Uncoupling of Ca2+ transport in sarcoplasmic reticulum as a result of labeling lipid amino groups and inhibition of Ca2+-ATPase activity by modification of lysine residues of the Ca2+-ATPase polypeptide

Limited labeling of amino groups with fluorescamine in fragmented sarcoplasmic reticulum vesicles inhibits Ca2+-ATPase activity and Ca2+ transport. Under the labeling conditions used, 80% of the label reacts with phosphatidylethanolamine and 20% with the Ca2+-ATPase polypeptide. This degree of labeling does not result in vesicular disruption or in loss of vesicular proteins and does not increase the membrane permeability to Ca2+. Fluorescamine labeling of a purified Ca2+-ATPase devoid of aminophospholipids also inhibits Ca2+-ATPase activity, suggesting that labeling of lysine residues of the enzyme polypeptide is responsible for the inhibition of Ca2+-ATPase activity in sarcoplasmic reticulum. Fluorescamine labeling interferes with phosphoenzyme formation and decomposition in both the native vesicles and the purified enzyme; addition of ATP during labeling, and with less effectiveness ADP or AMP, protects both partial reaction steps. Addition of a nonhydrolyzable ATP analog protects phosphoenzyme formation but not decomposition. The inhibition of Ca2+ transport but not of Ca2+-ATPase occurs in sarcoplasmic reticulum vesicles labeled in the presence of ATP, indicating that the transport reaction is uncoupled from the Ca2+-ATPase reaction. The inhibition of Ca2+ transport but not of Ca2+-ATPase activity is also found in sarcoplasmic reticulum vesicles in which only phosphatidylethanolamine has reacted with fluorescamine. Furthermore, the extent of labeling of phosphatidylethanolamine is correlated with the inhibition of Ca2+ transport rates. The inhibition of Ca2+ transport is a reflection of the inhibition of Ca2+ translocation and is not due to an increase in Ca2+ efflux. We propose that labeling of phosphatidylethanolamine perturbs the lipid environment around the enzyme, producing a specific defect in the Ca2+ translocation reaction.     

Antimicrotubular drugs binding to vinca domain of tubulin

The effect of oxidative stress on the Ca²⁺-ATPase activity, lipid peroxidation and protein modification of cardiac sarcoplasmic reticulum (SR) membranes was investigated. Isolated SR vesicles were exposed to FeSO₄/EDTA (0.2 mol Fe²⁺ per mg of protein) at 37°C for 1 h in the presence or absence of antioxidants. FeSO₄/EDTA decreased the maximum velocity of Ca²⁺-ATPase reaction without a change of affinity for Ca²⁺ or Hill coefficient. Treatment with radical-generating system led also to conjugated diene formation, loss of sulfhydryl groups, changes in tryptophan and bityrosine fluorescences and to production of lysine conjugates with lipid peroxidation end-products. Lipid antioxidants butylated hydroxytoluene (BHT) and stobadine partially prevented inhibition of Ca²⁺-ATPase and decrease in tryptophan fluorescence, while the loss of –SH groups and formation of bityrosines or lysine conjugates were completely prevented. Glutathione also partially protected Ca²⁺-ATPase activity and decreased formation of bityrosine, but it was not able to prevent oxidative modification of tryptophan and lysine. These findings suggest that combination of amino acid modifications, rather than oxidation of amino acids of one kind, is responsible for inhibition of SR Ca²⁺-ATPase activity.     

Increased Ca2+ -ATPase activity associated with methylation of phospholipids in human erythrocytes.

Ca²⁺-ATPase activity in human erythrocytes is increased by the enzymatic methylation of membrane phospholipids. Erythrocyte membranes incubated in the presence of the methyl donor, S-adenosyl-L-methionine, demonstrate increased Ca²⁺ stimulated ATP hydrolysis, increased [⁴⁵Ca²⁺] efflux from erythrocyte ghosts and synthesis of phosphatidyl-N-monomethylethanolamine. The increase in Ca²⁺-ATPase activity is due to an increase in Vmax, and not due to changes in affinity for ATP or Ca²⁺. The concentration of S-adenosyl-L-methionine needed to stimulate Ca²⁺-ATPase closely matches that needed for the methylation of phosphatidylethanolamine. Both the stimulation of Ca²⁺-ATPase and the methylation of phospholipids are inhibited by the methyltransferase inhibitor, S-adenosyl-L-homocysteine. Membrane fluidity is increased by phospholipid methylation, which may be the mechanism for Ca²⁺-ATPase stimulation.     

