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1.
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   

2.
The isolation and characterization of the receptor for vitellogenin from follicle membranes of the rainbow trout, Oncorhynchus mykiss, is described. Follicle membrane proteins subjected to SDS-polyacrylamide gel electrophoresis and subsequently to either protein staining or ligand blotting with radiolabelled vitellogenin (125iodine-vitellogenin) demonstrated that the vitellogenin receptor has an apparent molecular mass of 200 kD (probably comprising of two 100-kD subunits) under non-reducing conditions. The vitellogenin binding sites were identified as specific receptors: binding was saturable and the binding sites were both tissue specific to follicle membranes and exhibited ligand specificity. Scatchard analyses of specific binding data revealed a single class of binding sites with a high affinity for rainbow trout vitellogenin (K d=8.2·10-9 mol·1-1). Both brown trout, Salmo trutta, vitellogenin and carp, Cyprinus carpio, vitellogenin were able to displace the radiolabelled rainbow trout vitellogenin from its receptor, although they were less effective than rainbow trout vitellogenin.Abbreviations B max maximum number of binding sites available - BSA bovine serum albumin - bt-VTG brown trout vitellogenin - c-VTG earp vitellogenin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - K d dissociatian constant - NCM nitrocellulose membranes - PMSF phenylmethylsulphonylfluoride - rt-VTG rainbow trout vitellogenin - VTG vitellogenin  相似文献   

3.
Calmodulin (CaM) is a Ca2+ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the Kd for the interaction of CaM with the plasma membrane Ca2+-ATPase (PMCA), a Ca2+ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca2+ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant Kd in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a Kd value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (Kd = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca2+ dependence follows a simple Hill plot demonstrating cooperative binding of Ca2+ to the binding sites in CaM.  相似文献   

4.
ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na+ + K+)-ATPase in the absence of Mg2+ (EDTA present) with a homogeneous but 15-fold different affinity, the Kd values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na+ + K+)-ATPase, respectively. The Kd value for ATP is equal to the Km for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na+ + K+)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg2+, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The Kd and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg2+ concentration. The Kd increases from 0.06 mM at 0.5 mM Mg2+ to a maximum of 0.26 mM at 2 mM Mg2+ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg2+ to 3.3 nmol/mg protein at 4 mM Mg2+. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg2+ concentration yields a maximal binding capacity at infinite Mg2+ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na+ + K+)-ATPase. The Kd for Mg2+ at the sites, where it exerts this effect, is 0.8 mM. The Kd for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg2+ to a maximum of 4.2 μM at 2 mM Mg2+ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg2+ concentration. Hence, Mg2+ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na+ + K+)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.  相似文献   

5.
A particulate (Na + K)-ATPase preparation from dog kidney bound [48V]-ortho-vanadate rapidly at 37°C through a divalent cation-dependent process. In the presence of 3 mM MgCl2 theK d was 96 nM; substituting MnCl2 decreased theK d to 12 nM but the maximal binding remained the same, 2.8 nmol per mg protein, consistent with 1 mol vanadate per functional enzyme complex. Adding KCl in the presence of MgCl2 increased binding, with aK 0.5 for KCl near 0.5 mM; the increased binding was associated with a drop inK d for vanadate to 11 nM but with no change in maximal binding. Adding NaCl in the presence of MgCl2 decreased binding markedly, with anI 50 for NaCl of 7 mM. However, in the presence of MnCl2 neither KCl nor NaCl affected vanadate binding appreciably. Both the nonhydrolyzable, ,-imido analog of ATP and nitrophenyl phosphate, a substrate for the K-phosphatase reaction that this enzyme also catalyzes, decreased vanadate binding at concentrations consistent with their acting at the low-affinity substrate site of the enzyme; the presence of KCl increased the concentration of each required to decrease vanadate binding. Oligomycin decreased vanadate binding in the presence of MgCl2, whereas dimethyl sulfoxide and ouabain increased it. With inside-out membrane vesicles from red blood cells vanadate inhibited both the K-phosphatase and (Na + K)-ATPase reactions; however, with the K-phosphatase reaction extravesicular K+ (corresponding to intracellular K+) both stimulated catalysis and augmented vanadate inhibition, whereas with the (Na + K)-ATPase reaction intravesicular K+ (corresponding to extracellular K+) both stimulated catalysis and augmented vanadate binding.  相似文献   

