亲和层析纯化肌质网Ca2+－ATP酶

建立了一种亲和层析纯化肌质网Ca²⁺-ATP酶的方法．用非离子型去污剂C₁₂E₈ 溶解肌质网,再通过反应红－120琼脂糖亲和层析柱使肌质网Ca²⁺-ATP酶纯度从粗品中的65％提高到99％,并具有较高ATP水解活性．经SDS－聚丙烯酰胺凝胶电泳检测,为电泳纯．     

Solubilization,purification and reconstitution of Ca-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.     

Surfactin Induces Apoptosis and G<Subscript>2</Subscript>/M Arrest in Human Breast Cancer MCF-7 Cells Through Cell Cycle Factor Regulation

Surfactin, purified from Bacillus subtilis natto TK-1, inhibited proliferation of human breast cancer MCF-7 cells in a dose- and time-dependent manner, with IC₅₀ at 24, 48, and 72 h of 82.6, 27.3, and 14.8 μM, respectively. Surfactin-induced cell death was considered to be apoptotic by observing the typical apoptotic morphological change by acridine orange/ethidium bromide staining and Transferase-mediated dUTP Nick End-labeling assay. [Ca²⁺]i measurement revealed that surfactin induced a sustained increase in concentration of intracellular [Ca²⁺]i. Flow cytometric analysis also demonstrated that surfactin caused time-dependent apoptosis of MCF-7 cells through cell arrest at G₂/M phase. Western blot revealed that surfactin induced accumulation of the tumor suppressor p53 and cyclin kinase inhibitor p21^waf1/cip1, and inhibited the activity of the G₂-specific kinase, cyclin B1/p34^cdc2. Based on our findings, surfactin inhibited proliferation in MCF-7 cells by inducing apoptosis and the elevation of [Ca²⁺]i may play an important role in the apoptosis. The mechanism which surfactin caused G₂/M arrest seems to be through cell cycle factor regulation.     

Unfolding and partial refolding of a cellulase from the SDS-denatured state: From β-sheet to α-helix and back

Globular proteins are typically unfolded by SDS to form protein-decorated micelle-like structures. Several proteins have been shown subsequently to refold by addition of the nonionic surfactant octaethylene glycol monododecyl ether (C₁₂E₈). Thus SDS converts β-lactoglobulin, which has mainly β-sheet secondary structure, into a state rich in α-helicality, while addition of C₁₂E₈ leads to refolding and recovery of the original β-sheet structure. Here we extend these studies to the large β-sheet-rich cellulase Cel7b from Humicola insolens whose enzymatic activity provides a very sensitive refolding parameter. The enzymes widespread usage in the detergent industry makes it an obvious model system for protein-surfactant interactions. SDS-unfolding and subsequent refolding using C₁₂E₈ were investigated at pH 4.2 using near- and far-UV circular dichroism (CD), small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), size-exclusion chromatography (SEC) and activity measurements. The Cel7b:SDS complex can be described as a random configuration of 3–4 connected core-shell structures in which the protein is converted to a mainly α-helical secondary structure. Addition of C₁₂E₈ recovers almost all the secondary structure, part of the tertiary structure, about 50% of the activity and dissociates part of the protein population completely from detergent micelles. The lack of complete refolding may be due to charge neutralisation of Cel7b by SDS, kinetically trapping the enzyme into aggregated structures. In support of this, aggregates did not form when C₁₂E₈ was first mixed with Cel7b followed by addition of SDS. Formation of such aggregates may be a general phenomenon hampering quantitative refolding from the SDS-denatured state.     

ROS-Ca is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis

The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca²⁺ on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca²⁺ concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca²⁺ was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ_m). Then the cytoplasmic Ca²⁺ concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.     

