the presence of ADP lost its ability to be activated by actin. Subfragment 1 behaved similarly. About 2 moles of N-ethylmaleimide per mole of Subfragment 1 were required to eliminate actin activation of the Mg2+-ATPase activity. At the point at which actin activation was lost the K+-EDTA-ATPase activity was also lost, but the Ca2+-activated ATPase activity was increased. Kinetic measurements indicated that the labelling with N-ethylmaleimide in the presence of ADP reduced V (the ATPase activity at infinite actin concentration) but did not effect Kapp (which is related to the dissociation constant of the actin-Subfragment 1 complex). The Mg2+-activated activity of the reacted myosin alone remained unaltered and the ability to bind actin was retained. We propose that the N-ethylmaleimide labelling blocked the actin activation by preventing the accelerated release of hydrolysis products from the myosin. 相似文献
11.
The reaction pattern with N-[14C]ethylmaleimide served to follow conformational changes of 30 S ribosomal subunits that are induced by association with 50 S subunits and by the binding of aminoacyl-tRNA to 70 S ribosomes either enzymatically or non-enzymatically.The usefulness of the reaction with N-ethylmaleimide in discerning different conformational forms of the ribosome was previously demonstrated (Ginzburg et al., 1973) in an analysis of inactive and active 30 S subunits (as obtained at low Mg2+ and after heat reactivation, respectively). The reaction pattern of the 30 S moiety of 70 S ribosomes differs from the pattern of isolated active subunits (the only form capable of forming 70 S ribosomes) in both the nature of the labeled proteins and in being Mg2+-dependent. The reaction at 10 mm-Mg2+ reveals the following differences between isolated and reassociated 30 S subunits: (1) proteins S1, S18 and S21 that are not labeled in isolated active subunits, but are labeled in the inactive subunits, are highly reactive in 70 S ribosomes; (2) proteins S2, S4, S12 and S17 that uniquely react with N-ethylmaleimide in active subunits are all rendered inaccessible to modification after association; and (3) proteins S9, S13 and S19, that react in both active and inactive 30 S subunits, are labeled to a lesser extent in the 70 S ribosomes than in isolated subunits. This pattern is altered in two respects when the reaction with the maleimide is carried out at 20 mm-Mg2+; protein S18 is not modified while S17 becomes labeled.The differences in reaction pattern are considered as manifesting the existence of different conformational forms of the 30 S subunit in the dissociated and associated states as well as of different forms of 70 S ribosomes. The 30 S moiety of 70 S ribosomes at 10 mm-Mg2+ resembles the inactive subunit, while some of the features of the active subunit are preserved in the 70 S ribosome at 20 mmMg2+. The structural changes appear to be expressed in the functioning of the ribosome: non-enzymatic binding of aminoacyl-tRNA to active 30 S subunits is suppressed by 50 S subunits at 10 mm but not at 20 mm-Mg2+ (Kaufmann &; Zamir, 1972). The fact that elongation factor Tu-mediated binding is not suppressed by 50 S subunits raises the possibility that the function of the elongation factor might involve the facilitation of a conformational change of the ribosome. The analysis of different ribosomal binding complexes with N-ethylmaleimide showed that the binding of poly(U) alone results in a decrease in the labeling of S1 and S18. Binding of aminoacyl-tRNA, on the other hand, is closely correlated with the exposure of S17 for reaction with the maleimide. A model is outlined that accounts for this correlation as well as for the proposed role of elongation factor Tu. 相似文献
12.
A hepatic membrane-associated factor stimulates nuclear DNA synthesis in cultured fibroblastic cells
Subal Bishayee Manjusri Das 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,696(2):134-138
Nuclear DNA replication in cultured mouse fibroblasts is stimulated by isolated hepatic plasma membranes in a time- and concentration-dependent manner. The plasmalemmal activity is susceptible to trypsin treatment, and to treatment with protein modifying agents, N-ethylmaleimide, N-bromosuccinimide, and 2-hydroxy-5-nitro-benzylbromide. 相似文献
13.