6.
In this study, passive Ca2+ binding was determined in ventricular homogenates (VH) from neonatal (4–6 days) and adult rats, as well as in digitonin-permeabilized adult ventricular myocytes. Ca2+ binding sites, both endogenous and exogenous (Indo-1 and BAPTA) were titrated. Sarcoplasmic reticulum and mitochondrial Ca2+ uptake were blocked by thapsigargin and Ru360, respectively. Free [Ca2+] ([Ca2+]F was measured with Indo-1 and bound Ca2+ ([Ca2+]B) was the difference between [Ca2+]F and total Ca2+. Apparent Ca2+ dissociation constants (Kd) for BAPTA and Indo-1 were increased by 10–20 mg VH protein/ml (from 0.35 to 0.92 μM for Indo-1 and from 0.20 to 0.76 μM for BAPTA) and also by ruthenium red in the case of Indo-1. Titration with successive CaCl2 additions (2.5–10 nmoles) yielded δ[Ca2+]B/δ[Ca2+]F for the sum of [Ca2+]B at all three classes of binding sites. From this function, the apparent number of endogenous sites (Ben) and their Kd (Ken) were determined. Similar Ken values were obtained in neonatal and adult VH, as well as in adult myocytes (0.68 ± 0.14 μM, 0.69 ± 0.13 μM and 0.53 ± 0.10 μM, respectively). However, Ben was significantly higher in adult myocytes than in adult VH (1.73 ± 0.35 versus 0.70 ± 0.12 nmol/mg protein, P < 0.01), which correspond to ∼300 and 213 μmol/l cytosol. This indicates that binding sites are more concentrated in myocytes than in other ventricular components and that Ben determined in VH underestimates cellular Ben by 29%. Although Ben values in nmol/mg protein were similar in adult and neonatal VH (0.69 ± 0.12), protein content was much higher in adult ventricle (125 ± 7 versus 80 ± 1 mg protein/g wet weight, P < 0.01). Expressing Ben per unit cell volume (accounting for fractional mitochondrial volume, and 29% dilution in homogenate), the passive Ca2+ binding capacity at high-affinity sites is ∼300 and 176 mmol/I cytosol in adult and neonatal rat ventricular myocytes, respectively. Additional estimates suggest that passive Ca2+ buffering capacity in rat ventricle increases markedly during the first two weeks of life and that adult levels are attained by the end of the first month.  相似文献   

7.
An in vitro system for the uptake of 125l-vitellogenin (VG) or vitellin into isolated follicles of the tobacco hornworm, Manduca sexta, is described. After incubation with 125l-VG, follicles were disrupted and the internal yolk contents separated from the follicle membranes. The results showed that 125l-VG was associated principally with the membranes (92%) after incubation at 4°C. However, at 27°C, 125l-VG was mainly in the yolk (92%). Furthermore, trypsin treatment removed approximately 70% of VG bound to the follicles at 4°C. Labeled VG was shown to bind to sonicated follicle membranes with high specificity and affinity (KD ? 1.3 × 10?8 M). This binding was sensitive to pH and calcium concentration. The total binding sites were estimated at 4 × 1014 sites/g of membrane protein. Competition studies showed that binding of 125l-VG to follicle membranes was blocked by excess unlabeled vitellin and deglycosylated vitellogenin but not by lipophorin (the major hemolymph lipoprotein), microvitellogenin, a female-specific protein (Mr ~ 31,000) found in both hemolymph and eggs, and the smaller vitellogenin subunit, apovitellogenin-II (Mr ~ 45,000). These results suggest that selective uptake of M. sexta VG from the hemolymph involves binding to specific receptors located on the follicle membranes.  相似文献   

8.
The binding parameters of 125I-labeled calmodulin to bovine cerebellar membranes have been determined and correlted with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca2+, calmodulin binds to a finite number of specific membrane sites with a dissociation constant (Kd) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca2+-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be infered from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca2+ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca2+ atoms. The concentration of the active calmodulin ·Ca2+ species required for half-maximal activation of adenylate cyclase is very similar to the Kd of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca2+-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.  相似文献   

