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1.
Tet(L) and tet(K) tetracycline-divalent metal/H+ antiporters: characterization of multiple catalytic modes and a mutagenesis approach to differences in their efflux substrate and coupling ion preferences
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The Tet(L) protein encoded in the Bacillus subtilis chromosome and the closely related Tet(K) protein from Staphylococcus aureus plasmids are multifunctional antiporters that have three cytoplasmic efflux substrates: a tetracycline-divalent metal (TC-Me(2+)) complex that bears a net single positive charge, Na+, and K+. Tet(L) and Tet(K) had been shown to couple efflux of each of these substrates to influx of H+ as the coupling ion. In this study, competitive cross-inhibition between K+ and other cytoplasmic efflux substrates was demonstrated. Tet(L) and Tet(K) had also been shown to use K+ as an alternate coupling ion in support of Na+ or K+ efflux. Here they were shown to couple TC-Me(2+) efflux to K+ uptake as well, exhibiting greater use of K+ as a coupling ion as the external pH increased. The substrate and coupling ion preferences of the two Tet proteins differed, especially in the higher preference of Tet(K) than Tet(L) for K+, both as a cytoplasmic efflux substrate and as an external coupling ion. Site-directed mutagenesis was employed to test the hypothesis that some feature of the putative "antiporter motif," motif C, of Tet proteins would be involved in these characteristic preferences. Mutation of the A157 in Tet(L) to a hydroxyamino acid resulted in a more Tet(K)-like K+ preference both as coupling ion and efflux substrate. A reciprocal S157A mutant of Tet(K) exhibited reduced K+ preference. Competitive inhibition among substrates and the parallel effects of the single mutation upon K+ preference, as both an efflux substrate and coupling ion, are compatible with a model in which a single translocation pathway through the Tet(L) and Tet(K) transporters is used both for the cytoplasmic efflux substrates and for the coupling ions, in an alternating fashion. However, the effects of the A157 and other mutations of Tet(L) indicate that even if there are a shared binding site and translocation pathway, some elements of that pathway are used by all substrates and others are important only for particular substrates. 相似文献
2.
Krulwich TA Jin J Guffanti AA Bechhofer H 《Journal of molecular microbiology and biotechnology》2001,3(2):237-246
Tet(L) and Tet(K) are specific antibiotic-resistance determinants. They catalyze efflux of a tetracycline(Tc)-divalent metal complex in exchange for protons, as do other Tet efflux proteins. These Tet proteins also catalyze Na+ and K+ exchange for protons. Each of the "cytoplasmic substrates", Na+, K+ and the Tc-metal ion complex, can also be exchanged for K+, a catalytic mode that accounts for the long-recognized K+ uptake capacity conferred by some Tet proteins. The multiple catalytic modes of Tet(L) and Tet(K) provide potential new avenues for development of inhibitors of these efflux systems as well as avenues for exploration of structure-function relationships. The multiple catalytic modes of Tet(L), which is chromosomally encoded in Bacillus subtilis, also correspond to diverse physiological roles, including roles in antibiotic-, Na+-, and alkali-resistance as well as K+ acquisition. The use of K+ as an external coupling ion may contribute not only to the organism's K+ uptake capacity but also to its ability to exclude Na+ and Tc at elevated pH values. Regulation of the chromosomal tetL gene by Tc has been proposed to involve a translational re-initiation mechanism that is novel for an antibiotic-resistance gene and increases Tet expression seven-fold. Other elements of tetL expression and its regulation are already evident, including gene amplification and use of multiple promoters. However, further studies are required to clarify the full panoply of regulatory mechanisms, and their integration to ensure different levels of tetL expression that are optimal for its different functions. It will also be of interest to investigate the implications of Tet(L) and Tet(K) multifunctionality on the emergence and persistence of these antibiotic-resistance genes. 相似文献
3.
