共查询到20条相似文献,搜索用时 15 毫秒
1.
A fluorescence method was adapted to investigate active ion transport in membrane preparations of the SR-Ca-ATPase. The styryl
dye RH421 previously used to investigate the Na,K-ATPase was replaced by an analogue, 2BITC, to obtain optimized fluorescence
changes upon substrate-induced partial reactions. Assuming changes of the local electric field to be the source of fluorescence
changes that are produced by uptake/release or by movement of ions inside the protein, 2BITC allowed the determination of
electrogenic partial reactions in the pump cycle. It was found that Ca2+ binding on the cytoplasmic and on the lumenal side of the pump is electrogenic while phosphorylation and conformational transition
showed only minor electrogenicity. Ca2+ equilibrium titration experiments at pH 7.2 in the two major conformations of the protein indicated cooperative binding of
two Ca2+ ions in state E1 with an apparent half-saturation concentration, K
M
of 600 nm. In state P-E2 two K
M
values, 5 μm and 2.2 mM, were determined and are in fair agreement with published data. From Ca2+ titrations in buffers with various pH and from pH titrations in P-E2, it could be demonstrated that H+ binding is electrogenic and that Ca2+ and H+ compete for the same binding site(s). Tharpsigargin-induced inhibition of the Ca-ATPase led to a state with a specific fluorescence
level comparable to that of state E1 with unoccupied ion sites, independent of the buffer composition.
Received: 21 September 1998/Revised: 18 December 1998 相似文献
2.
Christine PeineltHans-Jürgen Apell 《Biophysical journal》2002,82(1):170-181
Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca2+ and H+ on binding of the other ion in the E1 and P-E2 states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg2+ a strictly competitive binding sequence, H2E1?HE1?E1?CaE1?Ca2E1, was found with two Ca2+ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K1/2(2 Ca) = 34 nM, K1/2(H) = 1 nM and K1/2(H2) = 1.32 μM. Up to 2 Mg2+ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K1/2(Mg) = 165 μM, K1/2(Mg2) = 7.4 mM). In the P-E2 state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca2+ and H+ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E2CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca2+ (0.4 mM and 25 mM) and H+ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E1 and P-E2 conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position. 相似文献
3.
Bartolommei G Gramigni E Tadini-Buoninsegni F Santini G Moncelli MR 《Biophysical journal》2010,99(7):2087-2096
The effect of Pb2+ ions on the Na+,K+-ATPase was investigated in detail by means of steady-state fluorescence spectroscopy. Experiments were performed by using the electrochromic styryl dye RH421. It is shown that Pb2+ ions can bind reversibly to the protein and do not affect the Na+ and K+ binding affinities in the E1 and P-E2 conformations of the enzyme. The pH titrations indicate that lead(II) favors binding of one H+ to the P-E2 conformation in the absence of K+. A model scheme is proposed that accounts for the experimental results obtained for backdoor phosphorylation of the enzyme in the presence of Pb2+ ions. Taken together, our results clearly indicate that Pb2+ bound to the enzyme stabilizes an E2-type conformation. In particular, under conditions that promote enzyme phosphorylation, Pb2+ ions are able to confine the Na+,K+-ATPase into a phosphorylated E2 state. 相似文献
4.
Gerhard Meissner 《生物化学与生物物理学报:生物膜》1973,298(4)
The binding of ATP and Ca2+ by the Ca2+ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca2+ pump protein has been found to contain one specific ATP and two specific Ca2+ binding sites per phosphorylation site. ATP binding is dependent on Mg2+ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca2+. In the presence of Mg2+ and the absence of Ca2+, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca2+ binding. In the absence of ATP, Ca2+ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg2+, Sr2+ and La3+, in that order, decrease Ca2+ binding by the Ca2+ pump protein. The affinity of the Ca2+ pump protein for both ATP and Ca2+ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca2+ pump protein-MgATP2− and Ca2+ pump protein-calcium complexes are approx. 0.25 and 0.5 μM−1, respectively. The unphosphorylated Ca2+ pump protein does not contain a Mg2+ binding site with an affinity comparable to those of the ATP and Ca2+ binding sites.The affinity of the Ca2+ pump protein for Ca2+ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca2+ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca2+. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca2+.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca22+-MgATP2− complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca2+ and probably Mg2+. 相似文献
5.