Solubilization and reconstitution of ca pump from corn leaf plasma membrane

The Ca²⁺ transport system of corn (Zea mays) leaf plasma membrane is composed of Ca²⁺ pump and Ca²⁺/H⁺ antiporter driven by H⁺ gradient imposed by a H⁺ pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca²⁺ transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca²⁺ pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C₁₂E₈) was the most effective detergent for a series of extraction and functional reconstitution of the Ca²⁺ pump among seven detergents examined. This was judged from activities of ATP-dependent ⁴⁵Ca²⁺ uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C₁₂E₈-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca²⁺-ATPase was separated from VO₄³⁻-sensitive Mg²⁺-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca²⁺ uptake was measured. The liposomes reconstituted with the Ca²⁺-ATPase, but not with the VO₄³⁻-sensitive Mg²⁺-ATPase, showed ATP-dependent Ca²⁺ uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca²⁺ uptake into liposomes reconstituted with the Ca²⁺-ATPase, suggesting that the Ca²⁺/H⁺ antiporter was not present in the preparation. These results indicate that the Ca²⁺-ATPase actually functions as Ca²⁺ pump in the corn leaf plasma membrane.     

Effect of Ca2+ on the self assembly of a nonionic peptide aggregate

Summary Conductometry, circular dichroism and fluorescence spectroscopy are the techniques employed to investigate the effect of added calcium ions and other monovalent and divalent metal ions on aqueous solutions of nonionic peptide aggregates, Boc-Leu-Asn-OEt (1). It is observed that among all the metal ions studied, Ca²⁺ ions facilitate the aggregation of the peptide. The interior dielectric constant of the micelles (ε) was found to depend upon the proportion of Ca²⁺ complexed peptide with the peptide mononers in the micelles. When Ca²⁺ ion becomes 1/4th of the peptide concentration, there is a structural transition leading to drastic change in the interior of the micro dielectric constant (ɛ _m).     

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca²⁺-pump (PMCA) in the membrane region upon interaction with Ca²⁺, calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [¹²⁵I]TID-PC/16, measuring the shift of conformation E₂ to the auto-inhibited conformation E₁I and to the activated E₁A state, titrating the effect of Ca²⁺ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca²⁺ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca²⁺ transport but does not modify the affinity for Ca²⁺ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E₁I to the activated E₁A conformation and thus modulating the transport of Ca²⁺. This is reflected in the different apparent constants for Ca²⁺ in the absence of CaM (calculated by Ca²⁺-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca²⁺ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca²⁺ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca²⁺ binding and activation.     

Binding,Activation, and Solubilization of the Ca2+-ATPase from Sarcoplasmic Reticulum by Nonionic Detergents

Interactions between delipidated Ca²⁺-ATPase from sarcoplasmic reticulum and four nonionic detergents—dodecyl octaoxyethyleneglycol monoether (C₁₂E₈), Triton X-100, Brij 58, and Brij 35—were characterized with respect to activation of ATPase activity, binding, and solubilization. C₁₂E₈ and Triton X-100 activated the delipidated ATPase to at least 80% of the original activity at the critical micelle concentrations (CMCs), whereas Brij 58 and Brij 35 activated no more than 10% of the original activity. The inability of Brij 58 and Brij 35 to activate the delipidated enzyme was probably a result of reduced binding of these detergents below the CMCs; both detergents exhibited a sixteenfold reduction in binding at the CMC compared with C₁₂E₈. The two Brij detergents were also unable to solubilize the delipidated enzyme and form monomers, as determined by sedimentation experiments. Thus the reduced binding levels of these detergents may result from an inability to overcome protein/protein interactions in the delipidated preparation. However, the Brij detergents were capable of solubilizing active enzyme from membrane vesicles, although with lower efficiency than C₁₂E₈ and Triton X-100. These results suggest that Brij 58 and 35 may be useful for solubilization of membrane proteins without disrupting protein/protein interactions, while Triton X-100 and C₁₂E₈ are more useful when bulk solubilization is the goal.     

