骨骼肌内质网Ca2+泵转运Ca2+的结构基础

Ca2 泵(Ca2 -ATPase)是调节细胞内Ca2 浓度的重要蛋白质之一.Ca2 泵在转运Ca2 的过程中经历一系列构象变化.其中,E1状态为外向的Ca2 高亲和状态,E2状态则为内向的Ca2 低亲和状态.目前,骨骼肌内质网Ca2 泵转运Ca2 过程中的几个中间状态,包括E1-2Ca2 ,E1-ATP,E1-P-ADP,E2-Pi和E2状态的三维晶体结构已经解析.介绍这几种状态的晶体结构,并分析Ca2 泵在执行功能过程中结构与功能的关系.     

Structural studies of a stabilized phosphoenzyme intermediate of Ca2+-ATPase

Ca(2+)-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca(2+). Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca(2+) gradient, and finally by removing Ca(2+) from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-A resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca(2+) and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated cross-link between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species.     

Structural and functional properties of a Ca2+-ATPase from human platelets

An antibody prepared against highly purified rabbit muscle Ca2+-ATPase from sarcoplasmic reticulum has been observed to cross-react with proteins in human platelet membrane vesicles. The antibody specifically precipitated Ca2+-ATPase activity from solubilized human platelet membranes and recognized two platelet polypeptides denatured in sodium dodecyl sulfate with Mr = 107,000 and 101,000. Ca2+-ATPase activity from Brij 78-solubilized platelet membranes was purified up to 10-fold. The purified preparation consisted mainly of two polypeptides with Mr approximately 100,000, and 40,000. The lower molecular weight protein appeared unrelated to Ca2+-ATPase activity. The Ca2+-ATPase in human platelet membrane vesicles exhibited "negative cooperativity" with respect to the kinetics of ATP hydrolysis. The apparent Km for Ca2+ activation of ATPase activity was 0.1 microM. Ca2+-dependent phosphorylation of platelet vesicles by [gamma-32P]ATP at 0 degrees C yielded a maximum of 0.2-0.4 nmol of PO4/mg of protein that was labile at pH 7.0 and 20 degrees C. This result suggests that only about 2-4% of the total protein in platelet membrane vesicles is the Ca2+-ATPase, which agrees with an estimate based on the specific activity of the Ca2+-ATPase in platelet membranes (20-50 nmol of ATP hydrolyzed/min/mg of protein at 30 degrees C). Calmodulin resulted in only a 1.6-fold stimulation of Ca2+-ATPase activity even after extensive washing of membranes with a calcium chelator or chlorpromazine. It is concluded that human platelets contain a Ca2+-ATPase immunochemically related to the Ca2+ pump from rabbit sarcoplasmic reticulum and that the enzymatic characteristics and molecular weight of the platelet ATPase are quite similar to those of the muscle ATPase.     

Comparison of (Ca2+-Mg2+)-ATPase and Ca2+-ATPase in rat cardiac sarcolemma

The calcium dependency of the Ca2+-pump ATPase of rat cardiac sarcolemma was investigated in the presence and absence of EGTA and EDTA in combination with two free Mg2+-ion concentrations. The results showed: that Mg2+-ions are not essential for the turnover of the Ca2+-pump ATPase; that the Ca2+-affinity is regulated by the concentration of the calcium-chelator complex present in the medium; that (Ca2+-Mg2+)-ATPase and Ca2+-ATPase are probably expressions of the same Ca2+-pump ATPase in the plasma membrane of the cell.     

Structural aspects of ion pumping by Ca2+-ATPase of sarcoplasmic reticulum

Ca²⁺-ATPase of muscle sarcoplasmic reticulum is an ATP-powered Ca²⁺-pump that establishes a >10,000-fold concentration gradient across the membrane. Its crystal structures have been determined for nine different states that cover nearly the entire reaction cycle. Presented here is a brief structural account of the ion pumping process, which is achieved by a series of very large domain rearrangements.     

Ca2+-mediated activation of human erythrocyte membrane Ca2+-ATPase

Ca2+-ATPase of human erythrocyte membranes, after being washed to remove Ca2+ after incubation with the ion, was found to be activated. Stimulation of the ATPase was related neither to fluidity change nor to cytoskeletal degradation of the membranes mediated by Ca2+. Activation of the transport enzyme was also unaffected by detergent treatment of the membrane, but was suppressed when leupeptin was included during incubation of the membranes with Ca2+. Stimulation of the ATPase by a membrane-associated Ca2+-dependent proteinase was thus suggested. Much less 138 kDa Ca2+-ATPase protein could be harvested from a Triton extract of membranes incubated with Ca2+ than without Ca2+. Activity of the activated enzyme could not be further elevated by exogenous calpain, even after treatment of the membranes with glycodeoxycholate. There was also an overlap in the effect of calmodulin and the Ca2+-mediated stimulation of membrane Ca2+-ATPase. While Km(ATP) of the stimulated ATPase remained unchanged, a significant drop in the free-Ca2+ concentration for half-maximal activation of the enzyme was observed.     

