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1.
Teresita Padilla-Benavides Alayna M. George Thompson Megan M. McEvoy José M. Argüello 《The Journal of biological chemistry》2014,289(30):20492-20501
Cellular copper homeostasis requires transmembrane transport and compartmental trafficking while maintaining the cell essentially free of uncomplexed Cu2+/+. In bacteria, soluble cytoplasmic and periplasmic chaperones bind and deliver Cu+ to target transporters or metalloenzymes. Transmembrane Cu+-ATPases couple the hydrolysis of ATP to the efflux of cytoplasmic Cu+. Cytosolic Cu+ chaperones (CopZ) interact with a structural platform in Cu+-ATPases (CopA) and deliver copper into the ion permeation path. CusF is a periplasmic Cu+ chaperone that supplies Cu+ to the CusCBA system for efflux to the extracellular milieu. In this report, using Escherichia coli CopA and CusF, direct Cu+ transfer from the ATPase to the periplasmic chaperone was observed. This required the specific interaction of the Cu+-bound form of CopA with apo-CusF for subsequent metal transfer upon ATP hydrolysis. As expected, the reverse Cu+ transfer from CusF to CopA was not observed. Mutation of CopA extracellular loops or the electropositive surface of CusF led to a decrease in Cu+ transfer efficiency. On the other hand, mutation of Met and Glu residues proposed to be part of the metal exit site in the ATPase yielded enzymes with lower turnover rates, although Cu+ transfer was minimally affected. These results show how soluble chaperones obtain Cu+ from transmembrane transporters. Furthermore, by explaining the movement of Cu+ from the cytoplasmic pool to the extracellular milieu, these data support a mechanism by which cytoplasmic Cu+ can be precisely directed to periplasmic targets via specific transporter-chaperone interactions. 相似文献
2.
3.
Properties of the P-Type ATPases Encoded by the copAP Operons of Helicobacter pylori and Helicobacter felis
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Denis Bayle Sabine Wngler Thomas Weitzenegger Wolfram Steinhilber Jürgen Volz Michael Przybylski Klaus P. Schfer George Sachs Klaus Melchers 《Journal of bacteriology》1998,180(2):317-329
The cop operons of Helicobacter pylori and Helicobacter felis were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd2+ and Cu2+ ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the copYZAB operon of Enterococcus hirae. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the H. pylori CopA ATPase bound to Cu2+ specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of H. pylori to Cu2+ but not to other divalent cations. As determined experimentally, H. pylori CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of H. pylori (K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446–457, 1996). The corresponding transmembrane segments of the H. felis CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu2+ binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that H. pylori and H. felis employ conserved mechanisms of ATPase-dependent copper resistance. 相似文献
4.
Daniel Raimunda Manuel González-Guerrero Blaise W. LeeberIII José M. Argüello 《Biometals》2011,24(3):467-475
Cu+-ATPases play a key role in bacterial Cu+ homeostasis by participating in Cu+ detoxification and cuproprotein assembly. Characterization of Archaeoglobus fulgidus CopA, a model protein within the subfamily of P1B-1 type ATPases, has provided structural and mechanistic details on this group of transporters. Atomic resolution structures
of cytoplasmic regulatory metal binding domains (MBDs) and catalytic actuator, phosphorylation, and nucleotide binding domains
are available. These, in combination with whole protein structures resulting from cryo-electron microscopy analyses, have
enabled the initial modeling of these transporters. Invariant residues in helixes 6, 7 and 8 form two transmembrane metal
binding sites (TM-MBSs). These bind Cu+ with high affinity in a trigonal planar geometry. The cytoplasmic Cu+ chaperone CopZ transfers the metal directly to the TM-MBSs; however, loading both of the TM-MBSs requires binding of nucleotides
to the enzyme. In agreement with the classical transport mechanism of P-type ATPases, occupancy of both transmembrane sites
by cytoplasmic Cu+ is a requirement for enzyme phosphorylation and subsequent transport into the periplasmic or extracellular milieus. Recent
transport studies have shown that all Cu+-ATPases drive cytoplasmic Cu+ efflux, albeit with quite different transport rates in tune with their various physiological roles. Archetypical Cu+-efflux pumps responsible for Cu+ tolerance, like the Escherichia coli CopA, have turnover rates ten times higher than those involved in cuproprotein assembly (or alternative functions). This
explains the incapability of the latter group to significantly contribute to the metal efflux required for survival in high
copper environments. 相似文献
5.
CopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu+ across the cell membrane. Millimolar concentration of Cys dramatically increases (≅ 800%) the activity of CopA and other PIB-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal (≅ 1 μM) together with the low Cu+-Cys KD (< 10− 10M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu+ chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2-20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu+, suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP Km suggesting no changes in the E1 ↔ E2 equilibrium. Characterization of Cu+ transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu+ can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu+-Cys complex. 相似文献
6.
《生物化学与生物物理学报:生物膜》2022,1864(2):183822
Cu+-ATPases are integral membrane proteins belonging to the IB subfamily of the P-type ATPases that couple Cu+ transport to the hydrolysis of ATP. As some structural and functional particularities arise for Cu+-ATPases, several authors suggest that some of the reaction steps of the Albers-Post model postulated for other P-ATPases may be different. In this work we describe a functional characterization of Legionella pneumophila Cu+-ATPase (LpCopA), the first PIB-ATPase whose structure was determined by X-ray crystallography. Cu+-ATPase activity of the enzyme presents a maximum at ~37 °C and pH 6.6–6.8. Phospholipids enhance LpCopA Cu+-ATPase activity in a non-essential mode where optimal activity is achieved at an asolectin molar fraction of 0.15 and an amphiphile-protein ratio of ~30,000. As described for other P-ATPases, Mg2+ acts as an essential activator. Furthermore, Cu+-ATPase activity dependence on [Cu+] and [ATP] can both be described by a sum of two hyperbolic functions. Based on that, and the [Cu+] and [ATP] dependencies of the best fitting parameters of the hyperbolae pointed above, we propose a minimal reaction scheme for the catalytic mechanism that shares the basic reaction steps of the Albers-Post model for P-type ATPases. The reaction scheme postulated contemplates two different binding affinities for a single ATP (apparent affinities of 0.66 and 550 μM at [Cu+] → ∞) and binding of at least 2 Cu+ with different affinities as well (apparent affinities of 1.4 and 102.5 μM at [ATP] → ∞). 相似文献
7.
The Enterococcus hirae paradigm of copper homeostasis: Copper chaperone turnover, interactions, and transactions 总被引:2,自引:0,他引:2
The cop operon is a key element of copper homeostasis in Enterococcus hirae. It encodes two copper ATPases, CopA and CopB, the CopY repressor, and the CopZ metallochaperone. The cop operon is induced by copper, which allows uncompromised growth in up to 5 mM ambient copper. Copper uptake appears to be accomplished by the CopA ATPase, a member of the heavy metal CPx-type ATPases and closely related to the human Menkes and Wilson ATPases. The related CopB ATPase extrudes copper when it reaches toxic levels. Intracellular copper routing is accomplished by the CopZ copper chaperone. Using surface plasmon resonance analysis, it was demonstrated that CopZ interacts with the CopA ATPase where it probably becomes copper loaded. CopZ in turn can donate copper to the copper responsive repressor CopY, thereby releasing it from DNA. In high copper, CopZ is proteolyzed. Cell extracts were found to contain a copper activated proteolytic activity that degrades CopZ in vitro. This post-translational control of CopZ expression presumably serves to avoid the accumulation of detrimental Cu-CopZ levels. 相似文献
8.
Daniel Mattle Limei Zhang Oleg Sitsel Lotte Thue Pedersen Maria Rosa Moncelli Francesco Tadini-Buoninsegni Pontus Gourdon Douglas C Rees Poul Nissen Gabriele Meloni 《EMBO reports》2015,16(6):728-740
Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. PIB-type Cu+-ATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu+ across cellular membranes. Crystal structures of a copper-free Cu+-ATPase are available, but the mechanism of Cu+ recognition, binding, and translocation remains elusive. Through X-ray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solid-supported membranes using wild-type and mutant forms of the Legionella pneumophila Cu+-ATPase (LpCopA), we identify a sulfur-lined metal transport pathway. Structural analysis indicates that Cu+ is bound at a high-affinity transmembrane-binding site in a trigonal-planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Met residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport. Additionally, the studies indicate essential roles of other conserved intramembranous polar residues in facilitating copper binding to the high-affinity site and subsequent release through the exit pathway. 相似文献
9.