Summary The ATP-dependent Ca2+ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum,Biochem. J.
227:239–245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg2+. Phosphate (5mm), but not oxalate (5mm), increased maximum Ca2+ accumulation by 50%. Half-maximal Ca2+ transport was achieved at 70nm Ca2+ in EGTA-buffered medium while maximal activity required >1 m Ca2+ (V
max=54 nmol/mg protein/min). Optimal rates of Ca2+ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca2+ transport could be significantly altered by preimposed membrane potentials generated by K+ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca2+ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca2+ transport in the absence of a pH gradient, pH
i
=7.5/pH
o
=7.5; the activity was 60% higher in the presence of an outwardly directed pH gradient, pH
i
=7.5/pH
o
=8.5; while it was 80% lower when an inwardly directed pH gradient was imposed, pH
i
=7.5/pH
o
=6.2. The data show that the ATP-dependent Ca2+ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca2+ uptake, which could be compensated by other ion movements. 相似文献
6.
The Ca2+-extruding ATPase pump of the human platelet was studiedin situ by measuring Ca2+ extrusion from quin2-overloaded platelets (Johansson, J.S., Haynes, D.H. 1988.J. Membrane Biol.
104:147–163). Cytoplasmic pH (pHcyt) was measured by BCECF fluorescence in parallel experiments. The pump was studied by raising the cytoplasmic free Ca2+ to 2.5 μM and monitoring active Ca2+ extrusion into a Ca2+-free medium. The pump was shown to perturb pHcyt, to not respond to changes in membrane potential and to respond to imposed changes in pHcyt in a manner consistent with the Ca2+ pump acting as a 2 Ca2+/nH+ exchanger. (i) Raising the external pH (pHext) from 7.40 to 7.60 lowers the Vmax of the pump in basal condition (Vmax,1) from 110±18 to 73±12 μM/min (=μmol/liter cell volume/min). (ii) Lowering pHext to 7.13 raised Vmax,1 to 150±15 μM/min. (iii) In an N-methyl-d-glucamine (NMDG+) medium, the pump operation against high [Ca2+]cyt acidifies the cytoplasm by −0.36±0.10 pH units, and the pump becomes self-inhibited. (iv) Use of nigericin to drive pHcyt down to 6.23 reduces the Vmax,1 to 18±11 μM/min. (v) Alkalinization of the cytoplasm by monensin in the presence of Na+ raises the Vmax,1 (basal state withK
m,1=80 nM) to 136±24 μM/min, but also activates the pump fourfold (Vmax,2=280±28 μM/min;K
m,2=502±36 nM). (vi) Transient elevation of pHcyt by NH4Cl at high [Ca2+]cyt activates the pump eightfold (Vmax,2≥671±350 μM/min). The large activation by alkaline pHcyt at high [Ca2+]cyt can be explained by Ca2+-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992.J. Membrane Biol.
130:63–82) and by increased Ca2+ affinity of calmodulin at high pH. 相似文献
7.
