Modulator binding protein antagonizes activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport of red blood cell membranes

Red blood cells contain a protein that activates membrane-bound (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ transport. The red blood cell activator protein is similar to a modulator protein that stimulates cyclic AMP phosphodiesterase. Wang and Desai [Journal of Biological Chemistry 252:4175–4184, 1977] described a modulator-binding protein that antagonizes the activation of cyclic AMP phosphodiesterase by modulator protein. In the present work, modulator-binding protein was shown to antagonize the activation of (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ transport by red blood cell activator protein. The results further demonstrate the similarity between the activator protein from human red blood cells and the modulator protein from bovine brain.     

Properties of (Mg2+ + Ca2+)-ATPase of erythrocyte membranes prepared by different procedures: Influence of Mg2+, Ca2+, ATP,and protein activator

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.     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca2+ transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca2+ transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mCICR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mCICR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mCICR and PTP opening. mCICR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca2+ transport and PTP opening are largely dependent on electron transport and energy coupling.     

高温胁迫对花生幼苗光合速率、叶绿素含量、叶绿体Ca2+-ATPase、Mg2+-ATPase及Ca2+分布的影响

将水培后盆栽的花生幼苗,置于培养箱42℃高温培养,定时测定幼苗叶光合速率、叶绿素含量和叶绿体Ca^2＋-ATPase、Mg^2＋-ATPase的相对活性,并观察幼叶细胞内Ca^2＋分布的变化。试验结果表明：高温胁迫过程中,光合速率及叶绿素含量都随处理时间的延伸而下降,并呈显著正相关;叶绿体Ca^2＋-ATPase和Mg^2＋-ATPase高温胁迫过程中相对活性呈先升后降趋势,Ca^2＋-ATPase热敏性高于Mg^2＋-ATPase;高温胁迫过程中,Ca^2＋具有从胞外转运到胞质内和叶绿体中的趋势,Ca^2＋能够稳定高温胁迫下叶肉细胞膜和叶绿体的超微结构。     

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.     

Diabetes-induced decrease in rat brain microsomal Ca2+-ATPase activity

The Ca(2+)-ATPase activity of rat brain microsomes was studied in streptozotocin (STZ)-induced diabetes. Male rats, 200-250 g, were rendered diabetic by injection of STZ (45 mg kg(-1) body weight) via the teil vein. Brain tissues were collected at 1, 4 and 10 weeks after diabetes was induced for determination of Ca(2+)-ATPase activity, lipid peroxidation and tissue calcium levels. Diabetic rats had significantly elevated blood glucose levels compared to controls. Blood glucose levels were 92.92 +/- 1.22 mg dl(-1) (mean +/- SEM) for the control group, 362.50 +/- 9.61 mg dl(-1) at 1 week and >500 mg dl(-1) at 4, 8 and 10 weeks for the diabetics. Enzyme activities were significantly decreased at 1, 4, 8 and 10 weeks of diabetes relative to the control group (p < 0.001). Ca(2+)-ATPase activity was 0.084 +/- 0.008 U l(-1), 0.029 +/- 0.005 U l(-1), 0.029 +/- 0.006 U l(-1), 0.033 +/- 0.003 U l(-1) and 0.058 +/- 0.006 U l(-1) (mean +/- SEM) at control, 1, 4, 8 and 10 week of diabetes respectively. The change in calcium levels in diabetic rat brain at 8 and 10 weeks of diabetes was significantly higher than that of the control group (p < 0.05). On the other hand lipid peroxidation measured as TBARS (thiobarbituric acid reactive substances) was significantly higher at 8 and 10 weeks of diabetes (p < 0.05). The increase in lipid peroxidation observed in diabetic rat brain may be partly responsible for the decrease in calcium ATPase activity.     