9.
The effect of taurine on Ca2+ binding and uptake was studied with rabbit brain cortical and hippocampal synaptosomes. Taurine (25 mM) increased by 25% the high affinity 45Ca2+ binding in the cortical fraction and by 55% in hippocampal synaptosomes but had no effect on low affinity Ca2+ binding. Taurine decreased significantly the fluorescence of the chlorotetracycline-hydrophobic Ca2+ chelate probe in both synaptosomal fractions which suggests a shift of bound Ca2+ from the hydrophobic to the hydrophilic part of the membranes. The uptake of 45Ca2+ by rabbit brain synaptosomes, when measured in control and 65 mK K+-containing media, was not influenced by taurine. However, taurine inhibited significantly the 45Ca2+ uptake in synaptosomes incubated in media containing moderately increased K+ concentrations (14 and 20 mM K+). The effects of taurine are discussed in conjunction with its stabilizing effect on excitable membranes.  相似文献   

10.
Summary The objectives of the title were accomplished by a four-step experimental procedure followed by a simple graphical and mathematical analysis. Platelets are (i) overloaded with the indicator quin2 to cytoplasmic concentrations of 2.9mm and (ii) are exposed to 2mm external Ca2+ and 1.0 m ionomycin to rapidly achieve cytoplasmic Ca2+ ([Ca2+]cyt) of ca. 1.5 m. (iii) The external Ca2+ is removed by EGTA addition, and (iv) the active Ca2+ extrusion process is then monitored as a function of time. Control experiments show that the ionophore shunts dense tubular uptake and does not contribute to the Ca2+ efflux process during phases iii–iv and that the extrusion process is sensitive to metabolic inhibitors.The progress curves for the decline of quin2 fluorescence (resulting from active Ca2+ extrusion) were analyzed as a function of [Ca2+]cyt using a mathematical model involving the probability that an exported Ca2+ was removed from a quin2 complex (vs. a cytoplasmic binding element). The observed rates of decline of quin2 fluorescence at a particular [Ca2+]cyt are dependent upon (i) the absolute rate of the extrusion system (a function of itsK m, Vm and Hill coefficient (n)), (ii) the intrinsic Ca2+ buffer capacity of the cytoplasm (a function of the total site concentration ([B] T ) and itsK d) and (iii) the buffer capacity of the intracytoplasmic quin2 (a function of its concentration andK d). The contribution of (iii) was known and varied and was used to determine (ii) and (i) as a function of [Ca2+]cyt.The Ca2+ binding data were verified by45Ca2+ experimentation. The data fit a single binding site ([B] T =730±200 m) with an averageK d of 140±10n m. This can be accounted for by platelet-associated calmodulin. The rate of the Ca2+ extrusionvs. [Ca2+]cyt curve can be described by two components: A saturable one withV m=2.3±0.3 nmol min–1 mg-membrane–1,K m=80±10 andn=1.7±0.3 (probably identified with a Ca2+-ATPase pump) and a linear one (probably identified with a Na+/Ca2+ exchanger).  相似文献   

11.
Anticoagulation factor I (ACF I) from the venom of Agkistrodon acutus forms a 1:1 complex with activated coagulation factor X (FXa) in a Ca2+-dependent fashion and thereby prolongs the clotting time. In the present study, the dependence of the binding of ACF I with FXa on the concentration of Ca2+ ions was quantitatively analyzed by HPLC, and the result showed that the maximal binding of ACF I to FXa occurred at concentration of Ca2+ ions of about 1 mM. The binding of Ca2+ ions to ACF I was investigated by equilibrium dialysis and two Ca2+-binding sites with different affinities were identified. At pH 7.6, the apparent association constants K1 and K2 for these two sites were (1.8 ± 0.5) × 105 and (2.7 ± 0.6) × 104 M–1 (mean ± SE, n = 4), respectively. It was evident from the observation of Ca2+-induced changes in the intrinsic fluorescence of ACF I that ACF I underwent a conformational change upon binding of Ca2+ ions. The occupation of both Ca2+-binding sites in ACF I required a concentration of Ca2+ ions of about 1 mM, which is equal to the effective concentration of Ca2+ ions required both for maximal binding of ACF I to FXa and for the maximal enhancement of emission fluorescence of ACF I. It could be deduced from these results that the occupation of both Ca2+-binding sites in ACF I with Ca2+ ions and subsequent conformational rearrangement might be essential for the binding of ACF I to FXa.  相似文献   

12.
The microsomal fraction isolated from sea urchin H. pulcherrimus eggs has the ability to actively accumulate Ca2+ in the presence of ATP. The Ca2+ uptake was sustained by addition of oxalate and was apparently insensitive to sodium azide. The sequestered microsomal Ca was readily released by the divalent cation ionophore A23187. The microsomal fraction obtained from fertilized eggs accumulated Ca2+ about five times more quickly than did that from unfertilized eggs. The increased Ca2+ uptake by microsomal fraction obtained from fertilized eggs was due to an increase in the maximum velocity of Ca2+ uptake and there was no difference in Km for calcium between the two fractions.  相似文献   