Mohamad Osman Harald Hiland Holm Holmsen Yutaka Ishigami 《Journal of peptide science》1998,4(7):449-458
We have studied the effects of extrinsic environmental conditions on the conformation of surfactin, a heptapeptide biosurfactant from Bacillus subtilis, in aqueous solutions. It has been made clear that temperature, pH, Ca2+ ions and the synthetic nonionic surfactant hepta-ethylene glycol (C12E7) affect the conformation of surfactin in aqueous solutions. The β-sheet formation reached a maximum at 40°C both in presence and absence of (C12E7) and the nonionic surfactant enhances the β-sheet formation even at 25°C. Ca2+ induced the formation of a-helices and caused this transition at 0.3 mm with surfactin monomers or at 0.5 mm with surfactin micelles, but above these transition concentrations of Ca2+ β-sheets were observed. In micellar solution the β-sheet structure was stabilized at pH values below 7 or upon addition of Ca2+ in concentrations above 0.5 mm . Our results indicated that the bioactive conformation of surfactin is most likely the β-sheets when the molecules are assembled in micelles. The β-sheet structure in micelles could be retained by tuning the micelles. Surfactin micelles could be tuned in the bioactive conformation by manipulating pH, temperature, Ca2+ or (C12E7) concentrations in surfactin solutions. Our results strongly indicated that Ca2+ and other molecules (such as C12E7) may function as directing templates in the assembly and conformation of surfactin in micelles. Thus, we suggest environmental manipulation and template-aided micellation (TAM) as a new approach for preparing predesigned micelles, microemulsions or micro-spheres for specific application purposes. © 1998 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
8.
Irene Mangialavori Mariela Ferreira-Gomes Mar��a F. Pignataro Emanuel E. Strehler Juan Pablo F. C. Rossi 《The Journal of biological chemistry》2010,285(1):123-130
The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca2+-pump (PMCA) in the membrane region upon interaction with Ca2+, calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [125I]TID-PC/16, measuring the shift of conformation E2 to the auto-inhibited conformation E1I and to the activated E1A state, titrating the effect of Ca2+ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca2+ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca2+ transport but does not modify the affinity for Ca2+ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E1I to the activated E1A conformation and thus modulating the transport of Ca2+. This is reflected in the different apparent constants for Ca2+ in the absence of CaM (calculated by Ca2+-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca2+ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca2+ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca2+ binding and activation. 相似文献
9.
Njanoor Narayanan 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,678(3):442-459
Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca2+ binding and accumulating activities as well as (Mg2+ + Ca2+)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca2+ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca2+ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca2+ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca2+ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca2+ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca2+ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca2+ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca2+ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca2+ accumulation (V (nmol Ca2+/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca2+ required for half-maximal velocities did not differ significantly with age (K0.5 for Ca2+ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca2+ binding also indicated that the velocity of Ca2+ binding but not the concentration of Ca2+ required for half-maximal binding was altered due to aging. At identical Ca2+ concentrations, the combined Ca2+ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca2+ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca2+ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg2+ + Ca2+)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca2+ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca2+ transport. Further, the age-related increment in the Ca2+ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca2+ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca2+ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart. 相似文献
10.
Biochemical and kinetic properties under identical substrate and reaction conditions were obtained for an ATP-dependent Ca2+ pump and (Ca2+ + Mg2+)-ATPase in synaptosome membrane vesicles prepared from the brain of the moth, Mamestra configurata. Both the ATP-dependent Ca2+ pump and (Ca2+ + Mg2+)-ATPase had single, high-affinity binding sites for ATP (Km = 14 and 116 μM, respectively), Ca2+free (Km = 0.13 nM and 0.072 nM, respectively), and Mg2+ (Km = 1.1 mM and 0.07 mM, respectively). Both systems were relatively little affected by K+ and were insensitive to ouabain, an inhibitor of (Na+ + K+)-ATPase. The results indicate that the ATP-dependent Ca2+ pump and (Ca2+ + Mg2+)-ATPase are functionally coupled in synaptic membranes and constitute a mechanism for Ca2+ transport in the brain of M. configurata. Although moth brain (Ca2+ + Mg2+)-ATPase is maximally active at nanomolar concentrations of free calcium ion, the enzyme retains at least one-half of its maximal activity at micromolar calcium concentrations, indicating either that the enzyme has two binding sites for calcium (a high-affinity site at nanomolar Ca2+free and a low-affinity site at micromolar Ca2+free), or that there are two enzymes with high and low affinity for calcium, respectively. Calcium extrusion from brain neurones of M. configurata may operate in a two-stage, concentration-dependent process in which a first stage, low-affinity pump reduces intraneuronal calcium to a concentration at which a second stage, high-affinity pump becomes activated. 相似文献
11.
