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1.
A fluorescence and absorption chemosensor (SAAT) based on 5-(hydroxymethyl)-salicylaldehyde (SA) and o-aminothiophenol (AT) was designed and synthesized. SAAT in DMSO–HEPES (20.0 mM, v/v, 1:99, pH = 7.0) solution shows a highly selective and sensitive absorption and an ‘on–off’ fluorescence response to Cu2+ ions in aqueous solutions over all other competitive metal ions including Na+, Ag+, Ba2+, Ca2+, Cd2+, Mg2+, Zn2+, Cr3+, Al3+, Hg2+, K+, Mn2+, Ni2+, Sr2+, Tb3+ and Co2+. SAAT exhibits ratiometric absorption sensing ability for Cu2+ ions. Importantly, SAAT also can sense Cu2+ ions using fluorescence quenching, the fluorescence intensity of SAAT showed a good linear relationship with Cu2+ concentration, and the detection limit of Cu2+ was 0.34 μM. The results of Job's plot, Benesi–Hildebrand plot, mass spectra, and density functional theory calculations confirmed that the selective absorption and fluorescence response were attributed to the formation of a 1:1 complex between SAAT and Cu2+. SAAT in test film could identify Cu2+ in water samples using the intuitive fluorescence colour change under a UV lamp. SAAT has great application value as a selective and sensitive chemosensor to discriminate and detect Cu2+ ions. 相似文献
2.
Antonio Gianguzza Francesco Maggio Silvio Sammartano 《Chemical Speciation and Bioavailability》2013,25(1-2):17-21
ABSTRACTProtonation constants and Na+-, Mg2+-, Ca2+-, Zn2+- and Cd2+-D-Glucuronate complex formation constants have been determined potentiometrically in different ionic strength conditions. Glucuronate forms a very weak complex species with Na+ (K ~ 1 dm3 mol?1), and weak species with Mg2+ and Ca2+ (K ~ 10 dm3 mol?1). When glucuronate interacts with Zn2+ and Cd2+, two species, M(gluc) and M(gluc)2 (glue = glucuronate) having a stability intermediate between that of mono and dicarboxylate complexes, are formed. This can be due to the involvement of the ethereal oxygen in the coordination. Medium effects are considered in the light of speciation problems. 相似文献
3.
Levels of Mg2+, Ca2+ and Fe2+/Fe3+ were determined in roots and shoots of sugar beet seedlings (Beta vulgaris L. cv. Monohill) cultured for 5 weeks in a complete nutrient solution to which either Cd2+ (0, 5 or 50 μM), EDTA (0, 10 or 100 μM) or a combination of both was added. The plants subjected to the various treatments showed a variety of deficiency symptoms. Leaves of the Cd2+-treated plants became thin and chlorotic (Mg- and Fe-deficiency symptoms). The plants showed reduced growth and developed only a few brownish roots with short laterals (Ca-deficiency symptoms). EDTA treatment resulted in green, stunted, hard leaves and reduced growth (Ca-deficiency symptoms). The deficiency symptoms observed correspond well with the observed uptake rates and distributions of Mg2+, Ca2+ and Fe2+/Fe3+. Increases in either Cd2+, EDTA or a combination of both in the growth medium, were correlated with increasing Mg2+ levels in the roots and with decreasing Mg2+ levels in the shoots. Cd2+ alone or in combination with EDTA had little influence on Ca2+ levels in the shoots but decreased Ca2+ levels in the roots. Thus, Cd2+ affects Mg2+ and Ca2+ transport in opposite ways: Mg2+ transport to the shoots is inhibited while that of Ca2+ is facilitated. Treatment with EDTA alone did not affect Ca2+ concentrations in either the shoots or the roots. Treatment with Cd2+ lowered Fe2+ concentrations in both roots and shoots. 相似文献
4.