Mads S. Toustrup-Jensen Anja P. Einholm Vivien R. Schack Hang N. Nielsen Rikke Holm María-Jesús Sobrido Jens P. Andersen Torben Clausen Bente Vilsen 《The Journal of biological chemistry》2014,289(6):3186-3197
The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na+,K+-ATPase α2 and α3 isoforms, expressed in glial and neuronal cells, respectively. Although these disorders are distinct, they overlap in phenotypical presentation. Two Na+,K+-ATPase mutations, extending the C terminus by either 28 residues (“+28” mutation) or an extra tyrosine (“+Y”), are associated with FHM2 and RDP, respectively. We describe here functional consequences of these and other neurological disease mutations as well as an extension of the C terminus only by a single alanine. The dependence of the mutational effects on the specific α isoform in which the mutation is introduced was furthermore studied. At the cellular level we have characterized the C-terminal extension mutants and other mutants, addressing the question to what extent they cause a change of the intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) in COS cells. C-terminal extension mutants generally showed dramatically reduced Na+ affinity without disturbance of K+ binding, as did other RDP mutants. No phosphorylation from ATP was observed for the +28 mutation of α2 despite a high expression level. A significant rise of [Na+]i and reduction of [K+]i was detected in cells expressing mutants with reduced Na+ affinity and did not require a concomitant reduction of the maximal catalytic turnover rate or expression level. Moreover, two mutations that increase Na+ affinity were found to reduce [Na+]i. It is concluded that the Na+ affinity of the Na+,K+-ATPase is an important determinant of [Na+]i. 相似文献
10.
Interaction of the CopZ copper chaperone with the CopA copper ATPase of Enterococcus hirae assessed by surface plasmon resonance 总被引:1,自引:0,他引:1
Multhaup G Strausak D Bissig KD Solioz M 《Biochemical and biophysical research communications》2001,288(1):172-177
Intracellular copper routing in Enterococcus hirae can be accomplished by the CopZ metallochaperone. Using surface plasmon resonance analysis, we show here that CopZ interacts with the CopA copper ATPase. The binding affinity of CopZ for CopA was increased in the presence of copper, due to a 15-fold lower dissociation rate constant. Mutating the N-terminal copper binding motif of CopA from CxxC to SxxS abolished this copper-induced effect. Moreover, CopZ failed to show an interaction with an unrelated copper binding protein used as a control. These results show that (i) the CopA copper ATPase specifically interacts with the CopZ chaperone, (ii) this interaction is based on protein-protein interaction, and (iii) surface plasmon resonance is a novel tool for quantitative analysis of metallochaperone-target interactions. 相似文献
11.
David Lewis Rajendra Pilankatta Giuseppe Inesi Gianluca Bartolommei Maria Rosa Moncelli Francesco Tadini-Buoninsegni 《The Journal of biological chemistry》2012,287(39):32717-32727
Ca2+ (sarco-endoplasmic reticulum Ca2+ ATPase (SERCA)) and Cu+ (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca2+, demonstrated by the addition of ATP and Ca2+ to SERCA deprived of Ca2+ (E2) as compared with ATP to Ca2+-activated enzyme (E1·2Ca2+). Activation by Ca2+ is slower at low pH (2H+·E2 to E1·2Ca2+) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca2+ translocation. A “H+-gated pathway,” demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca2+ release by H+/Ca2+ exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu+/H+ exchange. As opposed to SERCA after Ca2+ chelation, ATP7A/B does not undergo reverse phosphorylation with Pi after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms. 相似文献
12.
Chikashi Toyoshima 《The EMBO journal》2009,28(12):1782-1791
Heavy metal pumps constitute a large subgroup in P‐type ion‐transporting ATPases. One of the outstanding features is that the nucleotide binding N‐domain lacks residues critical for ATP binding in other well‐studied P‐type ATPases. Instead, they possess an HP‐motif and a Gly‐rich sequence in the N‐domain, and their mutations impair ATP binding. Here, we describe 1.85 Å resolution crystal structures of the P‐ and N‐domains of CopA, an archaeal Cu+‐transporting ATPase, with bound nucleotides. These crystal structures show that CopA recognises the adenine ring completely differently from other P‐type ATPases. The crystal structure of the His462Gln mutant, in the HP‐motif, a disease‐causing mutation in human Cu+‐ATPases, shows that the Gln side chain mimics the imidazole ring, but only partially, explaining the reduction in ATPase activity. These crystal structures lead us to propose a role of the His and a mechanism for removing Mg2+ from ATP before phosphoryl transfer. 相似文献
13.