13.
The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca2+-activated K+ channels (maxi-KCa) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-KCa channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (K d, 16.5 nM) and BPTI (K d, 1,490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (K d, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (K d, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (K d, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (K d, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (K d, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand–ligand interactions at distinct sites on the intracellular surface of maxi-KCa channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-KCa channel.  相似文献   

14.
This study has identified specific, stereoselective phenylalkylamine (PAA, (±)- [3H]verapamil) binding sites of low-affinity and high-density in cockroach (Periplaneta americana) nervous system and skeletal muscle membranes. Scatchard transformation of equilibrium binding data revealed a single population of binding sites in both tissues with dissociation constants (Kd) of 273 nM and 377 nM and binding capacities (Bmax) of 23 pmol·mg protein?1 and 37pmol·mg protein?1 for cockroach nervous tissue and skeletal muscle membranes, respectively. The PAA binding site in cockroach nervous tissue membranes was found to be dihydropyridine (DHP)-insensitive, whereas the corresponding site in cockroach skeletal muscle membranes was DHP-sensitive. This property of a DHP-sensitive PAA receptor distinguishes the binding sites identified in cockroach skeletal muscle from those in cockroach nervous tissue and indicates that pharmacologically distinct putative Ca2+ channel subtypes are present in insect nerve and muscle. © 1993 Wiley-Liss, Inc.  相似文献   

15.
Phosphate and a number of other compounds induce membrane permeability transition (MBT) in Ca2+-loaded mitochondria. 1-Anilino-8-naphthalene sulfonate (ANS) was used as a fluorescent probe to investigate perturbations on the inner membrane during MBT. Induction of MBT caused ANS fluoresence enhancement with a biphasic rate that reached a plateau. The enhancement is analogous to that reported for de-energization of mitochondria. The fluoresence level was independent of whether ANS was added before or at different times after phosphate. In the absence of ANS, fluorescence was low and remained unchanged. The initial time course of MBT, as followed by large-amplitude swelling, was similar to that of fluorescence enhancement. Ruthenium red, EGTA, ADP, and cyclosporin A inhibited the enhancement. Only EGTA + ADP (or ATP) reversed the enhancement when added after phosphate. Efflux of matrix Ca2+ by sodium acetate or A23187 did not alter ANS fluoresence. The binding parameters (K d and number of binding sites) were not significantly different, but the fluorescence maximum was more than doubled after MBT. Although the flourescence of bound ANS showed a nonlinear relationship, it was always higher (73.0 +/- 19.0%) after reaching the plateau. Since ANS binding to membranes is nonspecific, the exact mechanism of the enhanced fluorescence is not apparent. The dependence of the initial rate of fluorescence enhancement on Ca2+ concentration was nonlinear, with 45 µM at half-maximal rate. The dependence on phosphate was hyperbolic with 0.7 mM at half-maximal rate, which is close to theK m value of phosphate carrier. The kinetics is compatible with Ca2+ binding to some membrane component(s) during MBT and cause ANS fluorescence enhancement. It is suggested that the bilayer-nonbilayer (hexagonal11) transition consequent to Ca2+ binding to proteinphospholipid domains containing cardiolipin may play a role in fluorescence enhancement and MBT.  相似文献   

16.
Calmodulin (CaM) binding to the intracellular C-terminal tail (CTT) of the cardiac L-type Ca2+ channel (CaV1.2) regulates Ca2+ entry by recognizing sites that contribute to negative feedback mechanisms for channel closing. CaM associates with CaV1.2 under low resting [Ca2+], but is poised to change conformation and position when intracellular [Ca2+] rises. CaM binding Ca2+, and the domains of CaM binding the CTT are linked thermodynamic functions. To better understand regulation, we determined the energetics of CaM domains binding to peptides representing pre-IQ sites A1588, and C1614 and the IQ motif studied as overlapping peptides IQ1644 and IQ1650 as well as their effect on calcium binding. (Ca2+)4-CaM bound to all four peptides very favorably (Kd ≤ 2 nM). Linkage analysis showed that IQ1644-1670 bound with a Kd ~ 1 pM. In the pre-IQ region, (Ca2+)2-N-domain bound preferentially to A1588, while (Ca2+)2-C-domain preferred C1614. When bound to C1614, calcium binding in the N-domain affected the tertiary conformation of the C-domain. Based on the thermodynamics, we propose a structural mechanism for calcium-dependent conformational change in which the linker between CTT sites A and C buckles to form an A-C hairpin that is bridged by calcium-saturated CaM.  相似文献   