NaHCO3胁迫下星星草根中Ca2＋与Ca2＋-ATPase的超微细胞化学定位

利用焦锑酸盐和磷酸铅沉淀技术分别对NaHCO3胁迫条件下星星草（Puccinellia tenuiflora）根中Ca2＋和Ca2＋-ATPase进行超微细胞化学定位研究,旨在进一步探讨Ca2＋在NaHCO3胁迫诱导胞内信号转导过程中的作用,以及Ca2＋-ATPase活性定位变化与NaHCO3胁迫下星星草抗盐碱能力的关系。结果表明：在正常状态下,根毛区细胞质内Ca2＋较少,主要位于质膜附近和液泡中,Ca2＋-ATPase主要定位于质膜和液泡膜,有一定活性。在0.448%NaHCO3胁迫下,根毛区细胞质中Ca2＋增多,液泡中Ca2＋减少,且主要集中于液泡膜附近,质膜和液泡膜Ca2＋-ATPase活性明显升高。在1.054%NaHCO3胁迫下,细胞质中分布的Ca2＋增多,而液泡中Ca2＋极少,Ca2＋-ATPase活性也降低。以上结果表明,Ca2＋亚细胞定位和Ca2＋-ATPase活性变化在星星草响应NaHCO3胁迫的信号传递过程中具有重要作用。     

Changes in plasmalemma K+Mg2+ -ATPase dephosphorylating activity and H+ transport in relation to seasonal growth and freezing tolerance of Festuca pratensis Huds

Changes in plasmalemma K⁺Mg²⁺-ATPase dephosphorylating activity and H⁺ transport were examined in freezing-tolerant and non-tolerant genotypes of the perennial grass species Festuca pratensis Huds. Enzyme activity and ΔμH⁺ were measured in plasmalemma fractions isolated from basal nodes and roots. Three types of experiments were undertaken: (i) a field experiment, utilizing the seasonal growth and cessation cycle of a perennial plant; (ii) a cold acclimation experiment in hydroponics; and (iii) an instant freezing test. A specific fluctuation in K⁺Mg²⁺-ATPase activity was found throughout the seasonal growth of the plants (i). The K⁺Mg²⁺-ATPase activity peaks for both the basal node and the root plasmalemma were determined early in the spring before the renewal of growth. The lowest activity values in roots occurred at the time approaching flowering, and in basal nodes at the transition into the growth cessation. The K⁺Mg²⁺-ATPase activity was approximately 50% lower in the basal node plasmalemma of freezing-tolerant plants than of non-tolerant ones, when assessed at the optimal growth stage in hydroponics. In hydroponics (ii) and in the freezing test (iii), temperature stress was followed by a more pronounced change in the level of K⁺Mg²⁺-ATPase activity than in that of H⁺ transport, and this change was more clearly differentiated in the basal node plasmalemma of contrasting genotypes than in the roots. Stress response was manifested differently in freezing-tolerant and non-tolerant plants at cold acclimation (4–2 °C) and at freezing (−8 °C) temperatures. Proton transport regulation via coupled changes in the hydrolysed ATP/transported proton ratio, as an attribute of freezing-tolerant plants, is discussed.     

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca²⁺ binding isotherms obtained by equilibrium measurements in the presence of various H⁺ and Mg²⁺ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca²⁺ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.     

The Ca 2+ pumps and the Na + / Ca 2+ exchangers

The Ca 2+ ATPases or Ca 2+ pumps transport Ca 2+ ions out of the cytosol, by using the energy stored in ATP. The Na + / Ca 2+ exchanger uses the chemical energy of the Na + gradient (the Na + concentration is much higher outside than inside the cell) to remove Ca 2+ from the cytosol. Ca 2+ pumps are found in the plasma membrane and in the endoplasmic reticulum of the cells. The pumps are probably present in the membrane of other organelles, but little experimental information is available on this matter. The Na + / Ca 2+ exchangers are located on the plasma membrane. A Na + / Ca 2+ exchanger was found in the mitochondria, but very little is known on its structure and sequence. These transporters control the Ca 2+ concentration in the cytosol and are vital to prevent Ca 2+ overload of the cells. Their activity is controlled by different mechanisms, that are still under investigation. A number of the possible isoforms for both types of proteins has been detected.© Kluwer Academic Publishers     

