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Plasma membrane calcium ATPases (PMCA) are key proteins in the maintenance of calcium (Ca2+) homeostasis. Dysregulation of PMCA function is associated with several human pathologies, including neurodegenerative diseases, and, therefore, these proteins are potential drug targets to counteract those diseases. Gold compounds, namely of Au(I), are well-known for their therapeutic use in rheumatoid arthritis and other diseases for centuries. Herein, we report the ability of dichloro(2-pyridinecarboxylate)gold(III) (1), chlorotrimethylphosphinegold(I) (2), 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidenegold(I) chloride (3), and chlorotriphenylphosphinegold(I) (4) compounds to interfere with the Ca2+-ATPase activity of pig brain purified PMCA and with membranes from SH-SY5Y neuroblastoma cell cultures. The Au(III) compound (1) inhibits PMCA activity with the IC50 value of 4.9 µM, while Au(I) compounds (2, 3, and 4) inhibit the protein activity with IC50 values of 2.8, 21, and 0.9 µM, respectively. Regarding the native substrate MgATP, gold compounds 1 and 4 showed a non-competitive type of inhibition, whereas compounds 2 and 3 showed a mixed type of inhibition. All gold complexes showed cytotoxic effects on human neuroblastoma SH-SY5Y cells, although compounds 1 and 3 were more cytotoxic than compounds 2 and 4. In summary, this work shows that both Au (I and III) compounds are high-affinity inhibitors of the Ca2+-ATPase activity in purified PMCA fractions and in membranes from SH-SY5Y human neuroblastoma cells. Additionally, they exert strong cytotoxic effects. Projects BFU2017-85723-P (to A.M.M. and C.G.-M.), and PID2020-115512GB-I00 (to A.M.M.) funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”. We acknowledge Fundação para a Ciência e a Tecnologia (FCT) project UIDB/04326/2020, Associate Laboratory for Green Chemistry–LAQV, financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020) and Scientific Employment Stimulus-Institutional Call (CEECINST/00102/2018). info:eu-repo/semantics/publishedVersion

Given that the cadmium (Cd) toxicity could be due to its interference with the calcium (Ca) homeostasis, the aim of this work was to study the effect of Cd over the presence, distribution and volume density (Vv) of Ca and Ca-ATPase in the secretory cells of the pars preconvoluta (PPC) and the pars convoluta (pc) in Rhinella arenarum. The severe effect of the xenobiotic (CdCl2 2.5 mg/kg) in sexually matured females was evaluated. Co-localization, as well as a marked reduction of Ca and Ca-ATPase, was observed in treated animals, in the areas analyzed, compared to control. Low calcium deposits were found in the secreting granules (SG) of the epithelial (ESC) and glandular secretory cells (GSC), while an increase in their cytoplasm and intracellular space was observed. The Ca-ATPase in treated and control animals was detected at the SG and the plasmatic membrane of the ESC and GSC. In relation to the Vv estimates, a substantial reduction of Ca deposits and Ca-ATPase activity was observed in the treated group, with respect to the control. Both amounts of Vv of Ca and Ca-ATPase activity were higher in PPC than in pc, and, higher in ESC than in GSC. These results were associated with the Cd concentration in the oviductal PC, determining that it is a bioaccumulator organ. Thus, this work demonstrated that the Cd interacted with Ca-ATPase, leading to an increase of cytosolic Ca, which is responsible for the possible disruptions in cellular metabolism. Fil: Medina, Marcela Fatima. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Taboada, Luis Nicolás. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Gonzalez Alejandro, María Evangelina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Díaz, Miguel Ángel. Universidad Nacional de Tucumán. Facultad de Ciencias Exactas y Tecnología; Argentina Fil: Gelatti, Facundo Javier. Universidad Tecnológica Nacional; Argentina Fil: Torres, Mabel. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Romero, Cintia Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina

The therapeutic applications of gold are well-known for many centuries. The most used gold compounds contain Au(I). Herein, we report, for the first time, the ability of four Au(I) and Au(III) complexes, namely dichloro (2-pyridinecarboxylate) Au(III) (abbreviated as 1), chlorotrimethylphosphine Au(I) (2), 1,3-bis(2,6-diisopropylphenyl) imidazole-2-ylidene Au(I) chloride (3), and chlorotriphenylphosphine Au(I) (4), to affect the sarcoplasmic reticulum (SR) Ca2+-ATPase activity. The tested gold compounds strongly inhibit the Ca2+-ATPase activity with different effects, being Au(I) compounds 2 and 4 the strongest, with half maximal inhibitory concentration (IC50) values of 0.8 and 0.9 &micro, M, respectively. For Au(III) compound 1 and Au(I) compound 3, higher IC50 values are found (4.5 &micro, M and 16.3 &micro, M, respectively). The type of enzymatic inhibition is also different, with gold compounds 1 and 2 showing a non-competitive inhibition regarding the native substrate MgATP, whereas for Au compounds 3 and 4, a mixed type of inhibition is observed. Our data reveal, for the first time, Au(I) compounds with powerful inhibitory capacity towards SR Ca2+ATPase function. These results also show, unprecedently, that Au (III) and Au(I) compounds can act as P-type ATPase inhibitors, unveiling a potential application of these complexes.