Cd2+, Mn2+, and Al3+ inhibited synaptosomal amine uptake in a concentration-dependent and time-dependent manner. In the absence of Ca2+, the rank order of inhibition of noradrenaline uptake was: Cd2+ (IC50 = 250 μM) > Al3+ (IC50 = 430 μM) > Mn2+ (IC50 = 1.50 mM), the IC50 being the concentration of metal ions that gave rise to 50% inhibition of uptake. In the presence of 1 mM Ca2+, the rank order of inhibition of uptake was: Al3+ (IC50 = 330 μM) > Cd2+ (IC50 = 540 μM) > (IC50 = 1.5 mM). The rank order of inhibition of serotonin uptake without Ca2+ was: Al3+ (IC50 = 370 μM) > Cd2+ (IC50 = 610 μM) > Mn2+ (IC50 = 3.4 mM) and the rank order in the presence of 1 mM Ca2+ was: Al3+ (IC50 = 290 μM) > Cd2+ (IC50 = 1.5 mM) > Mn2+ (IC50 = 4.0 mM). Ca2+, at 1 mM, definitely antagonized the inhibitory actions of Cd2+ on noradrenaline and serotonin uptake. Al3+ stimulated noradrenaline uptake at concentrations around 20–250 μM but inhibited this uptake at concentrations exceeding 300 μM in a dose-related fashion. Ca2+, at 1 mM, enhanced both the stimulatory and inhibitory effects of Al3+. Ca2+ also enhanced the inhibitory actions of Al3+ on seotonin uptake. These results, in conjunction with those we have previously published, suggest that Cd2+, Mn2+, and Al3+ exert differential and selective effects on the structure and function of synaptosomal membranes. 相似文献
5.
6.
(1) The fluorescence of eosin Y in the presence of (Na+ + K+)-ATPase is enhanced by Mg2+. The enhancement by Mg2+ is larger than that obtained with Na+ (Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232–240). Mg2+ shifts the excitation maximum from 518 to 524 nm, the emission maximum from 538 to 542 nm. Also a shoulder appears at about 490 nm on the excitation curve, as was also observed with Na+. (2) The Mg2+-dependent enhancement of fluorescence can be reversed by K+ as well as by ATP. In the presence of Mg2+ + Pi (i.e. under conditions of phosphorylation), the fluorescence enhancement can be reversed by ouabain. With Mg2+ and a low concentation of K+ (i.e. conditions for vanadate binding), the enhancement of fluorescence can be reversed by vanadate. (3) There is a low-affinity binding of eosin which increases with the Mg2+ concentration. This is observed as a slight increase in the fluorescence when the excitation wavelength is above 520 nm. The low-affinity binding is K+-, ATP-, ouabain- and vanadate-insensitive. (4) Scatchard analysis of the binding experiments suggests that there are two high-affinity eosin-binding sites per 32P-labelling site in the presence of 5 mM Mg2+ both of which are ouabain-, vanadate- and ATP-sensitive. With 5 M Mg2+ + 0.25 Pi, the Kd values are 0.14 μM and 1.3 μM, respectively. With 5 mM Mg2+, 150 mM Na+, the Kd values are 0.45 μM and 3.2 μM, respectively. With 5 mM Mg2+, the addition of K+ gives a pronounced decrease in affinity but does not decrease the number of binding sites (which remains at two per 32P-labelling site). With 5 mM Mg2+ + 150 mM K+, the affinities of the two binding sites become identical, at a Kd of 17 μM. (5) The rate of conformational transitions was measured using the stopped-flow method. The rate of the transition from the Mg2+-form to the K+-form is high. Oligomycin has only a small (if any) effect on the rate. Addition of Na+ in the presence of Mg2+ does not appreciably change the rate of conversion to the K+-form, giving a rate constant of about 110 s?. However, the addition of oligomycin in the presence of Mg2+ + Na+ had a profound effect: the rate of conversion to the K+-form was decreased by a factor of 2000 to about 0.063 s?1. This suggests that the conformation with Mg2+ alone is different from the conformation with Na+ alone. (6) The effects of K+, ouabain, vanadate and ATP on the high-affinity binding of eosin suggest that the two eosin molecules bound per 32P-labelling site are bound to ATP sites. 相似文献
7.
Duong Tuan Quang Nguyen Van Hop Nguyen Dinh Luyen Ha Phuong Thu Doan Yen Oanh Nguyen Khoa Hien Nguyen Van Hieu Min Hee Lee Jong Seung Kim 《Luminescence》2013,28(2):222-225
We prepared an aminothiourea‐derived Schiff base (DA) as a fluorescent chemosensor for Hg2+ ions. Addition of 1 equiv of Hg2+ ions to the aqueous solution of DA gave rise to an obvious fluorescence enhancement and the subsequent addition of more Hg2+ induced gradual fluorescence quenching. Other competing ions, including Pb2+, Cd2+, Cr3+, Zn2+, Fe2+, Co3+, Ni2+, Ca2+, Mg2+, K+ and Na+, did not induce any distinct fluorescence changes, indicating that DA can selectively detect Hg2+ ions in aqueous solution. Copyright © 2012 John Wiley & Sons, Ltd. 相似文献
8.
