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1.
Isolated hepatocytes release 2–3 nmol Mg2+/mg protein or ~10% of the total cellular Mg2+ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg2+ release, a quantitatively similar amount of Ca2+ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg2+:1Ca2+. Calcium induced Mg2+ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg2+:1Ca2+ exchange ratio. The uptake of Ca2+ for the release of Mg2+ occurs in the absence of significant changes in Δψ as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP+) electrode or 3H-TPP+. Collapsing the Δψ by high concentrations of TPP+ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca2+-induced Mg2+ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca2+ uptake and Mg2+ release. These data demonstrate the operation of an electroneutral Ca2+/Mg2+ exchanger which represents a novel pathway for Ca2+ accumulation in liver cells following adrenergic receptor stimulation. This work was supported by National Institutes of Health Grant HL 18708.  相似文献   

2.
The plasma membrane (Ca2++Mg2+)ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca2+ ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca2+ strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca2+-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca2+ is related to opposite long-term hydration effects on the substrate binding domain and the Ca2+ binding domain.  相似文献   

3.
The Mg2+-dependency of Ca2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg2+- and Ca2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg2+ with a Km of 13 μ M in the absence or presence of 1 μ M free Ca2+. At free Mg2+ concentrations of 1 μ M and lower, ATP hydrolysis is Mg2+ -independent, but is strongly stimulated by submicromolar Ca2+ concentrations Km = 0.25 μM, Vmax = 24 μmol Pi/h per mg protein). The Ca2+-stimulated ATP hydrolysis strongly decreases at higher Mg2+ concentrations. The Ca2+-stimulated Mg2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca2+-pumping in renal basolaterals and on Ca2+-ATPase in erythrocyte ghosts suggest that the Ca2+-pumping enzyme requires Mg2+. In contrast, a role of the Ca2+-stimulated Mg2+-independent ATP hydrolysis in active Ca2+ transport across basolateral membranes is rather unlikely.  相似文献   

4.
Magnesium ions (Mg2+) play a fundamental role in cellular function, but the cellular dynamic changes of intracellular Mg2+ remain poorly delineated. The present study aims to clarify whether the concentration of intracellular Mg2+ possibly changes cyclically in association with rhythmic contraction and intracellular Ca2+ oscillation in cultured cardiac myocytes from neonatal rats. To do this, we performed a noise analysis of fluctuations in the concentration of intracellular Mg2+ in cardiac myocytes. The concentration was estimated by loading cells with either Mg‐fluo4/AM or KMG‐20/AM. Results revealed that the intensity of Mg‐fluo‐4 or KMG‐20 fluorescence fluctuated cyclically in association with the rhythmic contraction of cardiac myocytes. In addition, the simultaneous measurement of Fura2 and Mg‐fluo‐4 fluorescence revealed phase differences between the dynamics of the two signals, suggesting that the cyclic changes in the Mg‐fluo‐4 or KMG‐20 fluorescent intensity actually reflected the changes in intracellular Mg2+. The complete termination of spontaneous rhythmic contractions did not abolish Mg2+ oscillations, suggesting that the rhythmic fluctuations in intracellular Mg2+ did not result from mechanical movements. We suggest that the concentration of intracellular Mg2+ changes cyclically in association with spontaneous, cyclic changes in the concentration of intracellular Ca2+ of cardiac myocytes. A noise analysis of the fluctuation of subtle changes in fluorescence intensity could contribute to the elucidation of novel functional roles of Mg2+ in cells.  相似文献   

5.
The goal of the present study is to explore whether Ca2+ and Mg2+-binding properties of isomeric Kv channel-interacting proteins (KChIPs) have different effects on their molecular structure and the binding with Kv channel. 8-Anilinonaphthalene- 1-sulfonate fluorescence measurement showed that KChIP4.1 and KChIP2.2 possessed one and two types of Ca2+-binding sites, respectively, and only one type of Mg2+-binding site was noted in the two KChIP proteins. Removal of EF-hand 4 (EF-4) caused a marked drop in their high affinities for Ca2+, but the binding affinity for Mg2+ remained mostly the same. Unlike KChIP4.1, the intact EF-4 was essential for the Kv channel-binding ability of KChIP2.2 in a metal-free buffer. Nevertheless, the interaction of wild-type KChIPs and EF-4-truncated mutants with Kv channel was enhanced by the addition of Mg2+ and Ca2+. In contrast to KChIP4.1, the thermal stability of KChIP2.2 was decreased by the binding of Mg2+ and Ca2+. These results suggest that the conformational change with metal-bound KChIP4.1 is crucial for its interaction with Kv channel but not for KChIP2.2, and that the Mg2+- and Ca2+-binding properties of KChIP2.2 and KChIP4.1 have different effects on their molecular structure.  相似文献   