A fluorescence ratiometric method utilizing the probe eosin Y is presented for estimating the ATP binding site polarity of P-type ATPases in different conformational states. The method has been calibrated by measurements in a series of alcohols and tested using complexation of eosin Y with methyl-β-cyclodextrin. The results obtained with the Na+,K+-, H+,K+- and sarcoplasmic reticulum Ca2+-ATPases indicate that the ATP binding site, to which eosin is known to bind, is significantly more polar in the case of the Na+,K+- and H+,K+-ATPases compared to the Ca2+-ATPase. This result was found to be consistent with docking calculations of eosin with the E2 conformational state of the Na+,K+-ATPase and the Ca2+-ATPase. Fluorescence experiments showed that eosin binds significantly more strongly to the E1 conformation of the Na+,K+-ATPase than the E2 conformation, but in the case of the Ca2+-ATPase both fluorescence experiments and docking calculations showed no significant difference in binding affinity between the two conformations. This result could be due to the fact that, in contrast to the Na+,K+- and H+,K+-ATPases, the E2-E1 transition of the Ca2+-ATPase does not involve the movement of a lysine-rich N-terminal tail which may affect the overall enzyme conformation. Consistent with this hypothesis, the eosin affinity of the E1 conformation of the Na+,K+-ATPase was significantly reduced after N-terminal truncation. It is suggested that changes in conformational entropy of the N-terminal tail of the Na+, K+- and the H+,K+-ATPases during the E2-E1 transition could affect the thermodynamic stability of the E1 conformation and hence its ATP binding affinity.

形態学的な研究から，硬組織形成部位にアルカリ性至適pHのCa-ATPaseの存在が示唆されているが酵素学的な性質の報告は少ない．そこで骨芽細胞様細胞であるMC3T3-E1細胞が保持するCa-ATPase活性に関して研究を行った．細胞を石灰化時期まで培養後回収し，超音波破砕後に遠心分離操作を行って膜分画を得た．ATP加水分解により生じた無機リン酸をChifflet法で定量してATPase活性を測定し，以下の結果を得た．１．膜分画にはカルシウム（Ca）あるいはマグネシウム（Mg）により活性化されるATPaseが存在し両酵素ともthapsigarginによって阻害された．Ca存在下のATPase活性はMgによって拮抗されることと，Mg-ATPaseを阻害するazideによって阻害されないことから両酵素は別の酵素と示唆された．２．Ca-ATPase活性はCa濃度依存性に増加して1 mMの遊離Ca濃度で飽和し，50 ％活性化濃度は0.3 mMであった．３．活性はpH依存性に増加し，pH 9.1でpH 7.5のほぼ３倍の活性を示して最大となりpH 10.0までは同程度の活性を示した．４．活性はATP加水分解の過程においてリン酸化酵素を形成するP型ATPaseの阻害薬であるvanadateとエタクリン酸によっては阻害されなかった．５．活性は２価金属イオンのキレーターであるEGTAおよびEDTAにより濃度依存性に阻害されたが，ビスホスホネートによっては阻害されなかった．６．遊離Ca濃度100 nMでは， Ca-ATPase活性はほぼ検出されないが，カルモジュリンを添加すると濃度に依存して活性は増大し，50 ％活性化濃度は約6 μMであった．７．カルモジュリン非添加における活性は，カルモジュリン拮抗薬であるW7によって濃度依存性に抑制され，50 ％阻害濃度は0.3 mMであった．以上の結果は，E1細胞にはアルカリ性至適pHのP型ではないカルモジュリン依存性Ca-ATPaseが存在することを示唆する．本酵素は，形態学的に存在が示唆されるCa-ATPaseと類似しており，硬組織形成に関与する可能性がある．, Morphological studies suggest the presence of Ca-ATPase at alkaline pH optimum at the site of hard tissue formation, but there are few reports on enzymological properties. Therefore, we investigated Ca-ATPase activity using clonal osteoblastic cells (MC3T3-E1). The cells were cultured until calcification. The cells were then ultrasonically crushed. Further, they were centrifuged to obtain a membrane fraction. The ATPase activity was measured by measuring inorganic phosphate produced by ATP hydrolysis by the Chifflet method. The following results were obtained : 1. Ca-ATPase and Mg-ATPase exist in membrane fractionation. They are inhibited by thapsigargin. Ca-ATPase activity is inhibited by Mg. Mg-ATPase is inhibited by azide, but Ca -ATPase was not inhibited. Both are different enzymes. 2. Ca-ATPase activity increased with Ca concentration dependence, saturated at 1 mM free Ca concentration, and 50% activation was 0.3 mM. 3. Ca-ATPase activity increased in pH dependence and showed maximum activity at pH 9.1. It is nearly three times that of pH 7.5. It is the same value up to pH 10.0. 4. Ca-ATPase activity is not inhibited by vanadate, an inhibitor of P-type ATPase. Its activity is not inhibited by ethacrynic acid, an inhibitor of Mg-ATPase. 5. Ca-ATPase activity is inhibited in a concentration-dependent manner by EGTA and EDTA of bivalent metal chelators. Its activity is not inhibited by bisphosphonates. 6. At free Ca concentration of 100 nM, Ca-ATPase activity in the absence of calmodulin was not detected. Ca-ATPase activity increases with the addition of calmodulin depending on the concentration, and the 50 % activation concentration is about 6 μM. 7. Ca-ATPase activity is inhibited in a concentration-dependent manner by W7 which is a calmodulin antagonist when calmodulin is not added. The 50 % inhibitory concentration is 0.3 mM. The above results suggest that Ca-ATPase in MC3T3-E1 cells is calmodulin-dependent, which is optimal at alkaline pH and not P-type. This enzyme may be identical to Ca-ATPase which is morphologically suggested to be present, and it may be involved in hard tissue formation.