Background
Shigella flexneri is the major cause of bacillary dysentery in the developing countries. The lipopolysaccharide (LPS) O-antigen of S. flexneri plays an important role in its pathogenesis and also divides S. flexneri into 19 serotypes. All the serotypes with an exception for serotype 6 share a common O-antigen backbone comprising of N-acetylglucosamine and three rhamnose residues. Different serotypes result from modification of the basic backbone conferred by phage-encoded glucosyltransferase and/or acetyltransferase genes, or plasmid-encoded phosphoethanolamine transferase. Recently, a new site for O-acetylation at positions 3 and 4 of RhaIII, in serotypes 1a, 1b, 2a, 5a and Y was shown to be mediated by the oacB gene. Additionally, this gene was shown to be carried by a transposon-like structure inserted upstream of the adrA region on the chromosome.Results
In this study, a novel bacteriophage Sf101, encoding the oacB gene was isolated and characterised from a serotype 7a strain. The complete sequence of its 38,742 bp genome encoding 66 open reading frames (orfs) was determined. Comparative analysis revealed that phage Sf101 has a mosaic genome, and most of its proteins were >90% identical to the proteins from 12 previously characterised lambdoid phages. In addition, the organisation of Sf101 genes was found to be highly similar to bacteriophage Sf6. Analysis of the Sf101 OacB identified two amino acid substitutions in the protein; however, results obtained by NMR spectroscopy confirmed that Sf101-OacB was functional. Inspection of the chromosomal integration site of Sf101 phage revealed that this phage integrates in the sbcB locus, thus unveiling a new site for integration of serotype-converting phages of S. flexneri, and determining an alternative location of oacB gene in the chromosome. Furthermore, this study identified oacB gene in several serotype 7a isolates from various regions providing evidence of O-acetyl modification in serotype 7a.Conclusions
This is the first report on the isolation of bacteriophage Sf101 which contains the S. flexneri O-antigen modification gene oacB. Sf101 has a highly mosaic genome and was found to integrate in the sbcB locus. These findings contribute an advance in our current knowledge of serotype converting phages of S. flexneri.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-742) contains supplementary material, which is available to authorized users. 相似文献14.
Ei-Tora Yamamura Michio Sayama Makiko Kakikawa Masa-aki Mori Akira Taketo Ken-Ichi Kodaira 《Biochimica et Biophysica Acta (BBA)/General Subjects》2000,1475(1):10-16
The N-hydroxyarylamine O-acetyltransferase of Escherichia coli has been expressed as a histidine tagged fusion protein and purified using immobilized nickel column chromatography. The molecular mass of the histidine tagged N-hydroxyarylamine O-acetyltransferase was estimated to be 60.0 kDa by gel filtration and 34.0 kDa by SDS–PAGE and DNA sequence, suggesting that the native enzyme exists as homo dimer. The catalytic properties were investigated using o-aminobenzoic acid as a substrate. No difference in acetyltransfer activity was observed between histidine tagged protein and untagged enzyme. Kinetic studies indicated a ping-pong bi bi mechanism of the catalysis. Inhibition by N-ethylmaleimide and salicylic acid was competitive with o-aminobenzoic acid and non-competitive with acetyl-CoA. 相似文献
15.
Sadamu Kurono Yuka Kaneko Satomi Niwayama 《Bioorganic & medicinal chemistry letters》2013,23(10):3111-3118
We have developed a methodology for quantitative analysis and concurrent identification of proteins by the modification of cysteine residues with a combination of iodoacetanilide (IAA, 1) and 13C7-labeled iodoacetanilide (13C7-IAA, 2), or N-ethylmaleimide (NEM, 3) and d5-labeled N-ethylmaleimide (d5-NEM, 4), followed by mass spectrometric analysis using nano liquid chromatography/nanoelectrospray ionization ion trap mass spectrometry (nano LC/nano-ESI-IT-MS). The combinations of these stable isotope-labeled and unlabeled modifiers coupled with LC separation and ESI mass spectrometric analysis allow accurate quantitative analysis and identification of proteins, and therefore are expected to be a useful tool for proteomics research. 相似文献
16.