6.
Applications of intrinsic fluorescence measurements in the study of Ca2+-transport ATPases are reviewed. Since the initial reports showing that the fluorescence emission was sensitive to Ca2+ binding, a substantial amount of work has focused on the use of both steady-state and time-resolved fluorescence spectroscopy to investigate structure-function relationships in sarcoplasmic reticulum and plasma membrane Ca2+-ATPases. These studies have revealed ligand-induced conformational changes, as well as provided information on protein-protein, protein-solvent and/or protein-lipid interactions in different functional states of these proteins. The main results of these studies, as well as possible future prospects are discussed.  相似文献   

7.
Fedirko  N.  Vats  Yu.  Kruglikov  I.  Voitenko  N. 《Neurophysiology》2004,36(3):169-173
In a rat model of streptozotocin (STZ)-induced diabetes, we earlier showed that under these conditions the concentration of free cytosolic Ca2+ in input neurons of the nociceptive system increases, Ca2+ signals are prolonged, while Ca2+ release from intracellular calcium stores decreases. The aim of our study was to test the hypothesis that changes in the activities of Ca2+,Mg2+-ATPases of the endoplasmic reticulum (SERCA) and plasmalemma (PMCA) could be responsible for diabetes-induced disorders of calcium homeostasis in nociceptive neurons. We measured the Ca2+,Mg2+-ATPase activities in microsomal fractions obtained from tissues of the dorsal root ganglia (DRG) and spinal dorsal horn (DH) of control rats and rats with experimentally induced diabetes. The integral specific Ca2+,Mg2+-ATPase activity in microsomes from diabetic rats was lower than that in the control group. The activity of SERCA in samples of DRG and DH of diabetic rats was reduced by 50 ± 8 and 48 ± 12%, respectively, as compared with the control (P < 0.01). At the same time, the activity of PMCA decreased by 63 ± 6% in DRG and by 60 ± 9% in DH samples (P < 0.01). We conclude that diabetic polyneuropathy is associated with the reduction of the rate of recovery of the Ca2+ level in the cytosol of DRG and DH neurons due to down-regulation of the SERCA and PMCA activities.  相似文献   

8.
Quenching of the fluorescence of the (Ca2+ + Mg2+)-ATPase purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.  相似文献   

9.
The phospholipid requirement of the (Ca2+ + Mg2+)-ATPase present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the (Ca2+ + Mg2+)-ATPase activity. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.  相似文献   

10.
Cardiac plasma membrane Ca2+/Mg2+ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca2+ or Mg2+ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca2+/Mg2+ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca2+/Mg2+ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca2+-pump ATPase or sarcolemmal Ca2+-pump ATPase. On the other hand, the cardiac Ca2+/Mg2+ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca2+-pump ATPase or plasma membrane Ca2+-pump ATPase. Furthermore, the immune serum inhibited the Ca2+/Mg2+ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca2+/Mg2+ ecto-ATPase antibodies indicated the localization of Ca2+/Mg2+ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca2+/Mg2+ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca2+/Mg2+ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.  相似文献   

11.
Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca2+,Mg2+-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca2+ + Mg2+) > Mn2+ = (Ca2+ + Mn2+) > (Mg2+ + EGTA) > Ca2+. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn2+ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca2+,Mn2+-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca2+ + Mn2+) > (Ca2+ + Mg2+) > Mn2+ > (Mg2+ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed.  相似文献   

12.
Calmodulin (CaM) plays a very important role in many physiological processes and is highly conserved in different species. In a previous study, we successfully cloned CaM and a novel calmodulin-like protein (CaLP) with an extra C-terminal sequence from the pearl oyster Pinctada fucata and then expressed in Escherichia coli. In this research, we used fluorescence confocal microscopy to analyze the protein-protein interaction between CaM/CaLP and p21Cip1, which is cloned from mammalian cells, to show the different characteristics of these two proteins in vivo. The fluorescence confocal microscopy showed that the C-terminal globular domain together with the extra tail of CaLP is very important in CaLP's sequestration in cytoplasm. The most interesting phenomenon is that transfection of p21Cip1 can stimulate translocation of CaLP from the cytoplasm to the nucleus, but this is not the case for CaM. Fluorescence confocal microscopy and co-immunoprecipitation on different mutants of CaLP with p21Cip1 indicated that the C-terminal globular domain of CaLP is responsible for the trafficking of CaLP from cytoplasm to nucleus.  相似文献   