Lipid mixing between vesicles functionalized with SNAREs and the cytosolic C2AB domain of synaptotagmin-1 recapitulates the basic Ca2+ dependence of neuronal exocytosis. However, in the conventional ensemble lipid mixing assays it is not possible to discriminate whether Ca2+ accelerates the docking or the fusion of vesicles. Here we report a fluorescence microscopy-based assay to monitor SNARE-mediated docking and fusion of individual vesicle pairs. In situ measurement of the concentration of diffusing particles allowed us to quantify docking rates by a maximum-likelihood approach. This analysis showed that C2AB and Ca2+ accelerate vesicle-vesicle docking with more than two orders of magnitude. Comparison of the measured docking rates with ensemble lipid mixing kinetics, however, suggests that in most cases bilayer fusion remains the rate-limiting step. Our single vesicle results show that only ∼60% of the vesicles dock and only ∼6% of docked vesicles fuse. Lipid mixing on single vesicles was fast (tmix < 1 s) while an ensemble assay revealed two slow mixing processes with tmix ∼ 1 min and tmix ∼ 20 min. The presence of several distinct docking and fusion pathways cannot be rationalized at this stage but may be related to intrasample heterogeneities, presumably in the form of lipid and/or protein composition. 相似文献
12.
Sara E. Cnudde Mary Prorok Francis J. Castellino James H. Geiger 《Journal of biological inorganic chemistry》2010,15(5):667-675
Predatory sea snails from the Conus family produce a variety of venomous small helical peptides called conantokins that are rich in γ-carboxyglutamic acid (Gla)
residues. As potent and selective antagonists of the N-methyl-d-aspartate receptor, these peptides are potential therapeutic agents for a variety of neurological conditions. The two most
studied members of this family of peptides are con-G and con-T. Con-G has Gla residues at sequence positions 3, 4, 7, 10,
and 14, and requires divalent cation binding to adopt a helical conformation. Although both Ca2+ and Mg2+ can fulfill this role, Ca2+ induces dimerization of con-G, whereas the Mg2+-complexed peptide remains monomeric. A variant of con-T, con-T[K7γ] (γ is Gla), contains Gla residues at the same five positions
as in con-G and behaves very similarly with respect to metal ion binding and dimerization; each peptide binds two Ca2+ ions and two Mg2+ ions per helix. To understand the difference in metal ion selectivity, affinity, and the dependence on Ca2+ for dimer formation, we report here the structure of the monomeric Cd2+/Mg2+–con-T[K7γ] complex, and, by comparison with the previously published con-T[K7γ]/Ca2+ dimer structure, we suggest explanations for both metal ion binding site specificity and metal-ion-dependent dimerization. 相似文献
13.
The enzymatic basis for the Ca2+ pump in human red cells is an ATPase with hysteretic properties. The Ca2+-ATPase shifts slowly between a ground state deficient in calmodulin and an active state saturated with calmodulin, and rate constants for the reversible shifts of state were recently determined at different Ca2+ concentrations (Scharff, O. and Foder, B. (1982) Biochim. Biophys. Acta 691, 133–143). In order to study whether the Ca2+ pump in intact red cells also exhibits hysteretic properties we have analysed transient increases of intracellular calcium concentrations (Cai), induced by the divalent cation ionophore A23187. The time-dependent changes of Cai were measured by use of radioactive calcium (45Ca2+) and analysed with the aid of a mathematical model, based partly on the Ca2+-dependent parameters obtained from Ca2+-ATPase experiments, partly on the A23187-induced Ca2+ fluxes determined in experiments with intact red cells. According to the model a delay in the activation of the Ca2+ pump is a prerequisite for the occurrence of A23187-induced calcium transients in the red cells, and we conclude that the Ca2+ pump in human red cells responds hysteretically. It is suggested that Ca2+ pumps in other types of cell also have hysteretic properties. 相似文献
14.