Structural effects on the interaction of sterols with the (Ca2+ + Mg2+)-ATPase

The fluorescence quenching properties of a brominated derivative of androstenol 5 alpha,6 beta-dibromoandrostan-3 beta-ol have been used to study binding to phospholipid bilayers and to the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum of rabbit skeletal muscle. It is shown that androstenol is excluded from the phospholipid/protein interface of the ATPase but can bind to other (non-annular sites) on the ATPase. Binding to these sites increases in strength with decreasing chain length for the phospholipids present in the system. Binding is also stronger in the presence of phospholipids in the gel phase than in the liquid crystalline phase. Androstenol increases the ATPase activity of the ATPase reconstituted with phosphatidylcholines of chain lengths less than C18, but has no effect on activity for the ATPase reconstituted with phosphatidylcholines of chain lengths C18 or greater. The effects of cholestanols on the activity of the ATPase reconstituted with dimyristoleoylphosphatidylcholine depend on the configuration of the sterol, with 5 alpha-cholestan-3 alpha-ol having little effect but the other isomers causing a marked stimulation.     

Occlusion of Ca2+ in soluble monomeric sarcoplasmic reticulum Ca2+-ATPase

Sarcoplasmic reticulum Ca2+-ATPase solubilized in monomeric form by nonionic detergent was reacted with CrATP in the presence of 45Ca2+. A Ca2+-occluded complex formed, which was stable during high performance liquid chromatography in the presence of excess non-radioactive Ca2+. The elution position corresponded to monomeric Ca2+-ATPase. It is concluded that a single Ca2+-ATPase polypeptide chain provides the full structural basis for Ca2+ occlusion.     

Regulation of Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase at limiting [Ca2+

The factors regulating Ca2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca2+] at approximately 10 microM, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca2+] of 100 to 200 nM, and has been defined as [Ca2+]lim, whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca2+ fluophores, where it is presumed that sarcoplasmic free [Ca2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca2+]lim was decreased 70% to 80% by histidine, imidazole and cysteine. The K0.5 value for histidine was 3 mM, suggesting that residual [Ca2+]lim fluorescence is due to Zn2+. The level of Zn2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47+/-0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254-263). Histidine, 20 mM, included in the buffer, gave a corrected value for [Ca2+]lim of 49+/-1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible 'pump/leak' mechanism was tested by the effects of varying active Ca2+ transport 1 to 2 orders with temperature and pH. [Ca2+]lim remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca2+]lim levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca2+]lim was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca2+]lim was estimated by the unidirectional flux of 45Ca2+, and directly, following depletion of ATP, by measuring release of 40Ca2+, and was 0.02% of Vmax. Constant infusion of CaCl2 at [Ca2+]lim resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were Vmax=853 nmol/min per mg, K0.5(Ca)=279 nM, and nH(Ca)=1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca2+ transients, and in the relaxed state, will minimise contribution of Zn2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca2+]lim in the resting state is a characteristic feature of Ca2+ pump function, rather than a balance between active transport and passive leakage pathways.     

Lipid structure and Ca2+-ATPase function

Effects of lipid structure on the function of the Ca²⁺-ATPase of skeletal muscle of sarcoplasmic reticulum are reviewed. Binding of phospholipids to the ATPase shows little specificity. Phosphatidylcholines with short (C14) or long (C24) fatty acyl chains have marked effects on the activity of the ATPase, including a change in the stoichiometry of Ca binding. Low ATPase activity in gel phase lipid follows from low rate of phosphorylation. Phosphatidylinositol 4-phosphate increases ATPase activity by increasing the rate of dephosphorylation of the phosphorylated ATPase. Stimulation is not seen with other anionic phospholipids; phosphatidic acid decreases ATPase activity in a Mg²⁺-dependent manner.Abbreviations di(C141)PC dimyristoleoylphosphatidycholine - di(C160)PC dipalmitoylphosphatidylcholine - di(C181)PC dioleoylphosphatidylcholine - di(Br₂C180)PC dibromostearoylphosphatidylcholine - di(C241)PC dinervonylphosphatidylcholine - di(C181)PA dioleoylphosphatidic acid - di(C181)PE dioleoylphosphatidylethanolamine - Ptdlns phosphatidylinositol - PtdIns-4P phos-phatidylinositol 4-phosphate     