形態学的な研究から，硬組織形成部位にアルカリ性至適pH のCa-ATPase の存在が示唆されているが酵素学的な性質の報告は少ない．そこで骨芽細胞様細胞であるMC3T3-E1 細胞の保持するCa-ATPase 活性に関して研究を行った．細胞を石灰化時期まで培養後回収し，超音波破砕後遠心分離操作を行って膜分画を得た．ATP 加水分解により生じた無機リンをChifflet 法で定量してATPase 活性を測定し，以下の結果を得た．1．膜分画にはカルシウム（Ca）あるいはマグネシウム（Mg）により活性化されるATPase が存在し両酵素ともthapsigargin によって阻害された．Ca 存在下のATPase 活性はMg によって拮抗されることと，Mg-ATPase を阻害するazide によって阻害されないことから両酵素は別の酵素と示唆された．2．Ca-ATPase 活性はCa 濃度依存性に増加して1 mM の遊離Ca 濃度で飽和し，50％活性化濃度は0.3 mM であった．3．活性はpH 依存性に増加した．pH 9.1 でpH 7.5 のほぼ3 倍の活性を示して最大となりpH 10.0 までは同程度の活性を示した．4．活性はP 型ATPase の阻害薬であるvanadate とエタクリン酸によっては阻害されなかった．5．活性は2 価金属イオンのキレーターであるEGTA およびEDTA により濃度依存性に阻害されたが，ビスホスホネートによっては阻害されなかった．6．遊離Ca 濃度100nM では， Ca-ATPase 活性はほぼ検出されないが，カルモジュリンを添加すると濃度に依存して活性は増大し，50％活性化濃度は約6 μM であった．7．カルモジュリン非添加における活性は，カルモジュリン拮抗薬であるW7 によって濃度依存性に抑制され，50％阻害濃度は0.3 mM であった．以上の結果は，E1細胞にはアルカリ性至適pH のP 型ではないカルモジュリン依存性Ca-ATPaseが存在することを示唆する．本酵素は，形態学的に存在が示唆されるCa-ATPaseと類似しており，硬組織形成に関与する可能性がある．, (主査) 特任教授 鈴木 邦明, 教授 田村 正人, 教授 網塚 憲生, 歯学研究科（口腔医学専攻）

本研究は，骨芽細胞様MC3T3-E1（E1）細胞を用い，石灰化に伴う無機イオンの輸送に関与する可能性のある各種ATPaseに対するエストロゲンの作用を明らかにすることを目的として行なった．各種濃度の17 β-エストラジオール（17 β-E）存在下で10，20，30日間培養したE1細胞のホモジェネートを測定に使用した．アルカリ性ホスファターゼ（ALP）活性とATPase活性は，反応で遊離した無機リンを定量して測定した．１．ALP活性は，どの日数でも10－9Mから10－5Mの17 β-Eで濃度に依存して増加する傾向を示し，10－5Mでその作用は顕著であった．17 β-EはE1細胞の石灰化を促進し，10－5Mではその作用が強いことを示唆する．２．Na,K-ATPase活性を17 β-Eは20日目に約２倍増加したが，17 β-Eの濃度依存性は認められなかった．３．Ca-ATPase活性は，10－9M及び10－7Mの17 β-E存在下ではコントロールとの有意な差は認められなかったが，10－5Mの17 β-Eではどの日数でも有意に増加した．一方，Ca,Mg-ATPase活性も17 β-Eにより増加したが，17 β-Eの濃度依存性は顕著ではなかった．４．Mg-ATPase活性に対する17 β-Eの作用は，20及び30日ではALPに対する作用に類似していたが，20日での17 β-E濃度依存性は観察されなかった．以上の結果から，17 β-EによるE1細胞の各イオン輸送ATPase活性の活性化時期は異なったが，ATPaseに対する活性化作用は10－5Mという高濃度で強いことが示唆された．石灰化部位に存在すると推測される，アルカリ性至適pHで高濃度のCaを必要とするATPaseは，10－5Mの17 β-Eで顕著に促進されたことから，石灰化部位にCaを供給するATPaseとなる可能性があると考えられた．

The birds were developed to be 16 groups (4 groups of TT and 4 groups of RR, with male and female lines, respectively). The mating was conducted between similar groups of population (TT vs. TT, and RR vs. RR) to obtain second generation. The change in metabolism such as Ca–ATPase activity and Nτ–methylhistidine (Nτ–MH), also selection response between generations were analyzed. Parameters of metabolism were subjected to statistical analysis of T-test to compare between productions characteristic (TT and RR), especially for second generation. Body weight was also statistically analyzed by the same method. Selection response in TT group (3.10% or 2.74%) was higher than that in RR group (1.20% or 1.13%). Metabolism aspect in the quails of second generation either rate of protein turnover or activity of bone Ca-ATPase enzyme showed the change toward the productivity specification. Rate of muscle protein synthesis was higher and enzyme activity of Ca-ATPase was lower in group of TT population than those in group of RR population.

Plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger participate in regulating cell function by maintaining proper intracellular Ca2+ concentrations ([Ca2+]i). In renal epithelial cells these proteins have been additionally implicated in cellular calcium absorption. The purpose of the present studies was to determine the Ca2+ extrusion mechanisms in cells derived from the proximal tubule. Homology-based RT-PCR was used to amplify PMCA transcripts from RNA isolated from mouse cell lines originating from the S1, S2, and S3 proximal tubule segments. S1, S2, and S3 cells exhibited only PMCA1 and PMCA4 products. PCR product identity was confirmed by sequence analysis. Northern analysis of proximal tubule cell RNAs revealed appropriate transcripts of 7.5 and 5.5 kb for PMCA1 and 8.5 and 7.5 kb for PMCA4, but were negative for PMCA2 and PMCA3. Western analysis with a monoclonal antibody to PMCA showed that all proximal cell lines expressed a reacting plasma membrane protein of 140 kD, the reported PMCA molecular mass. Na+/Ca2+ exchanger (NCX1) mRNA expression, analyzed by RT-PCR, protein expression by Western analysis, and functional exchange activity were uniformly absent from all proximal tubule cell lines. These observations support the idea that immortalized cells derived from the proximal tubule express PMCA1 and PMCA4, which may serve as the primary mechanism of cellular Ca2+ efflux.