高温胁迫对花生幼苗光合速率、叶绿素含量、叶绿体Ca2+-ATPase、Mg2+-ATPase及Ca2+分布的影响

将水培后盆栽的花生幼苗,置于培养箱42℃高温培养,定时测定幼苗叶光合速率、叶绿素含量和叶绿体Ca2 -ATPase、Mg2 -ATPase的相对活性,并观察幼叶细胞内Ca2 分布的变化。试验结果表明:高温胁迫过程中,光合速率及叶绿素含量都随处理时间的延伸而下降,并呈显著正相关;叶绿体Ca2 -ATPase和Mg2 -ATPase高温胁迫过程中相对活性呈先升后降趋势,Ca2 -ATPase热敏性高于Mg2 -ATPase;高温胁迫过程中,Ca2 具有从胞外转运到胞质内和叶绿体中的趋势,Ca2 能够稳定高温胁迫下叶肉细胞膜和叶绿体的超微结构。     

Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary In reconstituted rabbit skeletal muscle (Ca²⁺ + Mg²⁺)-ATPase proteoliposomes, Ca²⁺-uptake is decreased by more than 90% with T₂ cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T₃ cleavage, which represents the cleavage of A₁ fragment to A_1a + A_1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca²⁺ efflux between control SR and tryptically digested SR in the absence of Mg⁺ ruthenium red or in the presence of ATP. However, the passive Ca²⁺ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg²⁺ + ruthenium red. These results show that the Ca²⁺ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca²⁺ channel inhibitors, Mg²⁺ and ruthenium red. In the reconstituted ATPase proteoliposomes, the Ca²⁺ efflux rates are the same regardless of digestion (T₂); also, efflux is not affected by the presence or absence of Mg²⁺ + ruthenium red. These results indicate that T₂ cleavage causes uncoupling of the Ca²⁺-pump from ATP hydrolytic activity.A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca²⁺ + Mg²⁺)-ATPase polypeptide with the loss of Ca²⁺-transport. Fits of the theoretical equations to the data are consistent with that Ca²⁺-transport system appears to require a dimer of the polypeptide (Ca²⁺ + Mg²⁺)-ATPase.     

钙肥对富士苹果品质及Ca2+-ATPase活性影响的研究

以盛果期的矮化富士为材料,研究了不同钙肥对富士苹果品质和Ca2+-ATPase活性的影响.结果表明,喷钙后单果重增加,Vc含量提高,可溶性固形物和花青苷含量增大,而成熟果实的叶绿素和可滴定酸含量下降.不同钙肥效果顺序为巨金钙>氨基酸钙>翠康钙宝>钙宝2000.钙肥对Ca2+-ATPase活性影响极为显著,其活性明显高于对照,不同钙肥间Ca2+-ATPase活性的变化趋势与果实品质的变化趋势相同.     

Association of (Ca+Mg)-ATPase activity with ATP-dependent Ca uptake in vesicles prepared from human erythrocytes

Ghost membranes prepared from human erythrocytes exhibit 2 distinct (Ca+Mg)-ATPase¹ activities (Quist and Roufogalis, Arch Biochem Biophys 168:240, 1975). (Ca+Mg)-ATPase activity dependent on a water soluble protein fraction is selectively lost from ghost membranes during preparation of vesicles under low ionic strength, slightly alkaline conditions. In this study, the Ca²⁺ dependence of the remaining membrane bound (Ca+Mg)-ATPase activity and ATP-dependent Ca uptake in vesicles were compared. The C²⁺ activation curves for (Ca+Mg)-ATPase activity and Ca uptake into vesicles were parallel over a Ca²⁺ range of 0.3–330 μM, and both curves have 2 apparent K_A values for Ca²⁺ of 0.45 and 100 μM. Addition of a concentrated soluble protein fraction containing predomintly spectrin to the vesicles increased (Ca+Mg)-ATPase activity over twofold but did not affect the rate of Ca uptake. These findings suggest that the (Ca+Mg)-ATPase activity remaining in vesicles after extraction of the water soluble proteins is associated with the Ca pump whereas (Ca+Mg)-ATPase activity dependent on the soluble protein fraction is associated with some other function.     

Calmodulin Stimulation of Ca2+-Dependent ATP Hydrolysis and ATP-Dependent Ca2+ Transport in Synaptic Membranes

We report here characterization of calmodulin-stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+-stimulated, Mg2+-dependent ATP hydrolysis coupled with ATP-dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synaptosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca2+-ATPase and ATP-dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 microM for Ca2+-ATPase and 0.4 microM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca2+ + Mg2+ ATPase as a regulator for cytosolic Ca2+.     