Rikke Holm Anja P. Einholm Jens P. Andersen Bente Vilsen 《The Journal of biological chemistry》2015,290(15):9801-9811
The Na+,K+-ATPase binds Na+ at three transport sites denoted I, II, and III, of which site III is Na+-specific and suggested to be the first occupied in the cooperative binding process activating phosphorylation from ATP. Here we demonstrate that the asparagine substitution of the aspartate associated with site III found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood causes a dramatic reduction of Na+ affinity in the α1-, α2-, and α3-isoforms of Na+,K+-ATPase, whereas other substitutions of this aspartate are much less disruptive. This is likely due to interference by the amide function of the asparagine side chain with Na+-coordinating residues in site III. Remarkably, the Na+ affinity of site III aspartate to asparagine and alanine mutants is rescued by second-site mutation of a glutamate in the extracellular part of the fourth transmembrane helix, distant to site III. This gain-of-function mutation works without recovery of the lost cooperativity and selectivity of Na+ binding and does not affect the E1-E2 conformational equilibrium or the maximum phosphorylation rate. Hence, the rescue of Na+ affinity is likely intrinsic to the Na+ binding pocket, and the underlying mechanism could be a tightening of Na+ binding at Na+ site II, possibly via movement of transmembrane helix four. The second-site mutation also improves Na+,K+ pump function in intact cells. Rescue of Na+ affinity and Na+ and K+ transport by second-site mutation is unique in the history of Na+,K+-ATPase and points to new possibilities for treatment of neurological patients carrying Na+,K+-ATPase mutations. 相似文献
14.
Chick brain microsomal ATPase was strongly inhibited by Cu2+. (Na+ + K+)-ATPase was more susceptible to low levels of Cu2+ than Mg2+-ATPase. The inhibition of (Na+ + K+)-ATPase could be partially protected from Cu2+ in the presence of ATP in the preincubation period. When Cu2+ (6 μM) was preincubated with the enzyme in the absence of ATP, only sulfhydryl-containing amino acids (d-penicillamine and l-cysteine) could reverse the inhibition. At lower concentrations of Cu2+ (< 1.4 μM), in the absence of ATP during preincubation, the inhibition could be completely reversed by the addition of 5 mM l-phenylalanine and l-histidine as well as d-penicillamine and l-cysteine.Kinetic analysis of action of Cu2+ (1.0 μM) on (Na+ + K+)-ATPase revealed that the inhibition was uncompetitive with respect to ATP. At a low concentration of K+ (5 mM), V with Na+ was markedly decreased in the presence of Cu2+ and Km was about twice that of the control. However, at high K+ concentration (20 mM), the Km for Na+ was not affected. At both low (25 mM) and high (100 mM) Na+, Cu2+ displayed non-competitive inhibition of the enzyme with respect to K+.On the basis of these data, we suggest that Cu2+ at higher concentrations (> 6 μM) inactivates the enzyme irreversibly, but that at lower concentrations (< 1.4 μM), Cu2+ interacts reversibly with the enzyme. 相似文献
15.
Anja Pernille Einholm Mads S. Toustrup-Jensen Rikke Holm Jens Peter Andersen Bente Vilsen 《The Journal of biological chemistry》2010,285(34):26245-26254
Rapid-onset dystonia parkinsonism (RDP), a rare neurological disorder, is caused by mutation of the neuron-specific α3-isoform of Na+,K+-ATPase. Here, we present the functional consequences of RDP mutation D923N. Relative to the wild type, the mutant exhibits a remarkable ∼200-fold reduction of Na+ affinity for activation of phosphorylation from ATP, reflecting a defective interaction of the E1 form with intracellular Na+. This is the largest effect on Na+ affinity reported so far for any Na+,K+-ATPase mutant. D923N also affects the interaction with extracellular Na+ normally driving the E1P to E2P conformational transition backward. However, no impairment of K+ binding was observed for D923N, leading to the conclusion that Asp923 is specifically associated with the third Na+ site that is selective toward Na+. The crystal structure of the Na+,K+-ATPase in E2 form shows that Asp923 is located in the cytoplasmic half of transmembrane helix M8 inside a putative transport channel, which is lined by residues from the transmembrane helices M5, M7, M8, and M10 and capped by the C terminus, recently found involved in recognition of the third Na+ ion. Structural modeling of the E1 form of Na+,K+-ATPase based on the Ca2+-ATPase crystal structure is consistent with the hypothesis that Asp923 contributes to a site binding the third Na+ ion. These results in conjunction with our previous findings with other RDP mutants suggest that a selective defect in the handling of Na+ may be a general feature of the RDP disorder. 相似文献
16.