Several effects of the neuroleptic agent haloperidol on the sarcoplasmic reticulum (SR) Ca-dependent adenosine triphosphatase (Ca-ATPase) and Ca transport are described. Haloperidol inhibits the Ca-ATPase activity in the presence of calcimycin. The effect depends on the conditions of preexposure of the membranes to the drug: the inhibition increases with the preincubation time; Ca and Mg protect the enzyme against the effect of the drug. The inhibitory effect of haloperidol decreases upon increasing [Ca2+], at constant [Mg], and disappears at 20 mM [Mg] for any [Ca2+], and at 0.5 mM [Ca2+] for any [Mg2+]. Haloperidol also inhibits phosphorylation of the enzyme by Pi, and ATP-dependent Ca2+ uptake, in both cases with apparent Ki = 0.10 – 0.15 mM, and increases the rate of Ca efflux from preloaded vesicles in this concentration range. The results suggest that haloperidol interacts with the catalytic site, interfering with the effect of the divalent catalytic cation, but not at other steps of the enzymatic cycle, where Mg2+ and Ca2+ are also activators. They are consistent with a reaction model where haloperidol interacts with the E2 conformers of the enzyme, with lower affinity for the phosphoenzyme than for the dephospho species. The inhibition of Ca uptake by SR vesicles is ascribed to an increased Ca2+ permeability rather than to the inhibition of the Ca-ATPase, which requires higher concentrations of the drug.

International audience; Electron microscopy (EM) has made possible to solve the structure of many proteins. However, the resolution of some of the EM maps is too low for interpretation at the atomic level, which is particularly important to describe function. We describe methods that combine low-resolution EM data with atomic structures for different conformations of the same protein in order to produce atomic models compatible with the EM map.We illustrate these methods with EM data from decavanadate-induced tubular crystals of a pseudo-phosphorylated intermediate of Ca-ATPase and the various atomic structures of other intermediates available in the Protein Data Bank (PDB). Determination of atomic structure permits not only to analyse protein-protein interactions in the crystals, but also to localize residues in the proximity of the crystallizing agent both within Ca-ATPase and between Ca-ATPase molecules.

A method for the flexible docking of high-resolution atomic structures into lower resolution densities derived from electron microscopy is presented. The atomic structure is deformed by an iterative process using combinations of normal modes to obtain the best fit of the electron microscopical density. The quality of the computed structures has been evaluated by several techniques borrowed from crystallography. Two atomic structures of the SERCA1 Ca-ATPase corresponding to different conformations were used as a starting point to fit the electron density corresponding to a different conformation. The fitted models have been compared to published models obtained by rigid domain docking, and their relation to the known crystallographic structures are explored by normal mode analysis. We find that only a few number of modes contribute significantly to the transition. The associated motions involve almost exclusively rotation and translation of the cytoplasmic domains as well as displacement of cytoplasmic loops. We suggest that the movements of the cytoplasmic domains are driven by the conformational change that occurs between nonphosphorylated and phosphorylated intermediate, the latter being mimicked by the presence of vanadate at the phosphorylation site in the electron microscopy structure.

A method for the flexible docking of high resolution atomic structures into lower resolution densities derived from electron microscopy is presented. The atomic structure is deformed by an iterative process using combinations of normal modes to obtain the best fit of the electron microscopical density. The quality of the computed structures have been evaluated by several techniques borrowed from crystallography. Two atomic structures of the SERCA1 Ca-ATPase correponding to different conformations were used as a starting point to fit the electron density corresponding to a different conformation. The fitted models have been compared to published models obtained by rigid domain docking, and their relation to the known crystallographic structures are explored by normal mode analysis. We find that only a few number of modes contribute significantly to the transition. The associated motions involve almost exclusively rotation and translation of the cytoplamic domains as well as displacement of cytoplasmic loops. We suggest that the movements of the cytoplasmic domains are driven by the conformational change that occurs between non-phosphorylated and phosphorylated intermediate, the latter being mimicked by the presence of vanadate at the phosphorylation site in the electron microscopy structure.