Effect of chemical modification on the crystallization of Ca2+-ATPase in sarcoplasmic reticulum

The influence of chemical modification on the morphology of crystalline ATPase aggregates was analyzed in sarcoplasmic reticulum (SR) vesicles. The Ca²⁺-ATPase forms monomer-type (P1) type crystals in the E₁ and dimer-type (P2) crystals in the E₂ conformation. The P1 type crystals are induced by Ca²⁺ or lanthanides; P2 type crystals are observed in Ca²⁺-free media in the presence of vanadate or inorganic phosphate. P1- and P2-type Ca²⁺-ATPase crystals do not coexist in significant amounts in native sarcoplasmic reticulum membrane. The crystallization of Ca²⁺-ATPase in the E₂ conformation is inhibited by guanidino-group reagents (2,3-butanedione and phenylglyoxal), SH-group reagents, phospholipases C or A₂, and detergents, together with inhibition of ATPase activity. Amino-group reagents (fluorescein 5′-isothiocyanate, pyridoxal phosphate and fluorescamine) inhibit ATPase activity but do not interfere with the crystallization of Ca²⁺-ATPase induced by vanadate. In fluorescamine-treated sarcoplasmic reticulum the vanadate-induced crystals contain significant P1-type regions in addition to the dominant P2 form.     

Rapid Desensitization of Serotonin 5-HT2C Receptor-Stimulated Intracellular Calcium Mobilization in CHO Cells Transfected with Cloned Human 5-HT2C Receptors

Abstract: Serotonin 5-HT_2C receptor-mediated intracellular Ca²⁺ mobilization was investigated in Chinese hamster ovary (CHO) cells transfected with 5-HT_2C receptors. Fura-2 acetoxymethyl ester was used to investigate the regulation of 5-HT_2C receptor function. CHO cells, transfected with a cDNA clone for the 5-HT_2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin-sensitive [³H]mesulergine binding (K_D = 0.49 nM). The addition of 5-HT mobilized intracellular Ca²⁺ in a dose-dependent fashion, ranging from a basal level of 99 ± 1.8 up to 379 ± 18 nM, with an EC₅₀ value for 5-HT of 0.029 µM. Exposure to 5-HT, 1-(3-chlorophenyl)piperazine dihydrochloride (a 5-HT_2C agonist), and 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (a 5-HT_2C and 5-HT_2A agonist) resulted in increased intracellular Ca²⁺ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5-HT-mediated intracellular Ca²⁺ mobilization more effectively than spiperone. The receptor was rapidly desensitized by preexposure to 5-HT in a time- and concentration-dependent manner. Mezerein and phorbol 12-myristate 13-acetate, protein kinase C activators, weakly inhibited the intracellular Ca²⁺ mobilization induced by 10 µM 5-HT. Furthermore, the protein kinase C inhibitor H-7 partially prevented the protein kinase C activator-induced inhibition of the 5-HT-mediated increase in intracellular Ca²⁺ concentration. The desensitization induced by pretreatment with 5-HT was blocked by W-7, added in conjunction with 5-HT, and partially inhibited by W-5, a nonselective inhibitor of protein kinases and weak analogue of W-7. Therefore, the 5-HT_2C receptor may be connected with protein kinase C and calcium/calmodulin turnover. These results suggest that 5-HT_2C receptor activation mobilizes Ca²⁺ in CHO cells and that the acute desensitization of the receptor may be due to calmodulin kinase-mediated feedback.     

Mechanisms of Na<Superscript>+</Superscript>-Ca<Superscript>2+</Superscript> Exchange Inhibition by Amphiphiles in CardiacMyocytes: Importance of Transbilayer Movement

The membrane lipid environment and lipid signaling pathways are potentially involved in the modulation of the activity of the cardiac Na⁺-Ca²⁺ exchanger (NCX). In the present study biophysical mechanisms of interactions of amphiphiles with the NCX and the functional consequences were examined. For this purpose, intracellular Ca²⁺ concentration jumps were generated by laser-flash photolysis of caged Ca²⁺ in guinea-pig ventricular myocytes and Na⁺-Ca²⁺ exchange currents (I_Na/Ca) were recorded in the whole-cell configuration of the patch-clamp technique. The inhibitory effect of amphiphiles increased with the length of the aliphatic chain between C₇ and C₁₀ and was more potent with cationic or anionic head groups than with uncharged head groups. Long-chain cationic amines (C₁₂) exhibited a cut-off in their efficacy in I_Na/Ca inhibition. Analysis of the time-course, comparison with the Ni²⁺-induced I_Na/Ca block and confocal laser scanning microscopy experiments with fluorescent lipid analogs (C₆- and C₁₂-NBD-labeled analogs) suggested that amphiphiles need to be incorporated into the membrane. Furthermore, NCX block appears to require transbilayer movement of the amphiphile to the inner leaflet (“flip”). We conclude that both, hydrophobic and electrostatic interactions between the lipids and the NCX may be important factors for the modulation by lipids and could be relevant in cardiac diseases where the lipid metabolism is altered.This revised version was published online in August 2005 with a corrected cover date.     