A modification of the method of Prescott and Jones (1) for the colorimetric determination of carbamyl aspartate has been developed to permit the assay of aspartate transcarbamylases in the presence of 2-mercaptoethanol. Interference by this compound is eliminated by means of N-ethylmaleimide. The usefulness of the modified method is illustrated by examination of the contrasting properties of the Escherichia coli and rat liver enzymes. 相似文献
17.
Reaction of isolated bovine rod outer segment membrane with radioactiveN-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule.On the basis of their reactivity towardsp-chloromercuribenzoate andp-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with thep-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and is probably located in the vicinity of the chromophoric center.The difference between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) orN-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of thep-chloromercuri-derivatives. 相似文献
18.
Agavni Petrosyan I-Hui Hsieh John P. Phillips Kourosh Saberi 《Genetics and molecular biology》2015,38(1):107-114
Mutation of the human gene superoxide dismutase (hSOD1) is
associated with the fatal neurodegenerative disease familial amyotrophic lateral
sclerosis (Lou Gehrig’s disease). Selective overexpression of hSOD1
in Drosophila motorneurons increases lifespan to 140% of normal. The
current study was designed to determine resistance to lifespan decline and failure of
sensorimotor functions by overexpressing hSOD1 in
Drosophila‘s motorneurons. First, we measured the ability to
maintain continuous flight and wingbeat frequency (WBF) as a function of age (5 to 50
days). Flies overexpressing hSOD1 under the D42-GAL4 activator were
able to sustain flight significantly longer than controls, with the largest effect
observed in the middle stages of life. The hSOD1-expressed line also
had, on average, slower wingbeat frequencies in late, but not early life relative to
age-matched controls. Second, we examined locomotor (exploratory walking) behavior in
late life when flies had lost the ability to fly (age ≥ 60 d).
hSOD1-expressed flies showed significantly more robust walking
activity relative to controls. Findings show patterns of functional decline
dissimilar to those reported for other life-extended lines, and suggest that the
hSOD1 gene not only delays death but enhances sensorimotor
abilities critical to survival even in late life. 相似文献
19.
Katia Herz Abraham Rimon Elena Olkhova Lena Kozachkov Etana Padan 《The Journal of biological chemistry》2010,285(3):2211-2220
The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na+/H+ antiporter. However, because NhaA is activated at physiological pH (pH 5.5–8.5), many questions related to the active state of NhaA have remained elusive. Our experimental results at physiological pH and computational analyses reveal that amino acid residues in transmembrane segment II contribute to the cation pathway of NhaA and its pH regulation: 1) transmembrane segment II is a highly conserved helix and the conserved amino acid residues are located on one side of the helix facing either the cytoplasmic or periplasmic funnels of NhaA structure. 2) Cys replacements of the conserved residues and measuring their antiporter activity in everted membrane vesicles showed that D65C, L67C, E78C, and E82C increased the apparent Km to Na+ and Li+ and changed the pH response of the antiporter. 3) Introduced Cys replacements, L60C, N64C, F71C, F72C, and E78C, were significantly alkylated by [14C]N-ethylmaleimide implying the presence of water-filled cavities in NhaA. 4) Several Cys replacements were modified by MTSES and/or MTSET, membrane impermeant, negatively and positively charged reagents, respectively, that could reach Cys replacements from the periplasm only via water-filled funnel(s). Remarkably, the reactivity of D65C to MTSES increased with increasing pH and chemical modification by MTSES but not by MTSET, decreased the apparent Km of the antiporter at pH 7.5 (10-fold) but not at pH 8.5, implying the importance of Asp65 negative charge for pH activation of the antiporter. 相似文献