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1.
High-affinity ouabain binding by yeast cells expressing Na+, K(+)-ATPase alpha subunits and the gastric H+, K(+)-ATPase beta subunit.
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K A Eakle K S Kim M A Kabalin R A Farley 《Proceedings of the National Academy of Sciences of the United States of America》1992,89(7):2834-2838
Recently, a beta subunit for the rat gastric H+,K(+)-ATPase (HK beta), which is structurally similar to the beta subunit of Na+, K(+)-ATPase, has been cloned and characterized. Using heterologous expression in yeast, we have tested the specificity of beta subunit assembly with different isoforms of the alpha subunit of Na+, K(+)-ATPase. Coexpression in yeast cells of the HK beta with both the sheep alpha 1 subunit and the rat alpha 3 subunit isoforms of Na+, K(+)-ATPase (alpha 1 and alpha 3, respectively) leads to the appearance of high-affinity ouabain-binding sites in yeast membranes. These ouabain-binding sites (alpha 1 plus HK beta, alpha 3 plus HK beta) have a high affinity for ouabain (Kd, 5-10 nM) and are expressed at levels similar to those formed with the rat beta 1 subunit of Na+, K(+)-ATPase (beta 1) (alpha 1 plus beta 1 or alpha 3 plus beta 1). Potassium acts as a specific antagonist of ouabain binding by alpha 1 plus HK beta and alpha 3 plus HK beta just like sodium pumps formed with beta 1. Sodium pumps formed with the HK beta, however, show quantitative differences in their affinity for ouabain and in the antagonism of K+ for ouabain binding. These data suggest that the structure of the beta subunit may play a role in sodium pump function. 相似文献
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D D Thomas C Hidalgo 《Proceedings of the National Academy of Sciences of the United States of America》1978,75(11):5488-5492
Using saturation transfer electron paramagnetic resonance, we have detected the rotational motion of a spin label rigidly attached to the sarcoplasmic reticulum Ca2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3). At 4 degrees C, the spectrum indicates an effective rotational correlation time of 60 microsec, determined by comparison with reference spectra obtained from theoretical calculations and from experiments on model systems. This motion appears to correspond to rotation of the enzyme with respect to the membrane, because the motion persists when the membrane fragments are immobilized by sedimentation and the motion stops when the polypeptide chains, but not the membrane vesicles, are crosslinked by glutaraldehyde. The rotational mobility of the enzyme increases with increasing temperature, and this increase becomes more gradual when the temperature exceeds 20 degrees C; the same kind of temperature dependence has been observed previously for lipid fluidity and enzymatic activity. 相似文献
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We investigated the role of singlet oxygen (generated from photoactivation of rose bengal) on the calcium transport and Ca(2+)-ATPase activity of cardiac sarcoplasmic reticulum (SR). Isolated cardiac SR exposed to rose bengal (10 nM) irradiated at 560 nm resulted in significant inhibition of Ca2+ uptake (from 2.27 +/- 0.05 to 0.62 +/- 0.05 mumol Ca2+/mg.min [mean +/- SEM], p less than 0.01) and Ca(2+)-ATPase activity (from 2.08 +/- 0.05 to 0.28 +/- 0.04 mumol Pi/min.mg [mean +/- SEM], p less than 0.01). The inhibition of calcium uptake and Ca(2+)-ATPase activity by rose bengal-derived activated oxygen (singlet oxygen) was dependent on the duration of exposure and intensity of light. Singlet oxygen scavengers ascorbic acid and histidine significantly protected SR Ca(2+)-ATPase against rose bengal-derived activated oxygen species, but superoxide dismutase and catalase did not attenuate the inhibition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of SR exposed to photoactivated rose bengal for up to 14 minutes demonstrated complete loss of the Ca(2+)-ATPase monomer band, which was significantly protected by histidine. The addition of dithiothreitol (5 mM) had a slight protective effect, showing that new disulfide bond formation was not a major cause of aggregation. The results were also confirmed by high-performance liquid chromatography of the SR exposed to irradiated rose bengal. Irradiation of rose bengal also caused an 18% loss of total sulfhydryl