Surfactin-Induced Apoptosis Through ROS–ERS–Ca2+-ERK Pathways in HepG2 Cells

Although surfactin is able to inhibit cancer cell proliferation and to induce cancer cell apoptosis, the molecular mechanism responsible for this process remain elusive. In this study, the signaling network underlying the apoptosis of human hepatoma (HepG2) cells induced by surfactin was investigated. It is found that the reaction oxygen species (ROS) production and intracellular calcium ([Ca²⁺]_i) accumulation are both induced HepG2 cells apoptosis. The [Ca²⁺]_i exaltation was partly depended on the Ca²⁺ release from inositol 1,4,5-trisphosphate (IP3) and ryanodine (Ry) receptors channels, which both triggered endoplasmic reticulum stress (ERS). The results showed that surfactin induced the ROS production and ROS production led to ERS. The occurrence of ERS increased the [Ca²⁺]_i level and the processes associated with blocking extracellular signal-regulated kinase (ERK) pathway. According to a comprehensive review of all the evidence, it is concluded that surfactin induces apoptosis of HepG2 cells through a ROS–ERS–Ca²⁺ mediated ERK pathway.     

Purification and properties of ATPase from an alkalophilic Bacillus

ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (?). The subunit structure is suggested to be α₃β₃γδ?. The enzyme is activated by Mg²⁺ and Ca²⁺. The pH optima of the enzyme with 0.1 and 2.0 mm Mg²⁺ are 9 and 6, and those with 1 and 10 mm Ca²⁺ are 8–9 and 7, respectively. Ca²⁺-ATPase hydrolyzes only ATP, whereas Mg²⁺-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and K_m of the enzyme with ATP in the presence of 10 mm Ca²⁺ or 0.6 mm Mg²⁺ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 mm, respectively. The enzyme with Mg²⁺ is appreciably activated by HCO^?₃. Relationship of the ATPase to the active transport system in the bacterium is suggested.     

Activation of protein kinase C inhibits ATP-induced [Ca2+]i elevation in rat osteoblastic cells: Selective effects on P2Y and P2U signaling pathways

Extracellular ATP elicits transient elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in osteoblasts through interaction with more than one subtype of cell surface P₂-purinoceptor. Elevation of [Ca²⁺]_i arises, at least in part, by release of Ca²⁺ from intracellular stores. In the present study, we investigated the possible roles of protein kinase C (PKC) in regulating these signaling pathways. [Ca²⁺]_i of indo-1-loaded UMR-106 osteoblastic cells was monitored by spectrofluorimetry. In the absence of extracellular Ca²⁺, ATP (100 μM) induced transient elevation of [Ca²⁺]_i to a peak 57 ± 7 nM above basal levels (31 ± 2 nM, means ± S. E. M., n = 25). Exposure of cells to the PKC activator 12-O-tetradecanoyl-β-phorbol 13-acetate (TPA, 100 nM) for 2 min significantly reduced the amplitude of the ATP response to 13 ± 4 nM (n = 11), without altering basal [Ca²⁺]_i. Inhibition was half-maximal at approximately 1 nM TPA. The Ca²⁺ response to ATP was also inhibited by the PKC activators 1,2-dioctanoyl-sn-glycerol or 4β-phorbol 12, 13-dibutyrate, but not by the control compounds 4α-phorbol or 4α-phorbol 12, 13-didecanoate. Furthermore, exposure of cells to the protein kinase inhibitors H-7 or staurosporine for 10 min significantly attenuated the inhibitory effect of TPA. However, these protein kinase inhibitors did not prolong the [Ca²⁺]_i response to ATP alone, indicating that activation of PKC does not account for the transient nature of this response. When the effects of other nucleotides were examined, TPA was found to cause significantly greater inhibition of the response to the P_2Y-receptor agonists, ADP and 2-methylthioATP, than the response to the P_2U-receptor agonist, UTP. These data indicate that activation of PKC selectively inhibits the P_2Y signaling pathway in osteoblastic cells. In vivo, endocrine or paracrine factors, acting through PKC, may regulate the responsiveness of osteoblasts to extracellular nucleotides. © 1995 Wiley-Liss, Inc.     