Ruiping Zhang Gan Liu Na Wu Mian Gu Houqing Zeng Yiyong Zhu Guohua Xu 《Plant and Soil》2011,349(1-2):3-11
The plasma membrane (PM) H+ ATPase is involved in the plant response to nutrient deficiency. However, adaptation of this enzyme in monocotyledon plants to phosphorus (P) deficiency lacks direct evidence. In this study, we detected that P deficient roots of rice (Oryza Sativa L.) could acidify the rhizosphere. We further isolated the PM from rice roots and analyzed the activity of PM H+ ATPase. In vitro, P deficient rice roots showed about 30% higher activity of PM H+ ATPase than the P sufficient roots at assay of pH 6.0. The P deficiency resulted in a decrease of the substrate affinity value (K m ) of PM H+ ATPase. The proton pumping activity of membrane vesicles from the P deficient roots was about 70% higher than that from P sufficient roots. Western blotting analysis indicated that higher activity of PM H+ ATPase in P deficient roots was related to a slightly increase of PM H+ ATPase protein abundance in comparison with that in P sufficient roots. Taken together, our results demonstrate that the P deficiency enhanced activities of both PM H+-ATPase and H+ pump, which contributed to the rhizosphere acidification in rice roots. 相似文献
17.
Kira Ekberg Alex G. Wielandt Morten J. Buch-Pedersen Michael G. Palmgren 《The Journal of biological chemistry》2013,288(14):9610-9618
The minimal proton pumping machinery of the Arabidopsis thaliana P-type plasma membrane H+-ATPase isoform 2 (AHA2) consists of an aspartate residue serving as key proton donor/acceptor (Asp-684) and an arginine residue controlling the pKa of the aspartate. However, other important aspects of the proton transport mechanism such as gating, and the ability to occlude protons, are still unclear. An asparagine residue (Asn-106) in transmembrane segment 2 of AHA2 is conserved in all P-type plasma membrane H+-ATPases. In the crystal structure of the plant plasma membrane H+-ATPase, this residue is located in the putative ligand entrance pathway, in close proximity to the central proton donor/acceptor Asp-684. Substitution of Asn-106 resulted in mutant enzymes with significantly reduced ability to transport protons against a membrane potential. Sensitivity toward orthovanadate was increased when Asn-106 was substituted with an aspartate residue, but decreased in mutants with alanine, lysine, glutamine, or threonine replacement of Asn-106. The apparent proton affinity was decreased for all mutants, most likely due to a perturbation of the local environment of Asp-684. Altogether, our results demonstrate that Asn-106 is important for closure of the proton entrance pathway prior to proton translocation across the membrane. 相似文献
18.
Johannes D. Clausen Jens Peter Andersen 《The Journal of biological chemistry》2010,285(27):20780-20792
The roles of Ser72, Glu90, and Lys297 at the luminal ends of transmembrane helices M1, M2, and M4 of sarcoplasmic reticulum Ca2+-ATPase were examined by transient and steady-state kinetic analysis of mutants. The dependence on the luminal Ca2+ concentration of phosphorylation by Pi (“Ca2+ gradient-dependent E2P formation”) showed a reduction of the apparent affinity for luminal Ca2+ in mutants with alanine or leucine replacement of Glu90, whereas arginine replacement of Glu90 or Ser72 allowed E2P formation from Pi even at luminal Ca2+ concentrations much too small to support phosphorylation in wild type. The latter mutants further displayed a blocked dephosphorylation of E2P and an increased rate of conversion of the ADP-sensitive E1P phosphoenzyme intermediate to ADP-insensitive E2P as well as insensitivity of the E2·BeF3− complex to luminal Ca2+. Altogether, these findings, supported by structural modeling, indicate that the E2P intermediate is stabilized in the mutants with arginine replacement of Glu90 or Ser72, because the positive charge of the arginine side chain mimics Ca2+ occupying a luminally exposed low affinity Ca2+ site of E2P, thus identifying an essential locus (a “leaving site”) on the luminal Ca2+ exit pathway. Mutants with alanine or leucine replacement of Glu90 further displayed a marked slowing of the Ca2+ binding transition as well as slowing of the dissociation of Ca2+ from Ca2E1 back toward the cytoplasm, thus demonstrating that Glu90 is also critical for the function of the cytoplasmically exposed Ca2+ sites on the opposite side of the membrane relative to where Glu90 is located. 相似文献
19.