groups of SR. On the other hand, superoxide radical (generated from xanthine oxidase action on xanthine) and hydroxyl radical (in the presence of Fe(3+)-EDTA or 0.5 mM H2O2 plus Fe(2+)-EDTA) as well as H2O2 (0.25-12 mM) were without any effect on the 97,000-d Ca(2+)-ATPase band of SR. Generation of radical species (superoxide and hydroxyl radical) from rose bengal was studied by electron paramagnetic resonance spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The results showed that irradiation of rose bengal formed a 1:2:2:1 quartet, characteristic of the DMPO-OH adduct, which was scavenged by ethanol but not by superoxide dismutase, catalase, or histidine. No radical species could be detected from irradiated rose bengal or irradiated DMPO under the assay conditions used. Peroxy adducts of DMPO might be produced but would be observed only at very low temperatures. Similarly, we could not detect any measurable.O2- anion from irradiation of rose bengal as indicated by either cytochrome c reduction at 550 nm or nitro blue tetrazolium reduction at 560 nm. These results show that SR is damaged most likely by singlet oxygen derived from rose bengal.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
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M M Shull J B Lingrel 《Proceedings of the National Academy of Sciences of the United States of America》1987,84(12):4039-4043
A human genomic library was constructed and screened with hybridization probes derived from sheep and rat cDNAs encoding the alpha and alpha(+) isoforms, respectively, of the Na+,K+-ATPase catalytic subunit. Genomic sequences spanning 150 kilobases were isolated. Four genes, designated alpha A, alpha B, alpha C, and alpha D, each 20-25 kilobases in length, were identified by restriction mapping, Southern blot hybridization analysis, and limited DNA sequencing. We present evidence that two of these genes, alpha A and alpha B, encode the alpha and alpha(+) isoforms, respectively. The other genes, alpha C and alpha D, one of which is physically linked to the alpha(+) gene, exhibit nucleotide and amino acid homology to Na+,K+-ATPase catalytic subunit cDNA sequences but do not correspond to any previously identified isoforms. 相似文献
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Crosslinking the active site of sarcoplasmic reticulum Ca(2+)-ATPase completely blocks Ca2+ release to the vesicle lumen.
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D B McIntosh D C Ross P Champeil F Guillain 《Proceedings of the National Academy of Sciences of the United States of America》1991,88(15):6437-6441
Intramolecular crosslinking of the active site of the sarcoplasmic reticulum Ca(2+)-ATPase with glutaraldehyde results in substantial inhibition of ATPase activity and stabilization of the ADP-sensitive E1 approximately P(2Ca) intermediate (E, enzyme) with occluded Ca2+ [Ross, D. C., Davidson, G. A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621]. We show here, using conditions of low passive vesicle permeability and absence of ADP, that Ca2+ "deoccludes" more rapidly than it leaks out of the vesicle lumen. Deocclusion is paralleled by dephosphorylation. Therefore, turnover of crosslinked E1 approximately P(Ca) (approximately 5 nmol/min per mg of protein at 25 degrees C) involves Ca2+ release to the vesicle exterior and concomitant phosphoenzyme hydrolysis. Ca2+ release to the lumen, the normal pathway, is apparently blocked completely. In the presence of ADP, Ca2+ is also released to the vesicle exterior, and this release is coupled to the synthesis of ATP. The results suggest that a tertiary structural change at the active site follows phosphorylation and is an absolute requirement for Ca2+ release from the native enzyme to the vesicle lumen. 相似文献
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肝硬化红细胞钠钾ATP酶、钙镁ATP酶及钠钾钙镁改变 总被引:1,自引:0,他引:1
目的:探讨肝硬化时细胞内钠钾钙镁的改变及细胞膜钠钾ATP酶(NKA)、钙镁ATP酶 (CMA)活性改变在细胞内钠钾钙镁改变中的作用.方法:测定了52例肝硬化失代偿期(实验组 A)、36例代偿期(实验组B)患者红细胞及血清钠钾钙镁(RNa、RK、RCa、RMg;SNa、 SK、SCa、SMg)含量和NKA和CMA活性.以 36名健康人为对照组.结果:与对照组比较,实验组A的NKA、 CMA、RK、RMg(t=5.92,P<0.001;t=7.21, P<0.001;t=2.32,P<0.02;t=4.79,P<0.001)和买验组B的NKA、CMA、RK、RMg(t=3.83, P<0.001;t=2.53,P<0.02;t=2.03,P<0.05;t= 3.33,P<0.002)均显著降低;与实验组B比较, 实验组A的NKA、CMA活性(t=2.29,P<0.05; t=4.14,P<0.005)显著降低.与对照组比较, 买验组A的SNa、SK、SCa、SMg(t=8.25, P<0.001;t=5.73,P<0.001;t=9.82,P<0.001; t=6.15,P<0.001)显著降低;与实验组B比较, 买验组A的SNa、SK、SCa、SMg(t=6.94, P<0.001;t=5.00,P<0.001;t=5.57,P<0.001; t=5.73,P<0.001)显著降低.与Child B级组比较,Child C级组的NKA、CMA、RK、RMg、 SNa、SK、SCa、SMg(P<0.05或P<0.01) 显著降低.与非肝性脑病组比较,肝性脑病组NKA、CMA、RK、RMg、SNa、SK、 SMg(P<0.05或P<0.01)显著降低.实验组A中, 低SMg者的NKA和CMA显著低于高SMg者 (16.87±3.19 vs 19.04±3.25;109.83±13.51 vs 120.13±13.27;P均<0.05).结论:肝硬化患者存在缺钾缺镁,且随病情加重而加重,缺钾缺镁可能为病情加重的原因之一.NKA和CMA活性降低可导致细胞内低钾低镁和钠钙蓄积.缺镁为ATP酶活性在失代偿期进一步降低的原因之一. 相似文献