Distinct Roles of the C-terminal 11th Transmembrane Helix and Luminal Extension in the Partial Reactions Determining the High Ca2+ Affinity of Sarco(endo)plasmic Reticulum Ca2+-ATPase Isoform 2b (SERCA2b)

The molecular mechanism underlying the characteristic high apparent Ca²⁺ affinity of SERCA2b relative to SERCA1a and SERCA2a isoforms was studied. The C-terminal tail of SERCA2b consists of an 11th transmembrane helix (TM11) with an associated 11-amino acid luminal extension (LE). The effects of each of these parts and their interactions with the SERCA environment were examined by transient kinetic analysis of the partial reaction steps in the Ca²⁺ transport cycle in mutant and chimeric Ca²⁺-ATPase constructs. Manipulations to the LE of SERCA2b markedly increased the rate of Ca²⁺ dissociation from Ca₂E1. Addition of the SERCA2b tail to SERCA1a slowed Ca²⁺ dissociation, but only when the luminal L7/8 loop of SERCA1 was simultaneously replaced with that of SERCA2, thus suggesting that the LE interacts with L7/8 in Ca₂E1. The interaction of LE with L7/8 is also important for the low rate of the Ca₂E1P → E2P conformational transition. These findings can be rationalized in terms of stabilization of the Ca₂E1 and Ca₂E1P forms by docking of the LE near L7/8. By contrast, low rates of E2P dephosphorylation and E2 → E1 transition in SERCA2b depend critically on TM11, particularly in a SERCA2 environment, but do not at all depend on the LE or L7/8. This indicates that interaction of TM11 with SERCA2-specific sequence element(s) elsewhere in the structure is critical in the Ca²⁺-free E2/E2P states. Collectively these properties ensure a higher Ca²⁺ affinity of SERCA2b relative to other SERCA isoforms, not only on the cytosolic side, but also on the luminal side.     

Two Distinct P2 Purinergic Receptors, P2Y and P2U, Are Coupled to Phospholipase C in Mouse Pineal Gland Tumor Cells

Abstract: We found that extracellular ATP can increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in mouse pineal gland tumor (PGT-β) cells. Studies of the [Ca²⁺]_i rise using nucleotides and ATP analogues established the following potency order: ATP, adenosine 5′-O-(3-thiotriphosphate) ≥ UTP > 2-chloro-ATP > 3′-O-(4-benzoyl)benzoyl ATP, GTP ≥ 2-methylthio ATP, adenosine 5′-O-(2-thiodiphosphate) (ADPβS) > CTP. AMP, adenosine, α,β-methyleneadenosine 5′-triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and UMP had little or no effect on the [Ca²⁺]_i rise. Raising the extracellular Mg²⁺ concentration to 10 mM decreases the ATP-and UTP-induced [Ca²⁺]_i rise, because the responses depend on the ATP^4? and UTP^4? concentrations, respectively. The P_2U purinoceptor-selective agonist UTP and the P_2Y purinoceptor-selective agonist ADPβS induce inositol 1,4,5-trisphosphate generation in a concentration-dependent manner with maximal effective concentrations of ～100 µM. In sequential stimulation, UTP and ADPβS do not interfere with each other in raising the [Ca²⁺]_i. Costimulation with UTP and ADPβS results in additive inositol 1,4,5-trisphosphate generation to a similar extent as is achieved with ATP alone. Pretreatment with pertussis toxin inhibits the action of UTP and ATP by maximally 45–55%, whereas it has no effect on the ADPβS response. Treatment with 1 µM phorbol 12-myristate 13-acetate inhibits the ADPβS-induced [Ca²⁺]_i rise more effectively than the ATP- and UTP-induced responses. These results suggest that P_2U and P_2Y purinoceptors coexist on PGT-β cells and that both receptors are linked to phospholipase C.     