Inhibition Mechanism of the Intracellular Transporter Ca2+-Pump from Sarco-Endoplasmic Reticulum by the Antitumor Agent Dimethyl-Celecoxib

Dimethyl-celecoxib is a celecoxib analog that lacks the capacity as cyclo-oxygenase-2 inhibitor and therefore the life-threatening effects but retains the antineoplastic properties. The action mechanism at the molecular level is unclear. Our in vitro assays using a sarcoplasmic reticulum preparation from rabbit skeletal muscle demonstrate that dimethyl-celecoxib inhibits Ca²⁺-ATPase activity and ATP-dependent Ca²⁺ transport in a concentration-dependent manner. Celecoxib was a more potent inhibitor of Ca²⁺-ATPase activity than dimethyl-celecoxib, as deduced from the half-maximum effect but dimethyl-celecoxib exhibited higher inhibition potency when Ca²⁺ transport was evaluated. Since Ca²⁺ transport was more sensitive to inhibition than Ca²⁺-ATPase activity the drugs under study caused Ca²⁺/P_i uncoupling. Dimethyl-celecoxib provoked greater uncoupling and the effect was dependent on drug concentration but independent of Ca²⁺-pump functioning. Dimethyl-celecoxib prevented Ca²⁺ binding by stabilizing the inactive Ca²⁺-free conformation of the pump. The effect on the kinetics of phosphoenzyme accumulation and the dependence of the phosphoenzyme level on dimethyl-celecoxib concentration were independent of whether or not the Ca²⁺–pump was exposed to the drug in the presence of Ca²⁺ before phosphorylation. This provided evidence of non-preferential interaction with the Ca²⁺-free conformation. Likewise, the decreased phosphoenzyme level in the presence of dimethyl-celecoxib that was partially relieved by increasing Ca²⁺ was consistent with the mentioned effect on Ca²⁺ binding. The kinetics of phosphoenzyme decomposition under turnover conditions was not altered by dimethyl-celecoxib. The dual effect of the drug involves Ca²⁺-pump inhibition and membrane permeabilization activity. The reported data can explain the cytotoxic and anti-proliferative effects that have been attributed to the celecoxib analog. Ligand docking simulation predicts interaction of celecoxib and dimethyl-celecoxib with the intracellular Ca²⁺ transporter at the inhibition site of hydroquinones.     

Singlet oxygen: a potential culprit in myocardial injury?

The purpose of this study was to explore the role of singlet oxygen in cardiovascular injury. To accomplish this objective, we investigated the effect of singlet oxygen [generated from photoactivation of rose-bengal] on the calcium transport and Ca²⁺-ATPase activity of cardiac sarcoplasmic reticulum and compared these results with those obtained by superoxide radical, hydrogen peroxide and hydroxyl radical. Isolated cardiac SR exposed to rose bengal (10 nM) irradiated at (560 nm) produced a significant inhibition of Ca ²⁺ uptake; from 2.27 ± 0.05 to 0.62 ± 0.05 µmol Ca⁺/mg.min (mean ± SE) (P < 0.01) and Ca²⁺-ATPase activity from 2.08 ± 0.05 µmol Pi/min. mg to 0.28 ± 0.04 µmol Pi/min. mg (mean ± SE) (P < 0.01). The inhibition of calcium uptake and Ca²⁺-ATPase activity by rose bengal derived activatedoxygen (singlet oxygen) was dependent on the duration of exposure and intensity of light. The singlet oxygen scavengers ascorbic acid and histidine significantly protected SR Ca²⁺-ATPase against rose bengal derived activated oxygen species but superoxide dismutase and catalase did not attenuate the inhibition. SDS-polyacrylamide gel electrophoresis of SR exposed to photoactivated rose bengal up to 14 min, demonstrated complete loss of Ca²⁺-ATPase monomer band which was significantly protected by histidine. Irradiation of rose bengal also caused an 18% loss of total sulfhydryl groups of SR. On the other hand, superoxide (generated from xanthine oxidase action on xanthine) and hydroxyl radical (0.5 mM H₂O₂ + Fe²⁺ -EDTA) as well as H₂O₂ (12 mM) were without any effect on the 97,000 dalton Ca²⁺-ATPase band ofsarcoplasmic reticulum. The results suggest that oxidative damage of cardiac sarcoplasmic reticulum may be mediated by singlet oxygen. This may represent an important mechanism by which the oxidative injury to the myocardium induces both a loss of tension development and arrhythmogenesis.     