Inge-Bert Täljedal 《Biochimica et Biophysica Acta (BBA)/General Subjects》1974,372(1):154-161
The fluorescence of microdissected pancreatic islets of ob/ob-mice was studied by microscope photometry after incubation with 10 μM chlorotetracycline. In Krebs-Ringer bicarbonate buffer, excitation at 390 nm yielded peak emission at 530 nm, suggesting that chelated Ca2+ was the major source of fluorescence. In support of this interpretation, incubation in Ca2+-free buffer markedly decreased the fluorescence, whereas withdrawal of Mg2+ increased it. Raising the Mg2+ concentration to 15 mM suppressed the fluorescence. In the presence of Ca2+, the substitution of choline ions for Na+ increased the fluorescence considerably; in the absence of Ca2+, however, Na+ deficiency had only little effect. Control experiments showed that Na+ or choline ions had no effect on the fluorescence of Ca2+-chlorotetracycline in 70 or 90% methanol. In 90%, but not in 70%, methanol 15 mM Mg2+ slightly quenched the fluorescence from 2.5 mM Ca2+ and 10 μM chlorotetracycline. It is suggested that Na+, and perhaps Mg2+, tends to decrease the amount of membrane-bound Ca2+ in the pancreatic islets. 相似文献
9.
1. Cadmium (≤ 50 μM) decreases the heat resistance (39°C) of the activity of frontal cilia in the Anodonta cygnea gills incubated in dechlorinated tap water, while in the presence of added 2 mM Ca2+ the minimal acting concentration of cadmium rises up to 100 μM.2. The inhibitory effect of Cd2+ (1.5 mM) on the ATPase activity measured in the gill microsomal fraction is temperature dependent and increases as follows: ouabain insensitive Na2+- or K+-ATPase (no inhibition), Ca2+-ATPase (50% inhibition), Mg2+-ATPase (100% inhibition).3. Cadmium itself (≤ 50 μM) added to microsomal suspension stimulates the H+-sensitive ATP hydrolysis resembling on its pH-dependence the Mg2+- but not Ca2+-ATPase activity.4. Cd2+ can mimic the effect of Mg2+ as a cofactor required for activation of the ouabain-insensitive Na+- or K+-ATPase. Monovalent cations fail to activate the ATPase when Mg2+ is substituted by Ca2+.5. One of the mechanisms underlying the toxicity of Cd2+ to Anodonta gills could be based upon an interaction of Cd2+ with Mg2+-ATPase followed by suppression of the ciliary activity. 相似文献
10.
A Ca2+-ATPase and a Mg2+/H+-antiporter are present on tonoplast membranes from roots of Zea mays L. 总被引:1,自引:0,他引:1
The primary or secondary energized transport of Ca2+, Mg2+ and H+ into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca2+ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca2+ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Ca2+-pumping and tonoplast H+-pyrophosphatase (PPiase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca2+-pumping activity could be detected. The ATP-dependent Ca2+ uptake into tonoplast vesicles (using Ca2+ concentrations from 0.8–1 μM) was completely inhibited by the Ca2+ ionophore ionomycin (1 μM) whereas the protonophore nigericin (1 μM) which eliminates ATP-dependent intravesicular H+ accumulation had no effect. Vanadate (IC50 = 43 μM) and diethylstilbesterol (IC50 = 5.2 μM) were potent inhibitors of this type of Ca2+ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%–50% of the ATP-dependent activity (K m = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca2+ transport mechanism is the only functioning Ca2+ transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca2+ concentration. In contrast, the secondary energized Ca2+-transport mechanism of the tonoplast, the low-affinity Ca2+/H+-antiporter, which was reported to allow the uptake of Ca2+ in exchange for H+, functions chiefly as an Mg2+ transporter under physiological conditions because cytosolic Mg2+ is several orders of magnitude higher than the Ca2+ concentration. This conclusion was deduced from experiments showing that Mg2+ ions in a concentration range of 0.01 to 1 mM triggered a fast efflux of H+ from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H+-ATPase and H+-PPiase was found to be influenced by Ca2+ differently from and independently of the Mg2+ concentration. Calcium was a strong inhibitor for the H+-PPiase (IC50 = 18 μM, Hill coefficient nH = 1.7) but a weak one for the H+-ATPase (IC50 = 330 μM, nH = 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca2+-and Mg2+-regulated H+-PPiase, (ii) the newly described high-affinity Ca2+-AT-Pase, (iii) the low-affinity Mg2+(Ca2+)/H+-antiporter and (iv) the H2+-ATPase. 相似文献
11.