Formation of the Stable Structural Analog of ADP-sensitive Phosphoenzyme of Ca2+-ATPase with Occluded Ca2+ by Beryllium Fluoride: STRUCTURAL CHANGES DURING PHOSPHORYLATION AND ISOMERIZATION*

As a stable analog for ADP-sensitive phosphorylated intermediate of sarcoplasmic reticulum Ca²⁺-ATPase E1PCa₂·Mg, a complex of E1Ca₂·BeF_x, was successfully developed by addition of beryllium fluoride and Mg²⁺ to the Ca²⁺-bound state, E1Ca₂. In E1Ca₂·BeF_x, most probably E1Ca₂·BeF₃⁻, two Ca²⁺ are occluded at high affinity transport sites, its formation required Mg²⁺ binding at the catalytic site, and ADP decomposed it to E1Ca₂, as in E1PCa₂·Mg. Organization of cytoplasmic domains in E1Ca₂·BeF_x was revealed to be intermediate between those in E1Ca₂·AlF₄⁻ ADP (transition state of E1PCa₂ formation) and E2·BeF₃⁻·(ADP-insensitive phosphorylated intermediate E2P·Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca₂·BeF_x in contrast to no superfluorescence of TNP-AMP bound to E1Ca₂·AlF_x. E1Ca₂·BeF_x with bound TNP-AMP slowly decayed to E1Ca₂, being distinct from the superfluorescent complex of TNP-AMP with E2·BeF₃⁻, which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca₂·BeF_x mimics E1PCa₂·Mg, and between those of E1Ca₂·AlF₄⁻·ADP and E2·BeF₃⁻. E1Ca₂·BeF_x at low 50–100 μm Ca²⁺ was converted slowly to E2·BeF₃⁻ releasing Ca²⁺, mimicking E1PCa₂·Mg → E2P·Mg + 2Ca²⁺. Ca²⁺ replacement of Mg²⁺ at the catalytic site at approximately millimolar high Ca²⁺ decomposed E1Ca₂·BeF_x to E1Ca₂. Notably, E1Ca₂·BeF_x was perfectly stabilized for at least 12 days by 0.7 mm lumenal Ca²⁺ with 15 mm Mg²⁺. Also, stable E1Ca₂·BeF_x was produced from E2·BeF₃⁻ at 0.7 mm lumenal Ca²⁺ by binding two Ca²⁺ to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P· Mg + 2Ca²⁺ → E1PCa₂·Mg.Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a),² a representative member of the P-type ion transporting ATPases, catalyze Ca²⁺ transport coupled with ATP hydrolysis (Fig. 1) (1–9). The enzyme forms phosphorylated intermediates from ATP or P_i in the presence of Mg²⁺ (10–13). In the transport cycle, the enzyme is first activated by cooperative binding of two Ca²⁺ ions at high affinity transport sites (E2 to E1Ca₂, steps 1–2) (14) and autophosphorylated at Asp³⁵¹ with MgATP to form the ADP-sensitive phosphoenzyme (E1P, step 3), which reacts with ADP to regenerate ATP in the reverse reaction. Upon this E1P formation, the two bound Ca²⁺ are occluded in the transport sites (E1PCa₂). Subsequent isomeric transition to the ADP-insensitive form (E2PCa₂), i.e. loss of ADP sensitivity at the catalytic site, results in rearrangement of the Ca²⁺ binding sites to deocclude Ca²⁺, reduce the affinity, and open the lumenal gate, thus releasing Ca²⁺ into the lumen (E2P, steps 4–5). Finally Asp³⁵¹-acylphosphate in E2P is hydrolyzed to form the Ca²⁺-unbound inactive E2 state (steps 6 and 7). Mg²⁺ bound at the catalytic site is required as a physiological catalytic cofactor in phosphorylation and dephosphorylation and thus for the transport cycle. The cycle is totally reversible, e.g. E2P can be formed from P_i in the presence of Mg²⁺ and absence of Ca²⁺, and subsequent Ca²⁺ binding at lumenally oriented low affinity transport sites of E2P reverses the Ca²⁺-releasing step and produces E1PCa₂, which is then decomposed to E1Ca₂ by ADP.Open in a separate windowFIGURE 1.Ca²⁺ transport cycle of Ca²⁺-ATPase.Various intermediate structural states in the transport cycle were fixed as their structural analogs produced by appropriate ligands such as AMP-PCP (non-hydrolyzable ATP analog) or metal fluoride compounds (phosphate analogs), and their crystal structures were solved at the atomic level (15–22). The three cytoplasmic domains, N, P, and A, largely move and change their organization state during the transport cycle, and the changes are coupled with changes in the transport sites. Most remarkably, in the change from E1Ca₂·AlF₄⁻·ADP (the transition state for E1PCa₂ formation, E1PCa₂·ADP·Mg^‡) to E2·BeF₃⁻ (the ground state E2P·Mg) (23–25), the A domain largely rotates by more than 90° approximately parallel to the membrane plane and associates with the P domain, thereby destroying the Ca²⁺ binding sites, and opening the lumenal gate, thus releasing Ca²⁺ into the lumen (see Fig. 2). E1PCa₂·Ca·AMP-PN formed by CaAMP-PNP without Mg²⁺ is nearly the same as E1Ca₂·AlF₄⁻·ADP and E1Ca₂·CaAMP-PCP in their crystal structures (17, 18, 22).Open in a separate windowFIGURE 2.Structure of SERCA1a and its change during processing of phosphorylated intermediate. E1Ca₂·AlF₄⁻·ADP (the transition state analog for phosphorylation E1PCa₂·ADP·Mg^‡) and E2·BeF₃⁻ (the ground state E2P analog (25)) were obtained from the Protein Data Bank (PDB accession code 1T5T (17) and 2ZBE (21), respectively). Cytoplasmic domains N (nucleotide binding), P (phosphorylation), and A (actuator), and 10 transmembrane helices (M1–M10) are indicated. The arrows on the domains, M1′ and M2 (Tyr¹²²) in E1Ca₂·AlF₄⁻·ADP, indicate their approximate motions predicted for E1PCa₂·ADP·Mg^‡ → E2P·Mg. The phosphorylation site Asp³⁵¹, TGES¹⁸⁴ of the A domain, Arg¹⁹⁸ (tryptic T2 site) on the Val²⁰⁰ loop (DPR¹⁹⁸AV²⁰⁰NQD) of the A domain, and Thr²⁴² (proteinase K site) on the A/M3-linker are shown. Seven hydrophobic residues gather in the E2P state to form the Tyr¹²²-hydrophobic cluster (Y122-HC); Tyr¹²²/Leu¹¹⁹ on the top part of M2, Ile¹⁷⁹/Leu¹⁸⁰/Ile²³² of the A domain, and Val⁷⁰⁵/Val⁷²⁶ of the P domain. The overall structure of E1Ca₂·AlF₄⁻·ADP is virtually the same as those of E1Ca₂·CaAMP-PCP and E1PCa₂·Ca·AMP-PN (17, 18, 22).Despite these atomic structures, yet unsolved is the structure of E1PCa₂·Mg, the genuine physiological intermediate E1PCa₂ with bound Mg²⁺ at the catalytic site without the nucleotide. Its stable structural analog has yet to be developed. E1PCa₂·Mg is the major intermediate accumulating almost exclusively at steady state under physiological conditions. Its rate-limiting isomerization results in Ca²⁺ deocclusion/release producing E2P·Mg as a key event for Ca²⁺ transport. In E1Ca₂·CaAMP-PCP, E1Ca₂·AlF₄⁻·ADP, and E1PCa₂·Ca·AMP-PN, the N and P domains are cross-linked and strongly stabilized by the bound nucleotide and/or Ca²⁺ at the catalytic site, thus they are crystallized (17, 18, 22). Kinetically, E1PCa₂·Ca formed with CaATP is markedly stabilized due to Ca²⁺ binding at the catalytic Mg²⁺ site, and its isomerization to E2P is strongly retarded in contrast to E1PCa₂·Mg (26, 27). Thus, the bound Ca²⁺ at the catalytic Mg²⁺ site likely produces a significantly different structural state from that with bound Mg²⁺.Therefore, it is now essential to develop a genuine E1PCa₂·Mg analog without bound nucleotide and thereby gain further insight into the structural mechanism in the Ca²⁺ transport process. It is also crucial to further clarify the structural importance of Mg²⁺ as the physiological catalytic cation. In this study, we successfully developed the complex E1Ca₂·BeF_x, most probably E1Ca₂·BeF₃⁻, as the E1PCa₂·Mg analog by adding beryllium fluoride (BeF_x) to the E1Ca₂ state without any nucleotides. For its formation, Mg²⁺ binding at the catalytic site was required and Ca²⁺ substitution for Mg²⁺ was absolutely unfavorable, revealing a likely structural reason for its preference as the physiological cofactor. In E1Ca₂·BeF₃⁻, two Ca²⁺ ions bound at the high affinity transport sites are occluded. It was also produced from E2·BeF₃⁻ by lumenal Ca²⁺ binding at the lumenally oriented low affinity transport sites, mimicking E2P·Mg + 2Ca²⁺ → E1PCa₂·Mg. All properties of the newly developed E1Ca₂·BeF₃⁻ fulfilled the requirements as the E1PCa₂·Mg analog, and hence we were able to uncover the hitherto unknown nature of E1PCa₂·Mg as well as structural events occurring in the phosphorylation and isomerization processes. Also, we successfully found the conditions that perfectly stabilize the E1Ca₂·BeF₃⁻ complex.     