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OBJECTIVE: Prolonged cardiac contraction and relaxation in hypothyroidism are in part related to diminished expression of the gene coding for the calcium pump of the sarcoplasmic reticulum (SERCA2a). Therefore, we examined whether or not transgenic SERCA2a gene expression in mice may compensate for the cardiac effects of hypothyroidism. METHODS: SERCA2a mRNA and protein were analyzed from hearts of euthyroid and hypothyroid mice of wild-type or SERCA2a transgene status. Contractile function was studied in isolated left ventricular papillary muscles. RESULTS: We found significant decreases of SERCA2a mRNA and protein levels in hearts of hypothyroid wild-type mice in comparison with euthyroid wild-type mice (controls). Papillary muscles from hypothyroid wild-type mice showed significant increases in time to peak contraction and relaxation times compared with controls. In contrast, SERCA2a mRNA and protein levels were significantly higher in hypothyroid SERCA2a transgenic mice than in hypothyroid wild-type mice. The transgene led to a functional improvement by compensating for the prolonged contraction and relaxation of papillary muscles. CONCLUSIONS: Our murine model of hypothyroidism revealed decreases in SERCA2a gene expression accompanied by prolonged contraction and relaxation of papillary muscles, and an improvement of the contractile phenotype due to compensated SERCA2a gene expression in SERCA2a transgenic mice. 相似文献
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Jean E. Ray Eva K. Ray Steven I. Baskin Zebulon V. Kendrick 《Age (Dordrecht, Netherlands)》1983,6(2):58-61
Reports have suggested that variation in phosopholipid and cholesterol content and/or ratio affects cellular membrane fluidity.
Quantitative changes in these lipids may provide evidence of age-associated changes in membrane structure which, in turn,
may influence functional parameters of the cell. Male, Fischer 344 rats (ages 3, 6, 12, 24, 28 months) were sacrificed and
spinal cords removed. Lipids were extracted and phospholipid and cholesterol content was measured. Significant increases in
the content of both lipids occurred between 3 and 6 months and between 24 and 28 months of age. The ratios of phospholipids
to cholesterol decreased significantly between 3 and 6 months of age. From 6 to 28 months of age the increase in phospholipids
generally paralleled those of cholesterol having a smaller effect on the ratio. The altered lipid content was accompanied
by increased sensitivity of spinal cord microsomal Na+, K+-ATPase to the cardiac glycoside ouabain. The changes in absolute content as well as ratios of phospholipids and cholesterol,
in addition to increased sensitivity of Na+,K+-ATPase to cardiac glycosides, suggest that membrane structure in rat spinal cord may change as a result of age. 相似文献
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Haruo Ogawa Takehiro Shinoda Flemming Cornelius Chikashi Toyoshima 《Proceedings of the National Academy of Sciences of the United States of America》2009,106(33):13742-13747
The sodium-potassium pump (Na+,K+-ATPase) is responsible for establishing Na+ and K+ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na+,K+-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2·2K+·Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K+, in marked contrast to previous models. Due to antagonism between ouabain and K+, the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity. 相似文献
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Teshima Y Takahashi N Saikawa T Hara M Yasunaga S Hidaka S Sakata T 《Journal of molecular and cellular cardiology》2000,32(4):655-664