Avinash A. Kamble Dattatray K. Dalavi Netaji K. Desai Prasad G. Mahajan Govind B. Kolekar Shivajirao R. Patil 《Luminescence》2023,38(11):1883-1891
Sodium dodecyl sulfate (SDS)-capped 1-pyrenecarboxaldehyde nanoparticles (PyalNPs) were prepared using a reprecipitation method in an aqueous medium and exhibited red-shifted aggregation-induced enhanced emission (AIEE). The dynamic light scattering (DLS) examination showed narrower particle size distribution with an average particle size of 41 nm, whereas −34.5 mV zeta potential value indicate the negative surface charge and good stability of nanoparticles (NPs) in an aqueous medium. The AIEE was seen at λmax = 473 nm in a fluorescence spectrum of a PyalNP suspension. In the presence of Cu2+ ions, the fluorescence of PyalNPs quenches very significantly, even in the presence of other metal ions like Ba2+, Ca2+, Cd2+, Co2+, Al3+, Fe2+, Hg2+, Ni2+ and Mg2+. The changes in the fluorescence lifetime of PyalNPs in the presence of Cu2+ ions suggested that the type of quenching was dynamic. The fluorescence quenching data for the NPs suspension fitted well into a typical Stern–Volmer relationship in the concentration range 1.0–25 μg/ml of Cu2+ ions. The estimated value of the correlation coefficient R2 = 0.9877 was close to 1 and showed the linear relationship between quenching data and Cu2+ ion concentration. The limit of detection (LOD) was found to be 0.94 ng/ml and is far below the tolerable intake limit value of 1.3 μg/ml accepted by the World Health Organization for Cu2+ ions in drinking water. The fluorescence quenching approach for a SDS-capped Pyal nanosuspension for copper ion quantification is of high specificity and coexisting ions were found to interfere very negligibly. The developed method was successfully applied for the estimation of copper ions in river water samples. 相似文献
12.
A highly sensitive and reversible chemosensor for Hg2+ detection based on porphyrin‐thymine conjugates
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Xiangzhu He Duanguang Yang Hongbiao Chen Wei Zheng Huaming Li 《Journal of molecular recognition : JMR》2015,28(5):293-298
In this study, we demonstrated a highly sensitive, selective, and reversible chemosensor for Hg2+ determination. This chemosensor was synthesized by direct condensation of thymin‐1‐ylacetic acid with zinc tetraaminoporphyrin, which has a porphyrin core as the fluorophore and four thymine (T) moieties as the specific interaction sites for Hg2+. The probe (4T‐ZnP) exhibited split Soret bands with a small peak at 408 nm and a strong band at 429 nm in a dimethylformamide/H2O (7/3, v/v) mixed solvent as well as a strong emission band at 614 nm. Upon the addition of Hg2+, the probe displayed strong fluorescence quenching due to the formation of T‐Hg2+‐T complexes. With the aid of the fluorescence spectrometer, the chemosensor in the dimethylformamide/H2O (7/3, v/v) mixed solvent (0.3 μM) exhibited a detection limit of 6.7 nM. Interferences from other common cations, such as Co2+, K+, Sn2+, Zn2+, Cu2+, Ni2+, Mn2+, Na+, Ca2+, Mg2+, Pb2+, and Cd2+, associated with Hg2+ analysis were effectively inhibited. Copyright © 2015 John Wiley & Sons, Ltd. 相似文献
13.