Eine neue Klasse starker Inhibitoren der Na,K-ATPase wurde von uns zuerst aus Helleborus purpurascens isoliert. Indem das Protein in einem bestimmten Zustand blockiert wird, können unbekannte Zwischenzustände des Reaktionszyklus sowie Struktur-Funktionsbeziehungen aufgedeckt werden. Im Gegensatz zu den wohlbekannten Herzglykosid-Inhibitoren der Na,K-ATPase (Digitalis aus dem Fingerhut), inhibieren diese neuen Inhibitoren nicht nur Kaninchen-Na,K-ATPase, sondern mit ähnlicher Wirksamkeit auch Na,K-ATPase aus der Rattenniere, die eine viel niedrigere Affinität für Herzglykoside hat und sogar SR-Ca-ATPase und H,K-ATPase aus dem Magen. Die chemische Struktur der neuen Inhibitoren kann vom einfachen anorganischen Gas Kohlensuboxid (C3O2) abgeleitet werden. Hexamere und Oktamere dieser Substanz bilden makrozyklische Ringe, die als makrozyklische Kohlenstoffsuboxid-Derivate (MCS) bezeichnet werden.Die Bindestelle der Na,K-ATPase für Herzglykoside ist stark konserviert, und könnte eine mögliche Bindestelle für endogene, digitalisartige Faktoren (EDLF auf Englisch) in Tieren sein. Wegen vielen gemeinsamen chemischen Eigenschaften von MCS-Faktoren und früher beschriebenen EDLFs wurde eine Untersuchungen des Aktionsmechanismus angeregt um eine mögliche Identität der beiden Substanzen zu klären.Die stark konservierte Herzglykosidbindestelle liegt auf der extrazellulären Seite der Na,K-ATPase. Deshalb war eine der Hauptanforderungen an die MCS-Faktoren, um als EDLF in Betracht zu kommen, dass sie an die extrazelluläre Seite der Na,K-ATPase binden. Experimente mit Na,K-ATPase enthaltenden Lipidvesikeln und Kompetitionsexperimente mit anderen bekannten Inhibitoren weisen darauf hin, dass MCS-Faktoren - im Gegensatz zu Ouabain - eher von der intrazellulären Seite auf die Na,K-ATPase wirken und somit nicht als typischer EDLF in Frage kommen.Bezüglich des Aktionsmechanismus wurde die Bindung von MCS-Faktoren an die Na,K-ATPase mit dem fluoreszierenden Styrylfarbstoff RH421 untersucht. Der Styrylfarbstoff detektiert Veränderungen in der Membrankapazität, die z.B. durch Ionen verursacht werden, die von Proteinen durch die Membranen transportiert werden. In sogenannten ´Standardexperimenten´ wurden sättigende Konzentrationen von MCS-Faktoren zur Na,K-ATPase hinzugegeben. In diesen Experimenten kann das Protein vornehmlich in den Zuständen H2E1, Na3E1, E2P und (K2)E2 vorliegen. Es wurde beobachtet, dass nur die Konformation E1 von dem Inhibitor beeinflusst wird. Nachfolgende Titrationen der MCS-Faktoren zum Zustand E1, zeigten eine Zunahme der Fluoreszenz in der Größe wie sie erwartet würde, wenn ein Kation aus dem Enzym freigegeben wird. Diese Fluoreszenzzunahme wurde auch in Anwesenheit von Na+- und K+-Ionen beobachtet. Die Größe der Fluoreszenzänderung war abhängig vom pH der Lösung. Ausgehend von einer einfachen Reaktionsgleichung für die Bindung von Protonen an die Na,K-ATPase konnte ein Modell einer Reaktionsgleichung erstellt werden, das in der Lage war, alle experimentell erlangten Daten zu simulieren. Das Modell beinhaltet vier Zustände in denen MCS-Faktoren an die Na,K-ATPase gebunden sind und in denen ebenfalls immer ein Kation an das Enzym gebunden ist. Die Abhängigkeit vom pH wurde darauf zurückgeführt, dass MCS-Faktoren bei niedrigem pH protoniert werden und dadurch in eine aktivere Form überführt werden. Ein ähnlicher, aber viel kleinerer Effekt wurde auch bei höheren Na+- und K+-Konzentrationen beobachtet. Auf diese Veränderungen der Inhibitoraktivität weisen auch die Aktivierungsprozedur und die Enzymaktivitätstests hin, bei denen Veränderungen der Aktivität in Abhängigkeit von Kationenkonzentrationen im Puffer registriert wurden.Abschließend kann man sagen, dass eine neue Klasse von Inhibitoren der P-Typ ATPasen charakterisiert wurde, die mehrere Typ II P-Typ ATPasen stark inhibiert. Die MCS-Faktoren binden bei der Na,K-ATPase an einer spezifischen Bindestelle auf der zytoplasmatischen Seite des Proteins. Dadurch wird eine Konformationsänderung induziert, die eine Veränderung in der Gleich­gewichts­dis­soziationskonstante für eine der ersten beiden intrazellulären Kationen­bindestellen bewirkt. Nur auf diese Studie basierend, kann den MCS-Faktoren keine physiologische Rolle zugesprochen werden. Die verblüffenden chemischen Ähnlichkeiten der MCS-Faktoren mit vorausgegangenen publizierten Eigenschaften von EDLFs, wurden durch weitere, gemeinsame Eigenschaften bestärkt. Die grundlegende Erwartung an EDLFs an die konservierte extrazelluläre Herzglykosidbindestelle zu binden, konnte von MCS-Faktoren nicht erfüllt werden. Trotzdem bleibt die Tatsache bestehen, dass diese Verbindung in essbaren Pflanzen vorkommt und somit in tierische Körper aufgenommen wird. Diese Studie zeigt eindeutig, dass eine Interaktion zwischen MCS-Faktoren und Typ II P-Typ-ATPasen prinzipiell möglich ist. Ob eine signifikante Interaktion dieser Art tatsächlich stattfindet, wird in physiologischen Studien untersucht werden müssen.

Control of smooth muscle is vital for health. The major route to contraction is a rise in intracellular [Ca2+], determined by the entry and efflux of Ca2+ and release and re-uptake into the sarcoplasmic reticulum (SR). We review these processes in myometrium, to better understand excitation-contraction coupling and develop strategies for preventing problematic labours. The main mechanism of elevating [Ca2+] is voltage-gated L-type channels, due to pacemaker activity, which can be modulated by agonists. The rise of [Ca2+] produces Ca-calmodulin and activates MLCK. This phosphorylates myosin and force results. Without Ca2+ entry uterine contraction fails. The Na/Ca exchanger (NCX) and plasma membrane Ca-ATPase (PMCA) remove Ca2+, with contributions of 30% and 70% respectively. Studies with PMCA-4 knockout mice show that it contributes to reducing [Ca2+] and relaxation. The SR contributes to relaxation by vectorially releasing Ca2+ to the efflux pathways, and thereby increasing their rates. Agonists binding produces IP3 which can release Ca from the SR but inhibition of SR Ca2+ release increases contractions and Ca2+ transients. It is suggested that SR Ca2+ targets K+ channels on the surface membrane and thereby feedback to inhibit excitability and contraction.