Voltage-dependence of Ca2+ uptake and ATP hydrolysis of reconstituted Ca2+-ATPase vesicles

Ca2+-ATPase from sarcoplasmic reticulum was reconstituted into phospholipid/cholesterol (9:1) vesicles (RO). Sucrose density gradient centrifugation of the RO vesicles separated a light layer (RL) with a high lipid/protein ratio and a heavy layer (RH). RH vesicles exhibited a high rate of Ca2+-dependent ATP hydrolysis but did not accumulate Ca2+. RL vesicles, on the other hand, showed an initial molar ratio of Ca2+ uptake to ATP hydrolysis of approximately 1.0. Internal trapping of transported Ca2+ facilitated studies over periods of several minutes. Ca2+ transport and ATP hydrolysis declined concomitantly, reaching levels near 0 with external Ca2+ concentrations less than or equal to 2 microM. Ca2+ uptake was inhibited by the Ca2+ ionophore A23187, the detergent Triton X-100, and the metabolic inhibitor quercetin. Ca2+ transport generated a transient electrical potential difference, inside positive. This finding is consistent with the hypothesis that the Ca2+ pump is electrogenic. Steady state electrical potentials across the membrane were clamped by using potassium gradients and valinomycin, and monitored with voltage-sensitive dyes. Over a range of +50 to -100 mV, there was an inverse relationship between the initial rate of Ca2+ uptake and voltage, but the rate of ATP hydrolysis was nearly constant. In contrast, lowering the external Ca2+ concentration depressed both transport and ATP hydrolysis. These findings suggest that the membrane voltage influences the coupling between Ca2+ transport and ATP hydrolysis.     

Na+,K+-ATPase and Ca2+-ATPase isozymes in malignant neoplasms

A current state of researches on mechanisms of ion homeostasis regulation in the specific conditions of the uncontrolled malignant tumor growth (mainly in carcinomas) concerning the contribution of Na+,K+-ATPase, plasma membrane and sarco(endo)plasmic reticulum Ca2+-ATPases has been reviewed. Particular attention has been focused on the molecular and biochemical links providing the redistribution of the transporting ATPases isozyme pattern for the regulatory requirements of the cell signaling pathways at stable proliferation and viability in malignancy.     

The structure and interactions of Ca2+-ATPase

Electron crystallographic studies on membrane crystals of Ca²⁺-ATPase reveal different patterns of ATPase-ATPase interactions depending on enzyme conformation. Physiologically relevant changes in Ca²⁺ concentration and membrane potential affect these interactions. Ca²⁺ induced difference FTIR spectra of Ca²⁺-ATPase triggered by photolysis of caged Ca²⁺ are consistent with changes in secondary structure and carboxylate groups upon Ca²⁺ binding; the changes are reversed during ATP hydrolysis suggesting that a phosphorylated enzyme form of low Ca²⁺ affinity is the dominant intermediate during Ca²⁺ transport. A two-channel model of Ca²⁺ translocation is proposed involving the membrane-spanning helices M2–M5 and M4, M5, M6 and M8 respectively, with separate but interacting Ca²⁺ binding sites.     

Solubilization and separation of Ca2+-ATPase from the Ca2+-ryanodine receptor complex

Heavy sarcoplasmic reticulum (SR) preparations of rabbit skeletal muscle, which are enriched in Ca2+-release vesicles from the terminal cisternae (TC) and [3H]ryanodine receptor density, exhibit 60% of the Ca2+-ATPase activity, 58% of the EP level, and 30% of the steady state Ca2+ loading compared to membrane vesicles from the longitudinal SR. The Ca2+-ATPase of TC SR is solubilized and separated from the Ca2+-ryanodine receptor complex in the insoluble fraction on treatment with the detergent C12E9. However, a 50% decrease in receptor density is observed upon removal of the Ca2+-ATPase, suggesting a significant contribution of this protein to maintaining optimal receptor complex density.     