The diabetic heart has an abnormal intracellular calcium ([Ca(2+)]i) metabolism. However, the responsible molecular mechanisms are unclear. The present study aimed to investigate mRNAs expressed in the proteins which regulate heart [Ca(2+)]i metabolism in streptozotocin (STZ)-induced diabetic rats. Expression of sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase (SR Ca(2+)-ATPase) mRNA was significantly less in the heart 3 weeks after STZ injection than that in the age-matched controls. Together with the down-regulation of SR Ca(2+)-ATPase, expression of ryanodine sensitive Ca(2+)channel (RYR) mRNA was also decreased 12 weeks after STZ injection. Insulin supplementation fully restored the decreased mRNAs expression of SR Ca(2+)-ATPase and RYR. The diminished expression and restoration with insulin supplementation of SR Ca(2+)-ATPase was further confirmed at the protein level. In contrast, expression of mRNAs coding the L-type Ca(2+)channel, Na(+)-Ca(2+)exchanger, or phospholamban were not affected 3 or 12 weeks after STZ injection. These results can be taken to indicate that the down-regulation of SR Ca(2+)-ATPase and RYR mRNAs is a possible underlying cause of cardiac dysfunction in STZ-induced diabetic rats. 相似文献
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Assays for complete quantification of Na+, K+-and Ca2+-ATPase in crude homogenates of rat ventricular myocardium by determination of K+-and Ca2+-dependentp-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K+-and Ca2+-dependentpNPPase activities in ventricular myocardium of 11–12 week-old rats were found to be 2.98±0.10 and 0.29±0.02 mol×min–1×g–1 wet wt. (mean±SEM) (n=5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na+, K+-and Ca2+-ATPase concentrations were estimated to 2 and 10 nmol×g–1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca2+-dependentpNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K+-dependentpNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple out-come of variations in water and protein content of myocardium. Expressed per heart, the K+-and Ca2+-dependentpNPPase activity gradually increased to a plateau. The present assay for Na+, K+-ATPase quantification has the advantage over [3H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K+-dependent 3-0-methylfluorescein phosphatase (3-0-MFPase) in crude tissue homogenates. Furthermore, with few modifications thepNPPase assay allows quantification of Ca2+-ATPase on crude myocardial homogenates. Age-dependent changes in K+-and Ca2+-dependentpNPPase activities are of developmental interest and indicate the importance of close age match in studies of quantitative aspects of Na+, K+-and Ca2+-ATPase in excitable tissues.Abbreviations Na+, K+-ATPase
sodium, potassium-dependent ATPase
- Ca2+-ATPase
caldium-dependent ATPase
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pNP
p-nitrophenyl
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pNPP
p-nitrophenyl phosphate
- 3-0-MFP
3-0 methylfluorescein phosphate
- DOC
sodium deoxycholate 相似文献
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The effect of pH on the Ca2+-Mg2+-dependent ATPase of sarcoplasmic reticulum (SR) was investigated with a rapid mixing quench-flow apparatus capable of measuring phosphorylation and dephosphorylation at times as rapid as 4 msec. The rates of formation and decomposition of the phosphorylated intermediate (E approximately P) of the Ca2+-Mg2+-ATPase were studied in the pH range between 7.6 and 6.0. At pH 6.8, the rates of formation of the phosphorylated intermediate of the Ca2+-Mg2+-ATPase of sarcoplasmic reticulum are the same (t1/2 = 10 msec) for cardiac and skeletal sarcoplasmic reticulum preloaded with calcium, but decrease as the pH is lowered. The effect of acid pH (6.0) is more pronounced for cardiac sarcoplasmic reticulum (t 1/2 = 47 msec) than for skeletal sarcoplasmic reticulum (t 1/2 = 17 msec), in agreement with studies showing that acidosis has a more pronounced effect on cardiac muscle than on skeletal muscle. In addition, a decrease in pH results in a decrease in the rate of the E approximately P decomposition step (the slowest step in the SR reaction sequence). The E approximately P decomposition half-lives were observed to be 97 and 77 msec, respectively for cardiac and skeletal SR at pH 6.8. At pH 6.0, the half-lives were increased to 136 and 178 msec for cardiac and skeletal SR, respectively. 相似文献