P-type ATPases are a large family of membrane proteins that perform active ion transport across biological membranes. In these proteins, the energy-providing ATP hydrolysis is coupled to ion transport of one or two ion species across the respective membrane. The pump function of the investigated pumps is described by a so-called Post-Albers cycle. Main features of the pumping process are (1) a Ping-Pong mechanism, i.e. both transported ion species are transferred successively and in opposite direction across the membrane, (2) the transport process for each ion species consists of a sequence of reaction steps, which are ion binding, ion occlusion, conformational transition of the protein, successive deocclusion of the ions and release to the other side of the membrane. (3) Recent experimental evidence shows that the ion-binding sites are placed in the transmembrane section of the proteins and that ion movements occur preferentially during the ion binding and release processes. The main features of the mechanism include narrow access channels from both sides, one gate per access channel, and an ion-binding moiety that is adapted specifically to the ions that are transported, and differently in both principal conformations.

Recently discovered macrocyclic carbon suboxide (MCS) factors with the general formula (C3O2)n were found to strongly inhibit rabbit and rat Na,K-ATPase as well as SR Ca-ATPase. Highly active MCS factors were obtained by a base/acid treatment of their lipophilic precursor isolated from plants. In the ESI-MS spectra, the dominant molar mass ion of 431 Da corresponds to a 1:1 complex of the carbon suboxide hexamer (n=6;Mr=408 Da) with a Na + ion. Additional mass ions identified in positive and negative ion mode were assigned as complexes of the MCS hexamer (n=6) and octamer (n=8) with Na + or with TFA in various ratios. The dominant mass ion values of these active MCS factors from plants are also found in mass spectra of previously described endogenous digitalis-like factors (EDLF) from animals. This would suggest that ubiquitously distributed MCS factors may function as putative endogenous regulatory substances of Na,KATPase and possibly of other ATPases. With the symmetric display of several equivalent carbonyl or hydroxy groups, the structure of MCS factors is particularly suited for interactions with proteins and other bio-molecules. This could explain the high biological activity and the unusual properties of the MCS factors. D 2002 Elsevier Science B.V. All rights reserved.

We investigated the possibility that, in hair cells mechanically isolated from frog semicircular canals, Ca2+ extrusion occurs via a Na+ : Ca2+ (cardiac type) or a Na+ : Ca2+,K+ (retinal type) exchanger. Cells concurrently imaged during whole-cell patch-clamp recordings using the Ca2+ sensitive fluorescent dye Oregon Green 488 BAPTA-1 (100 micro m) showed no voltage dependence of Ca2+ clearance dynamics following a Ca2+ load through voltage-gated Ca2+ channels. Reverse exchange was probed in hair cells dialyzed with a Ca2+- and K+-free solution, containing a Na+ concentration that saturates the exchanger, after zeroing the contribution to the whole-cell current from Ca2+ and K+ conductances. In these conditions, no reverse exchange current was detected upon switching from a Ca2+-free external solution to a solution containing concentrations of Ca2+ alone, or Ca2+ + K+ that saturated the exchanger. By contrast, the same experimental protocol elicited peak exchange currents exceeding 100 pA in gecko rod photoreceptors, used as positive controls. In both cell types, we also probed the forward mode of the exchanger by rapidly increasing the intracellular Ca2+ concentration using flash photolysis of two novel caged Ca2+ complexes, calcium 2,2'-([1-(2-nitrophenyl)ethane-1,2-diyl]bis(oxy))bis(acetate) and calcium 2,2'-([1-(4,5-dimethoxy-2-nitrophenyl)ethane-1,2-diyl]bis(oxy)) bis(acetate), in the presence of internal K+ and external Na+. No currents were evoked by UV-triggered Ca2+ jumps in hair cells, whereas exchanger conformational currents up to 400 pA, followed by saturating forward exchange currents up to 40 pA, were recorded in rod photoreceptors subjected to the same experimental conditions. We conclude that no functional electrogenic exchanger is present in this hair cell population, which leaves the abundant plasma membrane Ca2+-ATPases as the primary contributors to Ca2+ extrusion.

The sarcoplasmic reticulum Ca-ATPase is fully activated when approximately 1 microM [Ca2+] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub- or low microM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37 degreesC in 3 mM ATP, 3 mM MgCl2, 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl2, EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. Ki ranged between 20 and 100 microM, depending on the enzyme concentration. Pi production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca2+] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca2+] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 microM, is necessary for full activation of the ATPase.

Thirty‐nine moderately endurance trained males increased their normal training programme of 2.2 h week‐1 with an average training intensity of 65 % of maximum heart rate (HRmax) to 2.7 h week‐1 and a mean intensity of 78% of HRmax. Performance tests and measurements of the total concentrations of Na,K‐ATPase (3H‐ouabain binding) and Ca‐ATPase, fibre type distribution and fibre area were performed in biopsies from the vastus lateralis muscle before and after increased training. The 6 weeks of training elevated Vo2max from 54.9 + 3.1 to 58.3±3.0 ml 02 min‐1 kg‐1 (P < 0.0001). Exercise time to exhaustion at 86% of Fo2max (pre‐training) increased from 35 ±8 to 61 + 17 min (P < 0.0001). The concentration of Ca‐ATPase was unaffected by the intensified training (6.74 ± 1.03 vs. 6.68+ 1.07 nmol g wet wt‐1), but the concentration of Na,K‐ATPase increased from 307±43 to 354 + 59 pmol g wet wt ' (P < 0.0001). The relative distribution of FT‐fibres was correlated with the concentration of Ca‐ATPase (r = 0.72, P < 0.0001). The data support the view that intensive training induces an upregulation of the concentration of skeletal muscle Na,K‐ATPase, but no change in the total capacity for reaccumulation of Ca2+ into the SR. There was no correlation between the concentrations of Na,K‐ATPase, Ca‐ATPase and indices of endurance performance.