Active and passive cation transport and L antigen hertogeneity in low potassium sheep red cells
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Several lines of experimental evidence are presented suggesting that the L antigens in low potassium (LK) sheep red cells are associated with separate Na(+)K(+) pump flux is distinct from the action of anti-L(l) on K(+) leak flux, implying that K(+) leak transport sites may not be converted into active pumps by the L antiserum. Treatment of LK red cells with trypsin completely abolished both the stimulation of K(+) pump flux and the enhancement of the rate of ouabain binding brought about by anti- L. That this effect is due to a total destruction of the L(p) determinant associated with the LK pump was evident from the complete failure of anti-L(p) to bind to trypsinized LK red cells. The L(p) antigen can be effectively protected against the trypsin attack by prior incubation with anti-L, indicating that the sites for antibody binding and trypsin action may be closely adjacent at the structural level. Trypsin treatment, however, did not interfere with anti-L(l) reducing ouabain insensitive K(+) leak influx, nor did it prevent binding of anti-L(ly), the hemolytically active L antibody which is probably identical with anti-L(l). The functional independence of the L(p) and L(l) sites was documented by the observation that anti-L(l) still reduced K(+) leak influx in LK cells with experimentally induced high potassium concentrations, at which K(+) pump flux is fully suppressed, whether or not anti-L(p) was binding to the L(p) antigen associated with the LK pump. 相似文献
4.
Lingchun Kong Li Tan Ruitu Lv Zhennan Shi Lijun Xiong Feizhen Wu Kimberlie Rabidou Michael Smith Celestine He Lei Zhang Yanyan Qian Duan Ma Fei Lan Yang Shi Yujiang Geno Shi 《Nucleic acids research》2016,44(18):8682-8692
Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs’ role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT), but not catalytic inactive mutant (Mut), TET2 in low-TET-expressing cells results in an increase in the level of 5hmC with accompanying DNA demethylation at a subset of CGIs. Most importantly, this alteration is sufficient in making de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of essential developmental gene promoters, which are located within CGIs and are previously silenced due to DNA methylation. On the other hand, deletion of Tet1 and Tet2 in mouse embryonic stem (ES) cells results in an apparent loss of H3K27me3 at bivalent domains, which are associated with a particular set of key developmental gene promoters. Collectively, this study demonstrates the critical role of TET proteins in regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at CGIs associated with developmental genes. 相似文献
5.
Na(+),K(+)-ATPase, a basolateral transporter responsible for tubular reabsorption of Na(+) and for providing the driving force for vectorial transport of various solutes and ions, can also act as a signal transducer in response to the interaction with steroid hormones. At nanomolar concentrations ouabain binding to Na(+),K(+)-ATPase activates a signaling cascade that ultimately regulates several membrane transporters including Na(+),K(+)-ATPase. The present study evaluated the long-term effect of ouabain on Na(+),K(+)-ATPase activity (Na(+) transepithelial flux) and expression in opossum kidney (OK) cells with low (40) and high (80) number of passages in culture, which are known to overexpress Na(+),K(+)-ATPase (Silva et al., 2006, J Membr Biol 212, 163-175). Activation of a signal cascade was evaluated by quantification of ERK1/2 phosphorylation by Western blot. Na(+),K(+)-ATPase activity was determined by electrophysiological techniques and expression by Western blot. Incubation of cells with ouabain induced activation of ERK1/2. Long-term incubation with ouabain induced an increase in Na(+) transepithelial flux and Na(+),K(+)-ATPase expression only in OK cells with 80 passages in culture. This increase was prevented by incubation with inhibitors of MEK1/2 and PI-3K. In conclusion, ouabain-activated signaling cascade mediated by both MEK1/2 and PI-3K is responsible for long-term regulation of Na(+) transepithelial flux in epithelial renal cells. OK cell line with high number of passages is suggested to constitute a particular useful model for the understanding of ouabain-mediated regulation of Na(+) transport. 相似文献
6.