17.
《Cell calcium》2015,57(6):504-512
Imaging with Ca2+-sensitive fluorescent dye has provided a wealth of insight into the dynamics of cellular Ca2+ signaling. The spatiotemporal evolution of intracellular free Ca2+ observed in imaging experiments is shaped by binding and unbinding to cytoplasmic Ca2+ buffers, as well as the fluorescent indicator used for imaging. These factors must be taken into account in the interpretation of Ca2+ imaging data, and can be exploited to investigate endogenous Ca2+ buffer properties. Here we extended the use of Ca2+ fluorometry in the characterization of Ca2+ binding molecules within cells, building on a method of titration of intracellular Ca2+ binding sites in situ with measured amounts of Ca2+ entering through voltage-gated Ca2+ channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca2+ binding sites. The method was tested on simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca2+ indicator fluo-8. Focusing on data sets well described by a single endogenous Ca2+ buffer and dye, this method yielded estimates of the endogenous buffer concentration and Kd, the dye Kd, and the fraction of Ca2+ inaccessible cellular volume. The in situ Kd of fluo-8 thus obtained was indistinguishable from that measured in vitro. This method of calibrating Ca2+-sensitive fluorescent dyes in situ has significant advantages over previous methods. Our analysis of Ca2+ titration fluorometric data makes more effective use of the experimental data, and provides a rigorous treatment of multivariate errors and multiple Ca2+ binding species. This method offers a versatile approach to the study of endogenous Ca2+ binding molecules in their physiological milieu.  相似文献   

18.
红细胞在钙离子和离子载体A23187作用下的流变特性研究   总被引:1,自引:0,他引:1  
用新激光衍射法研究了钙离子及离子载体A23187对红细胞流变特性的影响.用不同浓度的钙离子及离子载体A23187分别处理红细胞后,测量其取向指数和小变形指数.结果表明离子载体A23187较细胞外钙离子浓度对红细胞流变特性的影响更大.而且,最大取向指数和最大小变形指数随着钙离子及离子载体A23187浓度的增加而降低.离子载体A23187浓度增加导致红细胞变形能力明显降低.  相似文献   

19.
We have studied the correlation between [3H]ouabain binding sites, (Na++K+)ATPase (EC 3.6.1.3) activity and acetylcholine (ACh) release in different subcellular fractions ofTorpedo marmorata electric organ (homogenate, synaptosomes, presynaptic plasma membranes). Presynaptic plasma membranes contained the greater number of [3H]ouabain binding sites in good agreement with the high (Na++K+)ATPase activity found in this fraction. Blockade of this enzymatic activity by ouabain dose-dependently induced ACh release from pure cholinergic synaptosomes, either in the presence or absence of extracellular calcium ions. We suggest that one of the mechanisms involved in the ouabain-induced ACh release in the absence of Ca2+ o may be an increase in Na+ i that could (a) evoke Ca2+ release from internal stores and (b) inhibit ATP-dependent Ca2+ uptake by synaptic vesicles.  相似文献   

20.
Adiponectin is secreted from adipose tissue and functions as a protein hormone in regulating glucose metabolism and fatty acid catabolism. Adiponectin plays an important role as a novel risk factor and potential diagnostic and prognostic biomarker in cancer. Crystal structures of globular adiponectin have been resolved with three calcium‐binding sites on the top of its central tunnel. However, the calcium‐binding property of adiponectin remains elusive. Mouse globular adiponectin was cloned into pET11a and expressed in Escherichia coli. The folding of adiponectin was indicated by the spread of resonances in HSQC spectrum. Luminescence resonance energy transfer was used to obtain the binding constant (Kd) of Tb3+ and the inhibitor constant (Ki) of Ca2+ for globular adiponectin. The obtained calcium‐binding affinity to adiponectin is relatively low (~2 mM), which indicates that the high concentration of adiponectin in circulating system may function as calcium storage bank and buffer the free calcium concentration. Copyright © 2012 John Wiley & Sons, Ltd.  相似文献   

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