The inhibition of Ca2+-ATPase of sarcoplasmic reticulum by thapsigargin has been reported to be associated with a suppression of calcium binding to the high affinity transport sites. We report here that thapsigargin also acts as an inhibitor of ATP binding by reducing its apparent affinity by about two orders of magnitude. This inhibition is non-competitive indicating that thapsigargin does not bind to the ATP binding site. This is confirmed by the fact that thapsigargin binding to the Ca2+-ATPase does not affect the binding of 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-ATP (TNP-ATP).

The presence and distribution of calsequestrin (CS), Ca2+ pump, and inositol 1,4,5-trisphosphate (IP3) receptor were investigated biochemically and immunologically in microsomal (P3) fractions isolated from chicken cerebrum and cerebellum. Two different batches of polyclonal antibodies specific for chicken skeletal muscle CS identified a Ca2+ binding, CS-like protein that was extremely enriched in cerebellum P3 fractions and absent from all cerebrum fractions. The cerebellum CS-like protein was deemed authentic CS because the N-terminal amino acid domain and peptide mapping were identical to those of skeletal muscle CS in the same species. CS was detected in striated muscles and cerebellum only. Cerebellum P3 fractions were also found to be considerably enriched in Ca2+ pump and IP3 receptor compared with the homologous cerebrum fractions, as judged by measurements of Ca2+ uptake, Ca2(+)-ATPase activity, IP3-induced Ca2+ release, and [3H]IP3 binding, respectively. Cerebellum microsomal fractions therefore appear to contain membrane fragments endowed with Ca2+ pump, IP3 receptor, and CS, i.e., three key components of a Ca2+ storage organelle.

Carp myofibrils (Mf) were added with various concentrations of sodium Lglutamate (Na-Glu), and examinations were made of the relationship between added concentrations of Na-Glu and bound water content in Mf or thermal stability with the rate constant (kD) for inactivation of myofibrillar Ca-ATPase as an index of the Mf in quality. At the concentration range used in this study, bound water content in Mf showed higher values for the addition of Na-Glu than no-additive Mf (control); it was gradually increased with an increase in concentration of Na-Glu, and reached the peak at the concentration of 2.0 mol, being slowly decreased at higher concentrations. Log kD for Mf was always smaller for the addition of Na-Glu than control; it was gradually decreased with an increase in concentration of Na-Glu, and reached the peak (highest thermal stability ) at the concentration of 2.0 mol, being slowly increased at higher concentrations. Some correlation was recognized to exist between bound water content in Mf and its thermal stability.

A Ca-stimulated ATPase activity (pH 9.5) associated with the tegumental membrane enriched (TME) fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.6) was histochemically visualized whithin the tegument of fixed worms on the cytoplasmic leaflet of both the doubel surface membrane and the basement membrane; this reaction was inhibited by 1 µM chloropromazine and it was also observed on the inner side of double membrane vesicles present in the TME fraction. No ATPase activity could be seen at alkaline pH with added Mg or Na/K ions. Without ATP, the addition of external Ca to the fixed worms induced the appearance of lead precipitates on the tegumental discoid bodies; this reaction was inhibited by molybdate and not by chlorpromazine. The intrategumentary regulation of calcium by the systems described and the possible use of phenothiazines against schistosimes are discussed. A atividade ATPse (pH 9.5) estimulada por ions de Ca associados a uma fração enriquecida de membranas do tegumento (fração EMT) de vermes adultos de Schistosoma mansoni, foi inibida pro NAP-taurina ou por concentrações crescentes de clorpromacina. Foi encontrada calmodulina enfogena associada principlamente a esta fração. Em vermes adultos fixados com glutaraldeido se detectou histoquimicamente uma atividade ATPase similar (pH 8.6) na face citoplasmática da dupla membrana de superfície e da membrana por 1 µM de clorpromacina e foi também observada na face interna de vesículas de dupla membrana presentes na fração EMT. Não se pôde detectar atividade ATpase em pH alcalino na presença de ions de Mg ou Na/K. A adição externa de Ca, sem ATP, aos vermes fixados induz ao aparecimento de precipitados nos corpos discóides do tegumento; esta reação foi inibida. Os resultados são discutidos em relação a uma possível regulação intrategumentária de Ca pelos sistemas descritos e o possível uso de fenotiacinas contra os esquistossomas.

A Ca-stimulated ATPase activity (pH 9.5) associated with the tegumental membrane enriched (TME) fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.6) was histochemically visualized whithin the tegument of fixed worms on the cytoplasmic leaflet of both the doubel surface membrane and the basement membrane; this reaction was inhibited by 1 µM chloropromazine and it was also observed on the inner side of double membrane vesicles present in the TME fraction. No ATPase activity could be seen at alkaline pH with added Mg or Na/K ions. Without ATP, the addition of external Ca to the fixed worms induced the appearance of lead precipitates on the tegumental discoid bodies; this reaction was inhibited by molybdate and not by chlorpromazine. The intrategumentary regulation of calcium by the systems described and the possible use of phenothiazines against schistosimes are discussed.A atividade ATPse (pH 9.5) estimulada por ions de Ca associados a uma fração enriquecida de membranas do tegumento (fração EMT) de vermes adultos de Schistosoma mansoni, foi inibida pro NAP-taurina ou por concentrações crescentes de clorpromacina. Foi encontrada calmodulina enfogena associada principlamente a esta fração. Em vermes adultos fixados com glutaraldeido se detectou histoquimicamente uma atividade ATPase similar (pH 8.6) na face citoplasmática da dupla membrana de superfície e da membrana por 1 µM de clorpromacina e foi também observada na face interna de vesículas de dupla membrana presentes na fração EMT. Não se pôde detectar atividade ATpase em pH alcalino na presença de ions de Mg ou Na/K. A adição externa de Ca, sem ATP, aos vermes fixados induz ao aparecimento de precipitados nos corpos discóides do tegumento; esta reação foi inibida. Os resultados são discutidos em relação a uma possível regulação intrategumentária de Ca pelos sistemas descritos e o possível uso de fenotiacinas contra os esquistossomas.