Twelve-transmembrane-segment (TMS) version (DeltaTMS VII-VIII) of the 14-TMS Tet(L) antibiotic resistance protein retains monovalent cation transport modes but lacks tetracycline efflux capacity
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A "Tet(L)-12" version of Tet(L), a tetracycline efflux protein with 14 transmembrane segments (TMS), was constructed by deletion of two central TMS. Tet(L)-12 catalyzed Na+/H+ antiport and antiport with K+ as a coupling ion as well as or better than wild-type Tet(L) but exhibited no tetracycline-Me2+/H+ antiport in Escherichia coli vesicles. 相似文献
7.
Krivoĭ II Drabkina TM Vasil'ev AN Kravtsova VV Mandel F 《Rossi?skii fiziologicheski? zhurnal imeni I.M. Sechenova / Rossi?skaia akademiia nauk》2006,92(2):191-203
The interaction between the nicotinic acetylcholine receptor and Na+,K(+)-ATPase described previously was further studied in isolated rat diaphragm and in a membrane preparation of Torpedo californica electric organ. Three specific agonists of the nicotinic receptor: acetylcholine, nicotine and carbamylcholine (100 nmol/L each), all hyperpolarized the non-synaptic membranes of muscle fibers by up to 4 mV. Competitive antagonists of nicotinic acetylcholine receptor, d-tubocurarine (2 mcmol/L) or alpha-bungarotoxin (5 nmol/L) completely blocked the acetylcholine-induced hyperpolarization indicating that the effect requires binding of the agonists to their specific sites. The noncompetitive antagonist, proadifen (5 mcmol/L), exerted no effect on the amplitude of hyperpolarized but decreased K0.5 for this effect from 28.3 +/- 3.6 nmol/L to 7.1 +/- 2.3 nmol/L. Involvement of the Na+,K(+)-ATPase was suggested by data demonstrating that three specific Na+,K(+)-ATPase inhibitors: ouabain, digoxin or marinobufagenin (100 nmol/L each), all inhibit the hyperpolarizing effect of acetylcholine. Acetylcholine did not affectation either the catalytic activity of the Na+,K(+)-ATPase purified from sheep kidney or the transport activity of the Na+,K(+)-ATPase in the rat erythrocytes, i. e. in preparations not containing acetylcholine receptors. Hence, acetylcholine does not directly affect the Na+,K(+)-ATPase. In a Torpedo membrane preparation, ouabain (< or = 100 nmol/L) increased the binding of the fluorescent ligand: Dansyl-C6-choline (DCC). No ouabain effect was observed either when the agonist binding sites of the receptor were occupied by 2 mmol/L carbamylcholine, or in the absence Mg2+, when the binding of ouabain to the Na+,K(+)-ATPase is negligible. These results indicate that ouabain only affects specific DCC binding and only when bound to the Na+,K(+)-ATPase. The data obtained suggest that, in two different systems, the interaction between the nicotinic acetylcholine receptor and the Na+,K(+)-ATPase specifically involve the ligand binding sites of these two proteins. 相似文献
8.
Ortiz-Acevedo A Rigor RR Maldonado HM Cala PM 《American journal of physiology. Cell physiology》2008,295(5):C1316-C1325
Alteration in cell volume of vertebrates results in activation of volume-sensitive ion flux pathways. Fine control of the activity of these pathways enables cells to regulate volume following osmotic perturbation. Protein phosphorylation and dephosphorylation have been reported to play a crucial role in the control of volume-sensitive ion flux pathways. Exposing Amphiuma tridactylu red blood cells (RBCs) to phorbol esters in isotonic medium results in a simultaneous, dose-dependent activation of both Na(+)/H(+) and K(+)/H(+) exchangers. We tested the hypothesis that in Amphiuma RBCs, both shrinkage-induced Na(+)/H(+) exchange and swelling-induced K(+)/H(+) exchange are activated by phosphorylation-dependent reactions. To this end, we assessed the effect of calyculin A, a phosphatase inhibitor, on the activity of the aforementioned exchangers. We found that exposure of Amphiuma RBCs to calyculin-A in isotonic media results in simultaneous, 1-2 orders of magnitude increase in the activity of both K(+)/H(+) and Na(+)/H(+) exchangers. We also demonstrate that, in isotonic media, calyculin A-dependent increases in net Na(+) uptake and K(+) loss are a direct result of phosphatase inhibition and are not dependent on changes in cell volume. Whereas calyculin A exposure in the absence of volume changes results in stimulation of both the Na(+)/H(+) and K(+)/H(+) exchangers, superimposing cell swelling or shrinkage and calyculin A treatment results in selective activation of K(+)/H(+) or Na(+)/H(+) exchange, respectively. We conclude that kinase-dependent reactions are responsible for Na(+)/H(+) and K(+)/H(+) exchange activity, whereas undefined volume-dependent reactions confer specificity and coordinated control. 相似文献
9.