O. Gateau-Roesch E. Pavlov A. V. Lazareva E. A. Limarenko C. Levrat N-E. L. Saris P. Louisot G. D. Mironova 《Journal of bioenergetics and biomembranes》2000,32(1):105-110
A hydrophobic, low-molecular weight component extracted from mitochondria forms aCa2+-activated ion channel in black-lipid membranes (Mironova et al., 1997). At pH 8.3–8.5, thecomponent has a high-affinity binding site for Ca2+ with a Kd of 8 × 10–6 M, while at pH7.5 this Kd was decreased to 9 × 10–5 M. Bmax for the Ca2+-binding site did not changesignificantly with pH. In the range studied, 0.2 ± 0.06 mmol Ca2+/g component were boundor one calcium ion to eight molecules of the component. The Ca2+ binding was stronglydecreased by 50–100 mM Na+, but not by K+. Treatment of mitochondria withCaCl2 priorto ethanolic extraction resulted in a high level of Ca2+-binding capacity of the partially purifiedcomponent. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition,when added to the mitochondrial suspension, decreased the Ca2+-binding activity of thepurified extract severalfold. The calcium-binding capability of the partially purified componentcorrelates with its calcium-channel activity. This indicates that the channel-forming componentmight be involved in the permeability transition that stimulates its formation. 相似文献
15.
Colindres-Rojas M Wolf MM Gross R Seidel S Dietzek B Schmitt M Popp J Hermann G Diller R 《Biophysical journal》2011,(1):260-267
To gain a better understanding of the light-induced reduction of protochlorophyllide (PChlide) to chlorophyllide as a key regulatory step in chlorophyll synthesis, we performed transient infrared absorption measurements on PChlide in d4-methanol. Excitation in the Q-band at 630 nm initiates dynamics characterized by three time constants: τ1 = 3.6 ± 0.2, τ2 = 38 ± 2, and τ3 = 215 ± 8 ps. As indicated by the C13′=O carbonyl stretching mode in the electronic ground state at 1686 cm−1, showing partial ground-state recovery, and in the excited electronic state at 1625 cm−1, showing excited-state decay, τ2 describes the formation of a state with a strong change in electronic structure, and τ3 represents the partial recovery of the PChlide electronic ground state. Furthermore, τ1 corresponds with vibrational energy relaxation. The observed kinetics strongly suggest a branched reaction scheme with a branching ratio of 0.5 for the path leading to the PChlide ground state on the 200 ps timescale and the path leading to a long-lived state (>>700 ps). The results clearly support a branched reaction scheme, as proposed previously, featuring the formation of an intramolecular charge transfer state with ∼25 ps, its decay into the PChlide ground state with 200 ps, and a parallel reaction path to the long-lived PChlide triplet state. 相似文献
16.
V. S. Ananthanarayanan S. K. Attah-poku P. L. Mukkamala P. H. Rehse 《Journal of biosciences》1985,8(1-2):209-221
We report here two sets of results on proline-containing linear peptides, one of which brings out the role of theβ-turn conformation in the structure of nascent collagen while the other points to the functional importance of the β-turn
in calcium-binding proteins. Based on the data on peptides containing the -Pro-Gly-sequence, we had proposed and experimentally
verified that theβ-turn conformation in these peptides is a structural requirement for the enzymic hydroxylation of the proline residues in
the nascent (unhydroxylated) procollagen molecule. Our recent data, presented here, on the conformation of peptides containing
both the -Pro-Gly- and -Gly-Pro-sequences reveal that while theβ-turn in the substrate molecule is required at the catalytic site of prolyl hydroxylase, the polyproline-II structure is necessary
for effective binding at the active site of the enzyme. Thus, peptides containing either theβ-turn or the polyproline-II structure alone are found to act only as inhibitors while those with the polyproline-II followed
byβ-turn serve as substrates of the enzyme. In another study, we have synthesized the two linear peptides: Boc-Pro-D-Ala-Ala-NHCH3 and Boc-Pro-Gly-Ala-NHCH3 each of which adopts, in solution, a structure with two consecutiveβ-turns, as judged from circular dichroism, infrared and nuclear magnetic resonance data. Drastic spectral changes are seen
in these peptides on binding to Ca2+. Both the peptides show a distinct specificity to Ca2+ over Mg2+, Na+ and Li+. A conformational change in the peptides occurs on Ca2+ binding which brings together the carbonyl groups to coordinate with the metal ion. These results imply a functional role
for theβ-turn in Ca2+ — binding proteins. 相似文献
17.