Proton Transport Activity of the Purified Vacuolar H-ATPase from Oats : Direct Stimulation by Cl
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To determine whether the detergent-solubilized and purified vacuolar H+-ATPase from plants was active in H+ transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K+-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K+ and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H+ pumping was electrogenic. Both H+ pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A1, a specific vacuolar type ATPase inhibitor. The reconstituted H+ pump was also inhibited by N,N′-dicyclohexylcarbodiimide or NO3− but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H+ pumping by the reconstituted ATPase in the presence of K+ and valinomycin. Hence, our results support the idea that the vacuolar H+-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl− concentration. The purified and reconstituted H+-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H+ pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H+ translocation. 相似文献
20.
The two major ATPase activities of intact and leaky cardiac membrane vesicles (microsomes) were characterized with respect to ionic activation requirements. The predominant ATPase activity of intact vesicles was (K+ + Ca2+)-ATPase, an enzymic activity localized to sarcoplasmic reticulum, whereas the predominant ATPase activity of leaky, sodium dodecyl sulfate-pretreated vesicles was (Na+ + K+)-ATPase, an enzymic activity localized to sarcolemma. The (K+ + Ca2+)-ATPase activity was stimulated 4- to 5-fold by 100 mM K+ in the presence of 50 μM Ca2+. Phosphorylation of the (K+ + Ca2+)-ATPase of intact vesicles with [γ-32P]ATP was Ca2+ dependent, and monovalent cations including K+ increased the level of [32P]phosphoprotein by up to 50% when phosphorylation was measured at 5°C. After the intact vesicles were treated with SDS (0.30 mg/ml), (K+ + Ca2+)-ATPase was inactivated, as was Ca2+-dependent 32P incorporation. The monovalent cation-stimulated ATPase activity of the particulate residue (SDS-extracted membrane vesicles) displayed the usual characteristics of ouabain-sensitive (Na+ + K+)-ATPase and the activity was increased 9- to 14-fold over the small amount of patent (Na+ + K+)-ATPase activity of intact membrane vesicles. 32P incorporation by the (Na+ + K+)-ATPase of SDS-extracted vesicles was Na+ dependent, and Na+-stimulated incorporation was increased 7- to 9-fold over that of intact vesicles.Slab gel polyacrylamide electrophoresis of both intact and SDS-extracted crude vesicle preparations revealed at least 40 distinct Coomassie Blue-positive protein bands and provided evidence for a possible heterogeneous membrane origin of the vesicles. Periodic acid-Schiff staining of the gels revealed at least two major glycoproteins. Simultaneous electrophoresis of the 32P-intermediates of the (K+ + Ca2+)-ATPase and the (Na+ + K+)-ATPase in the same gels did not resolve the two enzymes clearly. With sucrose gradient centrifugation of intact membrane vesicles, it was possible to physically resolve the two ATPase activities. Latent (Na+ + K+)-ATPase activity (unmasked by exposing the various fractions to SDS) was found in the higher regions of the gradient, whereas (K+ + Ca2+)-ATPase activity was primarily in the denser regions. A reasonable interpretation of the data is that cardiac microsomes consist of membrane vesicles derived both from sarcolemma and sarcoplasmic reticulum. (Na+ + K+)-ATPase is localized to intact vesicles of sarcolemma but is mainly latent, whereas (K+ + Ca2+)-ATPase is mostly patent and is localized to vesicles of sarcoplasmic reticulum. 相似文献