Characterization of pancreatic islet Ca2+-ATPase

Distribution of three isoenzymes of brain enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11) (alpha alpha, alpha gamma and gamma gamma forms) in clonal cell lines of neuroblastoma (NS20Y and N18TG-2), glioma (C6BU-1), and hybrid cells NG108-15, NCB20, Nbr10A, Nbr20A, N4G-B-a and N4G-C-a) was examined with a sensitive enzyme immunoassay system, that uses a rabbit antibody to rat brain enolase alpha alpha or gamma gamma. All cell lines tested were found to possess the enolase which contains gamma subunit (a neuron-specific protein), although the alpha alpha enolase (non-neuronal enolase) was the dominant from in these cells. A clonal rat glioma (C6BU-1) cell contained about 40, 1 and 0.07 microgram/mg protein of alpha alpha, alpha gamma and gamma gamma enolases, respectively, at the confluent stage. Inclusion of 1 mM dibutyryl cyclic AMP or 10 micrometers prostaglandin E1 plus 1 mM theophylline in the culture medium of a hybrid cell (NG108-15, mouse neuroblastoma x rat glioma) resulted in a more than 2-fold increase in the concentrations of alpha gamma and gamma gamma in the cell within a few days, with little change in the alpha alpha enolase concentration. A similar increase in the concentration of gamma subunit by the nucleotide (but not by prostaglandin E1 plus theophylline) was also observed in the glioma cell (C6BU-1) line. The results suggest that the gamma subunit or the neuron-specific protein can be regulated in NG108-15 and C6BU-1 cells in a cyclic AMP-dependent fashion.     

Induction of vacuolar Ca2+-ATPase and H+/Ca2+ exchange activity in yeast mutants lacking Pmr1, the Golgi Ca2+-ATPase.

We have analyzed Ca2+ transport activity in defined subcellular fractions of an isogenic set of wild-type and mutant yeast. The results, together with measurements of polypeptide expression levels and promoter::reporter gene activity, show that the Golgi Ca2+-ATPase, Pmr1, is the major Ca2+ pump under normal growth conditions. In the absence of Pmr1, we show a massive, calcineurin-dependent compensatory induction of the vacuolar Ca2+-ATPase, Pmc1. In addition, H+/Ca2+ exchange activity, that may be distinct from the vacuolar exchanger Vcx1, is also increased.     

Ca2+-binding sites and Ca2+-ATPase activity in barley root tip cells

Summary Different cytochemical methods were employed to demonstrate the existence of Ca²⁺-binding sites (Ca²⁺-bs) at the membranes of barley root tip cells, involving addition of CaCl₂ (10 mM or 1 mM) to all aqueous solutions used for tissue processing for electron microscopy, treatment of ultrathin sections by Ca-chelating agents, enzymic digestion of ultrathin sections and modification of Wachstein-Meisel procedure for localization of Ca²⁺-dependent ATPase activity. Addition of 10 mM CaCl₂ to the fixatives and rinsing solutions causes electron-dense globules (EDG) to be formed in a variety of cells, those in cortical cells being associated mainly with the plasma membranes, in root cap cells with the plasmalemma as well as with majority of intracellular membranes. The obligatory presence of EDG at the membranes of Golgi vesicles and secretory vesicles approaching plasmalemma was revealed in the secreting root cap cells. Besides, electron opaque connecting material was found between the plasmalemma and adjacent secretory vesicle membranes. In true meristematic cells Ca-supplemented solutions induce formation of EDG localized at the ER membranes, and nuclear and plastid envelopes. In root cells of seeds germinated in the presence of 1 mM CaCl₂ electron opaque deposits were found only in local areas of plasmalemma collars around plasmodesmata neck regions, contacting the terminals of subsurface ER channels. In control speciemens (germination, fixation and washing without added CaCl₂) EDG were absent in cortical and ground meristem cells, but present in root cap cells, although their number and average size were greatly reduced.Treatment of thin sections by 10mM EGTA or EDTA led to complete removing of EDGs, electron-transparent holes replacing them. Digestion by a variety of proteolytic enzymes and by phospholipase A induced partial destruction of EDG matrices, confirming the presence of protein as well as of phospholipid membrane components. Visualization of electron-dense granular product of cytochemical Ca-ATPase reaction at the same membrane areas where EDG were located suggests that one of the Ca-binding proteins in EDG may represent Ca-ATPase.It is proposed that EDG at plant cell membranes have a certain resemblance to the Ca²⁺-bs revealed by the same method on plasma membrane of a variety of animal cells. The data obtained are discussed regarding possible regulatory roles of calcium ions in plant cells, especially in exocytotic secretion.     

Structural properties of sarcoplasmic reticulum Ca2+-ATPase as studied by intrinsic protein fluorescence

1. From the intrinsic fluorescence spectral properties and fluorescence quenching experiments done with acrylamide and iodide, using native sarcoplasmic reticulum vesicles, purified ATPase and ATPase solubilized with 1% Triton X-100, it is deduced that practically all the fluorescent tryptophanyl residues of this protein belong to a single population showing similar hydrophobic microenvironments. 2. Both acrylamide and iodide seem to be able to penetrate through the sarcoplasmic reticulum membrane. 3. The intrinsic fluorescence of the Ca2+-ATPase due to tryptophan residues probably buried inside the membrane is used as a tool to follow thermotropic changes in membrane fluidity of reconstituted systems.