Voltage-gated Ca2+ channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca2+ over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca2+ permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca2+-channel α1 subunits, Cav3.1, Cav3.2 and Cav3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Cav3.1, Cav3.2, and Cav3.3 functionally expressed in Xenopus oocytes. Our data show that all Cav3 channels select Ca2+ over Na+ by affinity. Cav3.1 and Cav3.2 discriminate Ca2+, Sr2+ and Ba2+ based on the ion's effects on the open channel probability, whilst Cav3.3 discriminates based on the ion's intrapore binding affinity. All Cav3s were characterized by much smaller difference in the KD values for Na+ current blockade by Ca2+ (KD1 ∼ 6 μM) and for Ca2+ current saturation (KD2 ∼ 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na+/Ca2+ current at close to physiological Ca2+ concentrations, which was the strongest for Cav3.3, smaller for Cav3.2 and the smallest for Cav3.1. In addition to intrapore Ca2+ binding site(s) Cav3.2, but not Cav3.1 and Cav3.3, is likely to possess an extracellular Ca2+ binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca2+ current in native cells. 相似文献
18.
Gerhard Meissner 《生物化学与生物物理学报:生物膜》1973,298(4):906-926
The binding of ATP and Ca2+ by the Ca2+ pump protein of sarcoplasmic reticulum from rabbit skeletal muscle has been studied and correlated with the formation of a phoshorylated intermediate. The Ca2+ pump protein has been found to contain one specific ATP and two specific Ca2+ binding sites per phosphorylation site. ATP binding is dependent on Mg2+ and is severely decreased when a phosphorylated intermediate is formed by the addition of Ca2+. In the presence of Mg2+ and the absence of Ca2+, ATP and ADP bind completely to the membrane. Pre-incubation with N-ethylmaleimide results in inhibition of ATP binding and decrease of Ca2+ binding. In the absence of ATP, Ca2+ binding is noncooperative at pH 6–7 and negatively cooperative at pH 8. Mg2+, Sr2+ and La3+, in that order, decrease Ca2+ binding by the Ca2+ pump protein. The affinity of the Ca2+ pump protein for both ATP and Ca2+ increases when the pH is raised from 6 to 8. At the infection point (pH ≈ 7.3) the binding constants of the Ca2+ pump protein-MgATP2? and Ca2+ pump protein-calcium complexes are approx. 0.25 and 0.5 μM?1, respectively. The unphosphorylated Ca2+ pump protein does not contain a Mg2+ binding site with an affinity comparable to those of the ATP and Ca2+ binding sites.The affinity of the Ca2+ pump protein for Ca2+ is not appreciably changed by the addition of ATP. The ratio of phosphorylated intermediate formed to bound Ca2+ is close to 2 over a 5-fold range of phosphoenzyme concentration. The equilibrium constant for phosphoenzyme formation is less than one at saturating levels of Ca2+. The phosphoenzyme is thus a “high-energy” intermediate, whose energy may then be used for the translocation of the two Ca2+.A reaction scheme is discussed showing that phosphorylation of sarcoplasmic reticulum proceeds via an enzyme-Ca22+-MgATP2? complex. This complex is then converted to a phosphoenzyme intermediate which binds two Ca2+ and probably Mg2+. 相似文献
19.