Conformational structure of bombesin as studied by vibrational and circular dichroism spectroscopy

Raman and Fourier transform infrared (FTIR) spectroscopies and circular dichroism (CD) have been applied to investigate the secondary structure of bombesin in the solid state and in phosphate buffer solution (pH 3.8). At concentrations around 10⁻⁵ M, circular dichroism reveals that bombesin exists as an irregular or disordered conformation. However, the secondary structure of the peptide appears to be a mixture of disordered structure and intermolecular β-sheets in 0.01 M sodium phosphate buffer when the peptide concentrations are higher than around 6.5 mM. The tendency of bombesin to form aggregated β-sheet species seems to be originated mainly in the sequence of the residues 7–14, as supported by the Raman spectra and β-sheet propensities (P_β) of the amino-acid residues. It is the hydrophobic force of this amino-acid sequence, and not a salt bridge effect, that is the factor responsible for the formation of peptide aggregates.     

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg²⁺ plus K⁺. Mn²⁺ was less effective and Ca²⁺ could not substitute for Mg²⁺. However, adding Ca²⁺ with Mg²⁺ or substituting Mn²⁺ for Mg²⁺ activated it appreciably in the absence of added K⁺, and all three divalent cations decreased apparent affinity for K⁺. Inhibition by Na⁺ decreased with higher Mg²⁺ concentrations, when Ca²⁺ was added, and when Mn²⁺ was substituted for Mg²⁺. Dimethyl sulfoxide, which favorsE ₂ conformations of the enzyme, increased apparent affinity for K⁺, whereas oligomycin, which favorsE ₁ conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg²⁺ and Mn²⁺, favoring (under these conditions)E ₂ conformations, are effective, whereas Ca²⁺, favoringE ₁, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K⁺, and although Ca²⁺ and Mn²⁺ are fairly effective, Mg²⁺ is a poor substitute for K⁺, while Na⁺ at these sites favorsE ₁ conformations. K⁺ increases theK _m for substrate, but both Ca²⁺ and Mn²⁺ decrease it, perhaps by competing with K⁺. On the other hand, phosphatase activity in the presence of Na⁺ plus K⁺ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg²⁺ and Mn²⁺, but not by adding Ca²⁺; this is consistent with stimulation occurring through facilitation of an E₁ to E₂ transition, perhaps an E₁-P to E₂-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg²⁺ plus Na⁺ alone or Mg²⁺ plus Na⁺ plus low K⁺: this effect of oligomycin may reflect acceleration, in the absence of adequate K⁺, of an alternative E₂-P to E₁ pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.