Altered coupling states between calcium transport and (Ca2+, Mg2+)-ATPase in the AS-30D Ascites hepatocarcinoma plasma membrane

Plasma membrane fractions from normal, regenerating liver and the AS-30D ascites hepatocarcinoma exhibited a high degree of enrichment when a set of plasma membrane enzyme markers were studied in comparison to the ones associated to the mitochondrial and cytosolic compartments. While the (Ca²⁺, Mg²⁺)-ATPase observed for the plasma membrane fraction isolated from normal liver showed an activity of 1.2 µmoles/mg/min, the regenerating liver and the AS-30D plasma membrane fractions presented a much lower ATPase activity (0.3 and 0.22 µmoles/mg/min respectively). Despite the differences in ATPase activity observed between models, the plasma membrane fraction from the AS-30D hepatocarcinoma presented a calcium transport activity similar to the value observed for the normal system (5.9 and 5.5 nmoles Ca²⁺/mg/10min, respectively). Interestingly, the ATP Pi exchange experiments carried out with the different plasma membrane fractions revealed that the (Ca²⁺, Mg²⁺)-ATPase contained in the plasma membrane from the AS-30D cells shows an exchange activity of 26 nmoles ATP Pi/mg/min, similar to the one observed for the enzyme from normal liver (30 nmoles ATP Pi/mg/min). Our results suggest that the plasma membrane from the transformed model presents a more efficient mechanism to regulate the movement of calcium through the calcium pump, with an optimum expenditure of energy.Dedicated to the memory of Catalina Mas Oliva and Valentín Mas Morera.     

Disulfiram lowers Ca2+, Mg2+-ATPase activity of rat brain synaptosomes

The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase activity. In vitro studies indicated that DS (ID₅₀=20 M) produced a dose-dependent inhibition of Ca²⁺, Mg²⁺-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca²⁺, Mg²⁺-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID₅₀=20 M) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase specific activity was observed when treated with DS (30 M) and EGTA (100 M) respectively. The remaining 25–30% of total activity is suggested to be of Mg²⁺-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70–75% of total Ca²⁺, Mg²⁺-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca²⁺, Mg²⁺-ATPase activity could affect its function of maintaining intracellular Ca²⁺ concentration. This could contribute to the deleterious effects on CNS.     

Effect of phorbol 12-myristate 13-acetate on Ca2+-ATPase activity in rat liver nuclei

The effect of phorbol 12-myristate 13-acetate (PMA) on Ca²⁺-ATPase activity in rat liver nuclei was investigated. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺-Mg²⁺)-ATPase activity. The nuclear Ca²⁺-ATPase activity was significantly increased by the presence of PMA (2–20 μM) in the enzyme reaction mixture; the maximum effect was seen at 10 μM. The PMA (10 μM)-increased Ca²⁺-ATPase activity was not blocked by the presence of staurosporine (2 μM) or dibucaine (2 and 10 μM), an inhibitor of protein kinase. Meanwhile, vanadate (20 and 100 μM) caused a significant reduction in the nuclear Ca²⁺-ATPase activity increased by PMA (10 μM). The present finding suggests that PMA has an activating effect on liver nuclear Ca²⁺-ATPase independent of protein kinase. © 1994 Wiley-Liss, Inc.     

Ca2+预处理对热胁迫下辣椒叶肉细胞中Ca2+-ATP酶活性的影响

在常温下生长的辣椒(Capsicum annum L.)叶肉细胞中Ca2+-ATP酶主要分布于质膜、液泡膜上,叶绿体的基质和基粒片层上也有少量分布;在40℃下热胁迫不同的时间,酶活性逐渐下降,直至叶绿体超微结构解体.同样条件下,经过Ca2+预处理后,分布在上述细胞器膜或片层上的酶活性大大提高,表明Ca2+预处理对该酶活性具有激活作用;Ca2+预处理对热胁迫下的超微结构的完整性具有一定的保护作用,并且能使Ca2+-ATP酶在热胁迫下维持较高活性.结果表明,Ca2+预处理增强辣椒幼苗的抗热性,可能与其稳定细胞膜、从而使Ca2+-ATP酶在热胁迫下保持较高活性有一定关系.