The patch-clamp technique was used to investigate regulation of anion channel activity in the tonoplast of Chara corallina in response to changing proton and calcium concentrations on both sides of the membrane. These channels are known to be Ca2+-dependent, with conductances in the range of 37 to 48 pS at pH 7.4. By using low pH at the vacuolar side (either pHvac 5.3 or 6.0) and a cytosolic pH (pHcyt) varying in a range of 4.3 to 9.0, anion channel activity and single-channel conductance could be reversibly modulated. In
addition, Ca2+-sensitivity of the channels was markedly influenced by pH changes. At pHcyt values of 7.2 and 7.4 the half-maximal concentration (EC
50) for calcium activation was 100–200 μm, whereas an EC
50 of about 5 μm was found at a pHcyt of 6.0. This suggests an improved binding of Ca2+ ions to the channel protein at more acidic cytoplasm. At low pHcyt, anion channel activity and mean open times were voltage-dependent. At pipette potentials (V
p) of +100 mV, channel activity was approximately 15-fold higher than activity at negative pipette potentials and the mean
open time of the channel increased. In contrast, at pHcyt 7.2, anion channel activity and the opening behavior seemed to be independent of the applied V
p. The kinetics of the channel could be further controlled by the Ca2+ concentration at the cytosolic membrane side: the mean open time significantly increased in the presence of a high cytosolic
Ca2+ concentration. These results show that tonoplast anion channels are maintained in a highly active state in a narrow pH range,
below the resting pHcyt. A putative physiological role of the pH-dependent modulation of these anion channels is discussed.
Received: 14 March 2001/Revised: 16 July 2001 相似文献
20.
Cardiac cellular calcium (Ca2+) handling is the well-investigated mediator of excitation–contraction coupling, the process that translates cardiac electrical activation into mechanical events. The reverse—effects of mechanical stimulation on cardiomyocyte Ca2+ handling—are much less well understood, in particular during the inter-beat period, called ‘diastole’. We have investigated the effects of diastolic length changes, applied axially using a pair of carbon fibres attached to opposite ends of Guinea pig isolated ventricular myocytes, on the availability of Ca2+ in the main cellular stores (the sarcoplasmic reticulum; SR), by studying the rest-decay of SR Ca2+ content [Ca2+]SR, and the reloading of the SR after prior depletion of Ca2+ from the cell.Cells were loaded with Fura-2 AM (an indicator of the cytosolic ‘free’ Ca2+ concentration, [Ca2+]i), and pre-conditioned by field-stimulation (2 Hz) at 37 °C, while [Ca2+]i transients and sarcomere length (SL) were recorded simultaneously. After reaching a steady state in the behaviour of observed parameters, stimulation was interrupted for between 5 and 60 s, while cells were either held at resting length, or stretched (controlled to cause a 10% increase in SL, to aid inter-individual comparison). Thereafter, each cell was returned to its original resting length, followed by swift administration of 10 mM of caffeine (in Na+/Ca2+-free solution), which causes the release of Ca2+ from the SR (caffeine), but largely prevents extrusion of Ca2+ from the cytosol to the cell exterior (Na+/Ca2+-free solution). By comparing the [Ca2+]i in cells exposed/not exposed to diastolic stretch of different duration, we assessed the rest-decay dynamics of [Ca2+]SR. To assess SR reloading after initial Ca2+ depletion, the same stretch protocol was implemented after prior emptying of the cell by application of 10 mM of caffeine in normal Tyrode solution (which causes Ca2+ to be released from the SR and extruded from the cell via the Na+/Ca2+ exchanger; NCX).Axial stretch enhanced the rate of both rest-decay and reloading of [Ca2+]SR. Application of 40 μM streptomycin, a blocker of stretch-activated ion channels, did not affect the stretch-induced increase in SR reloading. This behaviour was reproduced in a computer simulation study, using a modified version of the 2006 Iribe–Kohl–Noble model of single cardiac myocyte Ca2+ handling, suggesting that stretch increases both Ca2+ leak from the SR and Ca2+ influx via the sarcolemma. This may have important implications for the mobilisation of Ca2+ in stretched cells, and could contribute to the regional ‘matching’ of individual cardiomyocyte contractility to dynamic, and regionally varying, changes in mechanical loads, such as diastolic pre-load, of cardiac tissue. 相似文献