Woodring J Lorenz MW Hoffmann KH 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2002,132(3):637-644
The transport of lysine has been investigated in epithelial cells isolated from chicken jejunum. The kinetics of lysine transport and the pattern of interaction with zwitterionic amino acids were consistent with system b(0,+) activity, the broad-spectrum and Na(+)-independent amino acid transporter. The half-saturation constant for lysine entry (K(m)+/-S.E.) was 0.029+/-0.002 mM and the flux was not affected significantly by Na(+) replacement with choline. Lysine influx was inhibited by L-leucine both in Na(+) and choline medium with inhibition constants (K(i)+/-S.E.) 0.068+/-0.006 mM (in Na(+)) and 0.065+/-0.009 mM (in choline). Other inhibitory amino acids (K(i)+/-S.E.) were (mM): L-tyrosine (0.073+/-0.018), L-methionine (0.15+/-0.015), L-cystine (0.42+/-0.04), L-cysteine (1.1+/-0.07), L-isoleucine (1.1+/-0.09), L-glutamine (1.8+/-0.16) and L-valine (2.5+/-0.13). Lysine exit was trans-accelerated (approx. 20 fold) by 2 mM L-lysine and L-leucine. The flux was resistant to pretreatment of the cells with p-chloromercuriphenylsulfonate (0.2 mM), which is an inhibitor of system y(+)L, the broad-spectrum and cation-modulated transporter. 相似文献
10.
We propose a reaction model for the palytoxin-sodium-potassium (PTX-Na(+)/K(+)) pump complex. The model, which is similar to the Albers-Post model for Na(+)/K(+)-ATPase, is used to elucidate the effect of PTX on Na(+)/K(+)-ATPase during the enzyme interactions with Na(+) and/or K(+) ions. Conformational substates and reactions for the pump are incorporated into the Albers-Post model to represent enzymes with or without bound PTX. A mathematical model based on the reaction scheme is used in simulations modeling experimental studies of PTX-induced ionic currents. Our simulations suggest that (i) extracellular Na(+) as well as K(+) promotes PTX-induced channel blockage; (ii) extracellular K(+) accelerates PTX unbinding; and (iii) K(+) occlusion in the PTX-pump complex is essential for describing the PTX-induced current dynamics. 相似文献
11.
12.
Yeast SMF1 mediates H(+)-coupled iron uptake with concomitant uncoupled cation currents 总被引:1,自引:0,他引:1
Chen XZ Peng JB Cohen A Nelson H Nelson N Hediger MA 《The Journal of biological chemistry》1999,274(49):35089-35094
Yeast membrane proteins SMF1, SMF2, and SMF3 are homologues of the DCT1 metal ion transporter family. Their functional characteristics and the implications of these characteristics in vivo have not yet been reported. Here we show that SMF1 expressed in Xenopus oocytes mediates H(+)-dependent Fe(2+) transport and uncoupled Na(+) flux. SMF1-mediated Fe(2+) transport exhibited saturation kinetics (K(m) = 2.2 microM), whereas the Na(+) flux did not, although both processes were electrogenic. SMF1 is also permeable to Li(+), Rb(+), K(+), and Ca(2+), which likely share the same uncoupled pathway. SMF2 (but not SMF3) mediated significant increases in both Fe(2+) and Na(+) transport compared with control oocytes. These data are consistent with the concept that uptake of divalent metal ions by SMF1 and SMF2 is essential to yeast cell growth. Na(+) inhibited metal ion uptake mediated by SMF1 and SMF2 expressed in oocytes. Consistent with this, we found that increased sensitivity of yeast to EGTA in the high Na(+) medium is due to inhibition of SMF1- and SMF2-mediated metal ion transport by uncoupled Na(+) pathway. Interestingly, DCT1 also mediates Fe(2+)-activated uncoupled currents. We propose that uncoupled ion permeabilities in metal ion transporters protect cells from metal ion overload. 相似文献
13.