Eugene E. Quist 《Biochemical and biophysical research communications》1980,92(2):631-637
In the presence of 1.0 mM ATP and MgCl2, the specific viscosity of suspensions of human erythrocyte ghosts decreases 35% in 20 minutes at 22°C. The changes in viscosity are a sensitive index of Mg-ATP dependent shape changes in these membranes. Low concentrations of Ca2+ (1 to 5 μM) inhibit Mg-ATP dependent viscosity changes. If ghosts were preincubated with 1 mM Mg-ATP and 20 μM A23187 to produce a maximal decrease in viscosity, addition of 10 μM Ca2+ to the preincubated ghosts increased the viscosity to levels observed in ghosts preincubated without ATP. Ca2+ (1 to 5 μM) also inhibited Mg2+ dependent phosphorylation 30% and stimulated dephosphorylation 25% in ghost membranes. These effects of Ca2+ on viscosity and phosphorylation may be due to a membrane bound Ca2+ phosphatase activity which dephosphorylates membranes phosphorylated by a Mg2+ dependent kinase activity. 相似文献
14.
(1) The effects of calmodulin binding on the rates of Ca2+-dependent phosphorylation and dephosphorylation of the red-cell Ca2+ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg2+-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg2+-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca2+ + Mg2+)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca2+ affinity for sustaining ATP hydrolysis is raised at least 20-fold, Km(Ca) ? 10 μM (—calmodulin) and Km(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca2+ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (Km(Ca) ? 3–4 · 10-8 M). (Ca2+ + Mg2+)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca2+ + Mg2+)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca2+ + Mg2+)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (Km(MgATP) ? 360 μM), and CaATP is an inhibitor (Ki(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca2+- pump active site, producing a high (Ca2+ + Mg2+)-ATPase activity, high Ca2+ affinity and regulation of activity by MgATP. 相似文献
15.
R.R. Preston 《The Journal of membrane biology》1998,164(1):11-24
The properties of Mg2+ conductances in Paramecium tetraurelia were investigated under two-electrode voltage clamp. When bathed in physiological Mg2+ concentrations (0.5 mm), depolarizing steps from rest elicited a prominent Mg2+-specific current (I
Mg) that has been noted previously. The dependence of this current on extracellular Mg2+ approximated that of Mg2+-induced backward swimming, demonstrating that I
Mg contributes to normal membrane excitation and behavior in this ciliate. Closer analysis revealed that the Mg2+ current deactivated biphasically. While this might suggest the involvement of two Mg2+-specific pathways, both tail-current components were affected similarly by current-specific mutations and they had similar
ion selectivities, suggesting a common pathway. In contrast, a Mg2+ current activated upon hyperpolarization could be separated into three components. The first, I
Mg, had similar properties to the current activated upon depolarization. The second was a nonspecific divalent cation current
(I
NS) that was revealed following suppression of I
Mg by eccentric mutation. The final current was relatively minor and was revealed following suppression of I
Mg and I
NS by obstinate A gene mutation. Reversal-potential analyses suggested that I
Mg and I
NS define two intracellular compartments that contain, respectively, low (0.4 mm) and high (8 mm) concentrations of Mg2+. Measurement of intracellular free Mg2+ using the fluorescent dye, Mag-fura-2, suggested that bulk [Mg2+]
i
rests at around 0.4 mm in Paramecium.
Received: 12 January 1998/Revised: 16 March 1998 相似文献
16.