Carp myofibrils (Mf) were added with various concentrations of sodium Lglutamate (Na-Glu), and examinations were made of the relationship between added concentrations of Na-Glu and bound water content in Mf or thermal stability with the rate constant (kD) for inactivation of myofibrillar Ca-ATPase as an index of the Mf in quality. At the concentration range used in this study, bound water content in Mf showed higher values for the addition of Na-Glu than no-additive Mf (control); it was gradually increased with an increase in concentration of Na-Glu, and reached the peak at the concentration of 2.0 mol, being slowly decreased at higher concentrations. Log kD for Mf was always smaller for the addition of Na-Glu than control; it was gradually decreased with an increase in concentration of Na-Glu, and reached the peak (highest thermal stability ) at the concentration of 2.0 mol, being slowly increased at higher concentrations. Some correlation was recognized to exist between bound water content in Mf and its thermal stability., 長崎大学水産学部研究報告, v.62, pp.23-28; 1987

Protein hydrolysates (SH, TH) were prepared from eel head by enzymatic treatment using protease. The eel head hydrolysates were used as a natural food preservative by adding to grass carp surimi at the 10% ranging. We compared the effect of protein hydrolysates of eel head with traditional antifreeze on the denatration, gel strength and contents of the salt soluble protein in grass carp surimi during frozen storage at -20°C for 80 days. The addition of eel head hydrolysate markedly decreased the activation rate of the Ca-ATPase. The contents of the salt soluble protein in the grass carp surimi containing eel head hydrolysates were higher than those without eel head hydrolysates (control).Therefore, the gel strength of grass carp surimi media with eel head hydrolysate lower than the others. The result suggests that eel head hydrolysate could suppress the denaturation of grass carp surimi during frozen storage.

Diallyl disulfide (DADS), an oil-soluble organosulfur compound in processed garlic, was more effective in inhibiting the in vitro growth of human tumor cell lines: HCT-15 (colon), A549 (lung), and SK MEL-2 (skin) than isomolar quantities of the water-soluble compound S-allyl cysteine (SAC). Addition of DADS (100 μM) was cytostatic to all three cell lines. The importance of the allyl and the disulfide groups were revealed by the lack of a comparable depression in the growth of HCT-15 cells exposed to its saturated analogue, dipropyl disulfide (DPDS). Treatment with DADS also resulted in a dose-dependent increase in intracellular free calcium in cells. A dose-dependent decrease in the activity of calcium-dependent ATPase enzyme occurred in HCT-15 cells exposed to increasing quantities of DADS. A correlation (r = −0.975) was found between the intracellular free calcium levels and the Ca-ATPase activity in DADS-treated cells. These studies document that DADS, a constituent of garlic oil, is an effective inhibitor of the growth of human neoplastic cells. Alterations in calcium homeostasis are likely involved in the growth inhibition/cytotoxicity caused by DADS.

The properties of the Ca-ATPase prepared from roots of wheat seedlings treated with benzylaminopurine were studied. The affinity of the ATPase towards Ca 2+ , plant or erythrocyte calmodulin increased after the hormonal treatment. It seems that in the membrane calmodulin-bonding sites were induced by benzylaminopurine, contributing to an increased affinity of the ATPase. The lower Ca-content of the hormone-treated plants suggests that in vivo the Ca-ATPase is involved in a Ca-extrusion process.

The sarcoplasmic reticulum Ca-ATPase was subjected to target size analysis by radiation inactivation in various buffer conditions and after solubilization in monomeric form in non-ionic detergent and in SDS. The target size was also determined for Ca-ATPase in bidimensional crystals formed in the presence of decavanadate or lanthanide. The standardization obtained with defined monomers of Ca-ATPase shows that the target size of Ca-ATPase in the functional membrane-bound state may be ascribed to a single peptide chain, possibly with surrounding lipid. Further analysis of the radiation inactivation sizes of various partial reactions of the pump cycle, including phosphorylation and Ca2+ occlusion, indicated much smaller values than the target size pertaining to decomposition of the whole peptide chain. This is consistent with the existence of separate functional domains within a single peptide chain.

Irreversible inactivation of membranous Na,K-ATPase by high-speed centrifugation in dilute aqueous solutions depends markedly on the protonation state of the protein. Pig kidney Na,K-ATPase is irreversibly inactivated at pH 5 but is fully protected at pH 7 and above. Shark rectal gland Na,K-ATPase is irreversibly inactivated at neutral or acidic pH and partially protected at an alkaline pH. The overall Na,K-ATPase activity and the K-dependent pNPPase activity were denatured in parallel. Cryoprotectants such as glycerol or sucrose at concentrations of 25–30% fully protect both enzymes against inactivation. The specific ligands NaCl and KCl protect the Na,K-ATPase activity partially and the pNPPase activity fully at concentrations of 0.2–0.3 M. Electron microscope analysis of the centrifuged Na,K-ATPase membranes revealed that the ultrastructure of the native membranes is preserved upon inactivation. It was also observed that the sarcoplasmic reticulum Ca-ATPase and hog gastric H,K-ATPase are susceptible to inactivation by high-speed centrifugation in a pH-dependent fashion. H,K-ATPase is protected at alkaline pH, whereas Ca-ATPase is protected only in the neutral pH range.