14.
R E Shetlar B Sch?lermann A I Morrison R K Kinne 《Biochimica et biophysica acta》1990,1023(2):184-190
In order to characterize the transport systems mediating K+ uptake into oocytes, flux studies employing 86Rb were performed on Xenopus oocytes stripped of follicular cells by pretreatment with Ca2(+)-Mg2(+)-free Barth's medium. Total Rb+ uptake consisted of an ouabain-sensitive and an ouabain-insensitive flux. In the presence of 100 mmol/l NaCl and 0.1 mmol/l ouabain the ouabain-insensitive flux amounted to 754.7 +/- 59.9 pmol/oocyte per h (n = 30 cells, i.e., 10 cells each from three different animals). In the absence of Na+ (Na+ substituted by N-methylglucamine) or when Cl- was replaced by NO3- the ouabain-insensitive flux was reduced to 84.4 +/- 42.9 and 79.2 +/- 12.1 pmol/oocyte per h, respectively (n = 50 cells). Furthermore, this Na(+)- and Cl(-)-dependent flux was completely inhibited by 10(-4) mol/l bumetanide, a specific inhibitor of the Na(+)-K(+)-2Cl- cotransport system. These results suggest that K+ uptake via a bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport system represents a major K+ pathway in oocytes. 相似文献
15.
硅改善盐胁迫下库拉索芦荟生长和离子吸收与分布 总被引:1,自引:0,他引:1
Si2.0mmol/L处理明显缓解NaCl 100、200mmol/L胁迫120d对库拉索芦荟(Aloevera)生长的抑制作用。Si可显著降低NaCl胁迫下芦荟植株中的Na^+和Cl^-含量,提高K^+含量,从而显著降低K^+/Na^+,促进根对K^+的选择性吸收(ASK,Na)和K^+向地上部的选择性运输(TSK,Na),以维持植株体内的离子稳态。根系和叶片横切面的X-射线能谱微区分析结果进一步证实了这一结果。Si改善盐胁迫下芦荟对K^+的选择性吸收和运输的机制之一是通过显著提高盐胁迫下芦荟根细胞质膜H^+ATPase、液泡膜H^+-ATPase和液泡膜H^+-PPase的活性。 相似文献
16.
17.
Vinagre TM Alciati JC Yunes JS Richards J Bianchini A Monserrat JM 《Physiological and biochemical zoology : PBZ》2002,75(6):600-608
Recent discoveries indicate that microcystins affect enzymes, such as Na(+),K(+)-ATPase, involved in ion regulation of aquatic animals, through K(+)-dependent phosphatase inhibition. In vitro studies showed the inhibitory effect of Microcystis aeruginosa extracts on Na(+),K(+)-ATPase and K(+)-dependent phosphatase activities in gills of Chasmagnathus granulata (Decapoda, Grapsidae). Extracts of M. aeruginosa were prepared from lyophilized or cultures cells of the cyanobacterium. For lyophilized cells, IC(50) values were estimated as 0.46 microg/L (95% confidence interval [CI]=0.40-0.52 microg/L) and 1.31 microg/L (95% CI=1.14-1.51 microg/L) for Na(+),K(+)-ATPase and K(+)-dependent phosphatase, respectively. However, extracts prepared from cultured cells presented a much lower inhibitory potency against both enzymes. Gas chromatography revealed long-chain fatty acids in the lyophilized cell extracts, indicating that they are in part responsible for the enzyme inhibition. In vivo studies showed that the toxin inhibited Na(+),K(+)-ATPase activity in anterior gills, whereas an increased augmented activity of glutathione-S-transferase was observed in both kind of gills, indicating that the crab has increased its ability to conjugate the toxin. No significant differences in hemolymph sodium or chloride concentration were detected. This result is in agreement with the lack of effects of microcystin on Na(+),K(+)-ATPase activity of posterior (osmoregulating) gills. 相似文献
18.