13.
The synthesis and characterisation of N-cyclohexyl-N′-(4-dimethylamino-α-naphthyl)carbodiimide (NCD-4) is described. Only the N-acetylurea and urea corresponding to NCD-4 are appreciably fluorescent: the O-phenylisourea and S-ethylisothiourea derivatives have negligible fluorescence. NCD-4 inhibits the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum irreversibly: Ca2+ protects against inhibition. Covalent incorporation of NCD-4 occurs into the Ca2+-protected sites, with a stoichiometry of approximately 1 mole/mole of ATPase. The modified enzyme has fluorescence emission properties similar to those of NCD-4 N-acetylurea in a relatively hydrophobic environment: it is concluded that NCD-4 has modified a carboxylate group (s) located in or near the Ca2+-binding sites of the ATPase.  相似文献   

14.
Eccentric is a newly-isolated mutant of Paramecium tetraurelia that fails to swim backwards in response to Mg2+. In the wild type, this backward swimming results from Mg2+ influx via a Mg2+-specific ion conductance (I Mg. Voltage-clamp analysis confirmed that, as suspected, step changes in membrane potential over a physiological range fail to elicit I Mg from eccentric. Further electrophysiological investigation revealed a number of additional ion-current defects in eccentric: (i) The Ca2+ current activated upon depolarization inactivates more slowly in eccentric than in the wild type, and it requires longer to recover from this inactivation. (ii) The Ca2+-dependent Na+ current deactivates significantly faster in the mutant, (iii) The two K+ currents observed upon hyperpolarization are reduced by >60% in eccentric. It is difficult to envision how these varied pleiotropic effects could result from loss of a single ion current. Rather, they suggest that the eccentric mutation affects a global regulatory system. Two plausible hypotheses are discussed.We are grateful to Dr. Yoshiro Saimi for his comments and suggestions on this work, and for the support of the Lucille P. Markey Charitable trust and the National Institutes of Health (GM22714 and GM38646).  相似文献   

15.
Tissue-specific age-dependent changes were observed in Na+K+-, Ca2+-, and Mg2+-ATPase activities in tropical tasar silkworm, Antheraea mylitta Drury. Maximum enzyme activity was recorded in all the tissues on day 12 (before spinning) in control group of animals. In testis, Na+K+-, Ca2+-, and Mg2+-ATPase activities gradually increased from day 2 to day 12 during fifth larval age and level was maintained up to adult eclosion while, in ovary, a marked decline was noted up to day of adult emergence. Further, a significant and sharp rise was found in ATPase activity in silk gland tissue up to day 12 and afterwards a drastic fall was noted on day 15 (end of spinning) during fifth larval age.Administration of T4 to fifth stage larvae (1 hr old) at doses 0.5–2.0 μg/g significantly elevated the Na+K+-, Ca2+-, and Mg2+-ATPase activities in larval and pupal gonads in a dose-dependent fashion. But, in moths, the enhancement was very much confined to Na+K+- and Ca2+-ATPase in testes and only Ca2+-ATPase in ovaries. Again, in silk glands thyroxine (0.5–2.0 μg/g) caused a significant rise in the all ion-dependent ATPase activities only during the fifth larval stage. Interestingly, higher doses of T4 (4.0 μg/g) caused a significant reduction in Na+K+-, Ca2+- and Mg2+-ATPase in all the tissues almost all the days studied so far. However, lower doses of T4 (0.1 and 0.25 μg/g) remained ineffective in altering the different ion-specific ATPase activities. This study suggests, that mammalian thyroxine has a metabolic influence showing biphasic nature of action in tasar silkworm ATPase system.  相似文献   

16.
(Ca2+ + Mg2+)-ATPase activator protein associated with human erythrocyte membranes could be extracted with EDTA under isotonic condition at pH 7.6. No activator was released, however, using isotonic buffer alone. Like calmodulin, the activator in the EDTA extract migrated as a fast moving band on polyacrylamide gel electrophoresis. It was also heat-stable, was capable of stimulating active calcium transport and could stimulate (Ca2+ + Mg2+)-ATPase to the same extent. When chromatographed on a Sephacryl S-200 column, it was eluted in the same position as calmodulin and a membrane associated (Ca2+ + Mg2+)-ATPase activator prepared according to Mauldin and Roufogalis (Mauldin, D. and Roufogalis, B.D. (1980) Biochem. J. 187, 507–513). Furthermore, both Mauldin and Roufogalis protein and the activator in the EDTA extract exhibited calcium-dependent binding to a fluphenazine-Sepharose affinity column. On the basis of these data, it is concluded that the activator protein released from erythrocyte membranes by EDTA is calmodulin. A further pool of the ATPase activator could be released by boiling but not by Triton X-100 treatment of the EDTA-extracted membranes. This pool amounted to 8.9% of the EDTA-extractable pool.  相似文献   