Vijai S. Shankar Christopher M. R. Bax Bridget E. Bax A. S. M. Towhidul Alam Baljit S. Moonga Bruce Simon Michael Pazianas Christopher L.-H. Huang Mone Zaidi 《Journal of cellular physiology》1993,155(1):120-129
Earlier studies have demonstrated that a high (mM) extracellular Ca2+ concentration triggers intracellular [Ca2+] signals with a consequent inhibition of bone resorptive activity. We now report that micromolar concentrations of the divalent cation, Ni2+, elicited rapid and concentration-dependent elevations of cytosolic [Ca2+]. The peak change in cytosolic [Ca2+] increased monotonically with the application of [Ni2+] in the 50–5,000 μM range in solutions containing 1.25 mM-[Ca2+] and 0.8 mM-[Mg2+]. The resulting concentration-response function suggested Ni2+-induced activation of a single class of binding site (Hill coefficient = 1). The triggering process also exhibited a concentration-dependent inactivation in which conditioning Ni2+ applications in the range 5–1,500 μM-[Ni2+] inhibited subsequent responses to a maximally effective [Ni2+] of 5,000 μM. Ni2+-induced cytosolic [Ca2+] responses were not dependent on extracellular [Ca2+]. Thus, when 5,000 μM-[Ni2+] was applied to osteoclasts in Ca2+-free, ethylene glycol bis-(aminoethyl ether) tetraacetic acid (EGTA)-containing medium (≤5 nM-[Ca2+] and 0.8 mM-[Mg2+]), cytosolic [Ca2+] responses resembled those obtained in the presence of 1.25 mM-[Ca2+]. Prior depletion of intracellular Ca2+ stores by ionomycin prevented Ni2+-induced cytosolic [Ca2+] responses, suggesting a major role for intracellular Ca2+ redistribution in the response to Ni2+. The effects of Ni2+ were also modulated by the extracellular concentration of the divalent cations, Ca2+ and Mg2+. When these cations were not added to the culture medium (0 μM-[Ca2+] and [Mg2+]), even low [Ni2+] ranging between 5 pM and 50 μM elicited progressively larger cytosolic [Ca2+] transients. However, the response magnitude decreased at higher, 250–5,000 μM-[Ni2+], resulting in a “hooked” concentration-response curve. Furthermore, increasing extracellular [Mg2+] or [Ca2+] (0–1 mM) diminished the response to 50 μM-[Ni2+], a concentration on the rising phase of the “hook.” Similar increases (0–10 mM) in extracellular [Mg2+] or [Ca2+] increased the response to 5,000 μM-[Ni2+], a concentration on the falling phase of the “hook”. These findings are consistent with the existence of a membrane receptor strongly sensitive to Ni2+ as well as the divalent cations, Ca2+ and Mg2+. Receptor occupancy apparently activates intracellular Ca2+ release followed by inactivation. Furthermore, repriming is independent of intracellular Ca2+ stores, suggesting that such inactivation operates at a transduction step between receptor occupancy and intracellular Ca2+ release. © 1993 Wiley-Liss, Inc. 相似文献
17.
Betty L. Black Jay M. McDonald Leonard Jarett 《Archives of biochemistry and biophysics》1980,199(1):92-102
The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg2+-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca2+ + Mg2+-ATPase) of relatively low activity. Mg2+-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca2+ + Mg2+)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg2+-ATPase activity revealed apparent Km values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg2+-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg2+-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca2+ + Mg2+)-ATPase was 13.7 nmol 32P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol 32P/mg/min. Characterization of (Ca2+ + Mg2+)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent Kms for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca2+ + Mg2+)-ATPase activity was stimulated by potassium with an apparent Km of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca2+ + Mg2+)-ATPase are similar to those of the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca2+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme. 相似文献
18.
Sidney Katz Basil D. Roufogalis Amiram D. Landman Larry Ho 《Journal of cellular biochemistry》1979,10(2):215-225
Erythrocyte membranes prepared by three different procedures showed (Mg2+ + Ca2+)-ATPase activities differing in specific activity and in affinity for Ca2+. The (Mg2+ + Ca2+)-ATPase activity of the three preparations was stimulated to different extents by a Ca2+-dependent protein activator isolated from hemolystes. The Ca2+ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2–200 μM or lowering the Mg:ATP ratio to less than one shifted the (Mg2+ + Ca2+)-ATPase activity in stepwise hemolysis membranes from mixed “high” and “low” affinity to a single high Ca2+ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strengh, or membranes prepared by the EDTA (1–10 mM) procedure, did not undergo the shift in the Ca2+ affinity with changes in ATP and MgCl2 concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg2+ + Ca2+)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form. 相似文献
19.
Aravind P Chandra K Reddy PP Jeromin A Chary KV Sharma Y 《Journal of molecular biology》2008,376(4):1100-1115
Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca2+ signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca2+-specific) and structural (Ca2+- or Mg2+-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca2+ binding using NMR and EF-hand mutants. Ca2+ titration, as monitored by [15N,1H] heteronuclear single quantum coherence, suggests that Ca2+ binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg2+ binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca2+-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca2+ and not Mg2+. Ca2+ binding induces conformational rearrangements in the protein by reversing Mg2+-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg2+ with Ca2+ reduces the Ca2+-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca2+-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg2+-bound NCS-1 are similar, the early unfolding transitions of Ca2+-bound NCS-1 are partially influenced in the presence of Mg2+. This study demonstrates the importance of Mg2+ as a modulator of calcium homeostasis and active-state behavior of NCS-1. 相似文献