Relationships between adenylate cyclase and Na+, K(+)-ATPase in rat pancreatic islets 总被引:4,自引:0,他引:4
P Tung G Pai D G Johnson R Punzalan S R Levin 《The Journal of biological chemistry》1990,265(7):3936-3939
We tested the hypothesis that the adenylate cyclase system and Na+, K(+)-ATPase are reciprocally related in rat pancreatic islets. We studied the effect of theophylline, caffeine, and dibutyryl cyclic AMP on Na+, K(+)-ATPase activity in a membrane preparation from collagenase-isolated rat islets. Theophylline, caffeine, or dibutyryl cyclic AMP, in concentrations of 1 mM, all inhibited Na+, K(+)-ATPase activity (44,62, and 43%, respectively). Kinetic analysis indicated that theophylline and dibutyryl cAMP inhibit Na+, K(+)-ATPase by different mechanisms; theophylline decreased Vmax and decreased apparent Km (ATP), whereas dibutyryl cAMP decreased Vmax and increased apparent Km (ATP). Similar inhibition of Na+, K(+)-ATPase by theophylline or dibutyryl cAMP was noted in a particulate fraction from rat kidney and in a purified porcine brain Na+, K(+)-ATPase preparation. The adenylate cyclase system and Na+, K(+)-ATPase may act reciprocally in pancreatic islets and in other tissues. In the beta cell this relationship may be essential in coordinating consumption of ATP in the stimulated, as opposed to the rest, state. 相似文献
19.
Kraemer DM Strizek B Meyer HE Marcus K Drenckhahn D 《European journal of cell biology》2003,82(2):87-92
Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1). 相似文献
20.
Shainskaya A Schneeberger A Apell HJ Karlish SJ 《The Journal of biological chemistry》2000,275(3):2019-2028
Based on the following observations we propose that the cytoplasmic loop between trans-membrane segments M6 and M7 (L6/7) of the alpha subunit of Na(+),K(+)-ATPase acts as an entrance port for Na(+) and K(+) ions. 1) In defined conditions chymotrypsin specifically cleaves L6/7 in the M5/M6 fragment of 19-kDa membranes, produced by extensive proteolysis of Na(+),K(+)-ATPase, and in parallel inactivates Rb(+) occlusion. 2) Dissociation of the M5/M6 fragment from 19-kDa membranes is prevented either by occluded cations or by competitive antagonists such as Ca(2+), Mg(2+), La(3+), p-xylylene bisguanidinium and m-xylylene bisguanidinium, or 1-bromo-2,4, 6-tris(methylisothiouronium)benzene and 1,3-dibromo-2,4,6-tris (methylisothiouronium)benzene (Br(2)-TITU(3+)). 3) Ca(2+) ions raise electrophoretic mobility of the M5/M6 fragment but not that of the other fragments of the alpha subunit. It appears that negatively charged residues in L6/7 recognize either Na(+) or K(+) ions or the competitive cation antagonists. Na(+) and K(+) ions are then occluded within trans-membrane segments and can be transported, whereas the cation antagonists are not occluded and block transport at the entrance port. The cytoplasmic segment of the beta subunit appears to be close to or contributes to the entrance port, as inferred from the following observations. 1) Specific chymotryptic cleavage of the 16-kDa fragment of the beta subunit to 15-kDa at 20 degrees C (Shainskaya, A., and Karlish, S. J. D. (1996) J. Biol. Chem. 271, 10309-10316) markedly reduces affinity for Br(2)-TITU(3+) and for Na(+) ions, detected by Na(+) occlusion assays or electrogenic Na(+) binding, whereas Rb(+) occlusion is unchanged. 2) Na(+) ions specifically protect the 16-kDa fragment against this chymotryptic cleavage. 相似文献