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The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) is the third element of capacitative calcium entry. It colocalizes with STIM1 and Orai1 at puncta, where couples plasma membrane store-operated Ca(2+) channels (SOC) to Ca(2+) pumping into the ER. The efficiency of this calcium entry-calcium refilling (CECR) coupling is comparable to the classic excitation-response transduction mechanisms. This allows efficient filling of the endoplasmic reticulum (ER) with the Ca(2+) entering through SOC channels with little progression of the Ca(2+) wave towards the cell core. CECR coupling is very sensitive to changes in stoichiometry among STIM, Orai and SERCA, with excess Orai antagonizing ER refilling. ER takes up most of the calcium load that enters through SOC, whereas mitochondria take up a very small fraction. This difference is due to the spatial positioning with regard to SOC, the amplitude of the high Ca(2+) microdomains, and the differences in the Ca(2+) affinity of the uptake mechanisms. 相似文献
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OBJECTIVE: Catecholamines that accompany acute physiological stress are also involved in mediating the development of hypertrophy and failure. However, the cellular mechanisms involved in catecholamine-induced cardiac hypertrophy, particularly Ca2+ handling, are largely unknown. We therefore investigated the effects of cardiac hypertrophy, produced by isoprenaline, on I(Na/Ca) and sarcoplasmic reticulum (SR) function in isolated myocytes. METHODS: I(Na/Ca) was studied in myocytes from Wistar rats, using descending (+80 to -110 mV) voltage ramps under steady state conditions. Myocytes were also loaded with fura-2 and either field stimulated or voltage clamped to assess [Ca2+]i and SR Ca2+ content. RESULTS: Ca2+-dependent, steady state I(Na/Ca) density was increased in hypertrophied myocytes (P<0.05). Ca2+ release from the SR was also increased, whereas resting [Ca2+]i and the rate of decline of [Ca2+]i to control levels were unchanged. SR Ca2+ content, estimated by using 10.0 mmol/l caffeine, was also significantly increased in hypertrophied myocytes, but only when myocytes were held and stimulated from their normal resting potential (-80 mV) but not from -40 mV. However, the rate of decline of caffeine-induced Ca2+ transients or I(Na/Ca) was not significantly different between control and hypertrophied myocytes. Ca2+-dependence of I(Na/Ca), examined by comparing the slope of the descending phase of the hysteresis plots of I(Na/Ca) vs. [Ca2+]i, was also similar in the two groups of cells. CONCLUSION: Data show that SR Ca2+ release and SR Ca2+ content were increased in hypertrophied myocytes, despite an increase in the steady state I(Na/Ca) density. The observation that increased SR function occurred only when myocytes were stimulated from -80 mV suggests that Na+ influx may play a role in altering Ca2+ homeostasis in hypertrophied cardiac muscle, possibly through increased reverse Na+/Ca2+ exchange, particularly at low stimulation frequencies. 相似文献
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Leszek P Szperl M Klisiewicz A Janas J Biederman A Rywik T Piotrowski W Kopacz M Korewicki J 《European journal of heart failure》2007,9(6-7):579-586
BACKGROUND: Reduced myocardial contractility is often attributed to altered Ca(2+) transients and expression of Ca(2+)-ATPase of the SR (SERCA) and Na+/Ca(2+)exchanger (NCX) genes. AIMS: To assess myocardial expression of SERCA and NCX protein levels in left ventricular (LV) remodelling due to chronic severe mitral regurgitation (MR). METHODS: Myocardial expression of SERCA/NCX in biopsy specimens obtained during mitral surgery was assessed in 36 MR patients with LV remodelling and plasma neurohumoral/cytokine activation and in four non-failing hearts (NFH). RESULTS: Myocardial protein levels of SERCA were significantly (20%) lower in the MR group than in NFH group (p=0.016). No significant changes in NCX were observed. However, a lack of homogeneity with regard to SERCA/NCX proteins was observed. Moreover, SERCA was negatively correlated with BNP (r=-0.49, p=0.02), TNFalpha (r=-0.68, p=0.0005) and IL-6 (r=-0.52, p=0.02), whereas NCX was only negatively correlated with TNFalpha (r=-0.62, p=0.002). CONCLUSIONS: MR patients showed wide variations in SERCA/NCX protein expression. Myocardial protein levels of SERCA were significantly lower in the MR population. Moreover, a correlation between BNP, cytokines (IL-6, TNFalpha) and the expression of SERCA/NCX proteins was observed. 相似文献