17.
A rapid loss of accumulated Ca2+ is produced by addition of H+ to isolated heart mitochondria. The H+-dependent Ca+ efflux requires that either (a) the NAD(P)H pool of the mitochondrion be oxidized, or (b) the endogenous adenine nucleotides be depleted. The loss of Ca2+ is accompanied by swelling and loss of endogenous Mg2–. The rate of H+-dependent Ca2+ efflux depends on the amount of Ca2+ and Pi taken up and the extent of the pH drop imposed. In the absence of ruthenium red the H+-induced Ca2+-efflux is partially offset by a spontaneous re-accumulation of released Ca2+. The H+-induced Ca2+ efflux is inhibited when the Pi transporter is blocked withN-ethylmaleimide, is strongly opposed by oligomycin and exogenous adenine nucleotides (particularly ADP), and inhibited by nupercaine. The H+-dependent Ca2+ efflux is decreased markedly when Na+ replaces the K+ of the suspending medium or when the exogenous K+/H+ exchanger nigericin is present. These results suggest that the H+-dependent loss of accumulated Ca2+ results from relatively nonspecific changes in membrane permeability and is not a reflection of a Ca2+/H+ exchange reaction.  相似文献   

18.
We reported previously that a Ca2+-ATPase in rat testes and goat spermatozoa could be activated by Ca2+ alone without Mg2+, though it has a lot of similarities with the well known Ca2+, Mg2+-ATPase. Recently, we were successful in isolating the phosphorylated intermediate of the former enzyme under control conditions i.e., in the presence of low concentration of Ca2+ and at low temperature. Increase of the concentration of Ca2+ and/or temperature lead to dephosphorylation. Based on our observations, we proposed a reaction scheme comparable to that of Ca2+, Mg2+-ATPase. The findings strengthened our previous report that Mg2+-independent Ca2+-ATPase is involved in Ca2+ transport and Ca2+ uptake like Ca2+, Mg2+-ATPase.  相似文献   

19.
Effects of intracellular Mg2+ on a native Ca2+-and voltage-sensitive large-conductance K+ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca2+ ([Ca2+]i) of 10−5–10−4 M, addition of 1–10 mM Mg2+ increased the open probability (Po) of the channel, which shifted the Po –membrane potential (Vm) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg2+-induced increase in Po was suppressed at a relatively low [Ca2+]i (10−5.5–10−6 M). Dwell-time histograms have revealed that addition of Mg2+ mainly increased Po by extending open times at 10−5 M Ca2+ and extending both open and closed times simultaneously at 10−5.5 M Ca2+. Since our data showed that raising the [Ca2+]i from 10−5 to 10−4 M increased Po mainly by shortening the closed time, extension of the closed time at 10−5.5 M Ca2+ would result from the Mg2+-inhibited Ca2+-dependent activation. At a constant Vm, adding Mg2+ enhanced the sigmoidicity of the Po–[Ca2+]i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg2+ on this channel is to elevate Po by lengthening the open time, while extension of the closed time at a relatively low [Ca2+]i results from a lowering of the sensitivity to Ca2+ of the channel by Mg2+, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.  相似文献   

20.
In order to address the mechanism whereby Ca2+ wad crucial for the manifestation of the enzymatic activity of phospholipase A2 (PLA2), four divalent cations were used to assess their influences on the catalytic activity and the fine structures ofNaja naja atra PLA2. It was found that substitution of Mg2+ or Sr2+ for Ca2+ in the substrate solution caused a decrease in the PLA2 activity to 77.5% or 54.5%, respectively, of that in the presence of Ca2+. However, no PLA2 activity was observed with the addition of Ba2+. With the exception of Mg2+, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA2 markedly increased with the binding of cations to PLA2. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate andp-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca2+, Sr2+, and Ba2+, but not by Mg2+. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba2+> Sr2+> Ca2+> Mg2+, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA2 and that Tyr-3, Lys-6, and Tyr-63 of PLA2 are involved in the binding with the substrate, suggest that the binding of Ca2+ to PLA2 induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg2+ or the bigger atomic radii with Sr2+ and Ba2+ might render the conformation improperly rearranged after their binding to PLA2 molecule.  相似文献   

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