Transmembrane Ca2+ gradient is essential for high anion transport activity of human erythrocytes

The role of a transmembrane Ca²⁺ gradient in anion transport by Band 3 of human resealed erythrocyte ghosts has been studied. The results show that a transmembrane Ca²⁺ gradient is essential for the conformation of erythrocyte Band 3 with higher anion transport activity. The dissipation of the transmembrane Ca²⁺ gradient by the ionophore A23187 inhibits the anion transport activity. The extent of this inhibition approaches 90% as the Ca²⁺ concentration on both sides of the ghost membrane is increased to 1.0 mM and half-maximum inhibition is observed at 0.25 mM Ca²⁺. Addition of ATP (0.4 mM) to the resealing medium can partly reestablish the transmembrane Ca²⁺ gradient by activation of Ca²⁺-ATPase and alleviate the inhibition to some extent. N-ethylmaleimide, an inhibitor of erythrocyte Ca²⁺-ATPase, prevents such restoration. Electron micrographs reveal that numerous larger intramembranous particles can be observed on the P-faces of freeze-fractured resealed ghosts in the absence of a transmembrane Ca²⁺ gradient.Abbreviations DPA dipicolinic acid - EITC eosin 5-isothiocyanate - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - TES N-Tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid - PMSF phenylmethyl-sulfonylfluoride - NEM N-ethylamaleimide - BSA bovine serum albumin - EGTA ethyleneglycol-bis (aminoethylether)-tetra-acetic acid - EITC-Band 3 Band 3 labeled with EITC - Ca_i Ca²⁺ inside resealed ghosts - Ca_o Ca²⁺ outside resealed ghosts     

Lysophospholipid-mediated alterations in the calcium transport systems of skeletal and cardiac muscle sarcoplasmic reticulum

Summary The effects of various lysophospholipids on the calcium transport activity of sarcoplasmic reticulum (SR) from rabbit skeletal and canine cardiac muscles were examined. The lipids decreased calcium transport activity in both membrane types; the effectiveness being in the order lysoPC > lsyoPS, lysoPG > lysoPE. The maximum inhibition induced by lysoPC, lysoPG and lysoPS was greater than 85% of the normal Ca²⁺-transport rate. In cardiac SR lysoPE had a maximal inhibition of about 50%. Half maximal inhibition of calcium transport by lysoPC was achieved at 110 nmoles lysoPC/mg SR. At this concentration of lysoPC, the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺-uptake activities were inhibited to the same extent (about 60%) in skeletal sarcoplasmic reticulum, while in cardiac sarcoplasmic reticulum, there was less than 20% inhibition of the Ca²⁺ + Mg²⁺-ATPase activity. Studies with EGTA-induced passive calcium efflux showed that up to 200 nmoles lysoPC/mg SR did not alter calcium permeability significantly in cardiac sarcoplasmic reticulum. In skeletal muscle membranes the lysophospholipid mediated decrease in calcium uptake correlated well with the increase in passive calcium efflux due to lysophosphatidylcholine. The difference in the lysophospholipid-induced effects on the sarcoplasmic reticulum from the two muscle types probably reflects variations in protein and other membrane components related to the respective calcium transport systems.     

Synthesis of the calcium transport ATPase of sarcoplasmic reticulum and other muscle proteins during development of muscle cells in vivo and in vitro

The effect of medium Ca²⁺ concentration upon the concentration and the rate of synthesis of muscle proteins was investigated in chicken pectoralis muscle cultures.There is an easily identifiable class of muscle protein which includes the Ca²⁺-ATPase of sarcoplasmic reticulum, myosin, troponin C, ATP : creatine phosphotransferase, muscle specific actin, tropomyosin 1 and 2, and muscle hemagglutinin, which show a large increase in concentration during normal development. The increased synthesis of these proteins was inhibited, without inhibition of cell proliferation, in culture media of relatively low Ca²⁺ concentration, 0.05–0.3 mM, where fusion was prevented. Similar medium Ca²⁺ concentration was required for the expression of all these proteins, suggesting their coordinate regulation. The proteins are denoted as ‘calcium-modulated proteins’. The increased Ca²⁺ transport activity of sarcoplasmic reticulum in cultured chicken pectoralis muscle cells during development at 1.8 mM medium calcium concentration represents de novo synthesis of the Ca²⁺ transport ATPase, as shown by immunoprecipitation, active site labeling and direct identification of the Ca²⁺ transport ATPase on two-dimensional gel electropherograms of whole muscle homogenates.The concentration and the turnover rate of the majority of the muscle proteins is not affected significantly by medium Ca²⁺ concentration between 0.06 and 1.8 mM.It is proposed that increase in cytoplasmic free Ca²⁺ concentration during fusion plays a central role in the regulation of the synthesis of calcium-modulated proteins.     

Effect of long chain unsaturated fatty acids on the calcium transport of sarcoplasmic reticulum

The effect of archidonic, oleic and linoleic acid on calcium uptake and release by sarcoplasmic reticulum isolated from longissimus dorsi muscle was investigated using a Ca²⁺ electrode. All three long chain fatty acids stimulated the release of Ca²⁺ from sacroplasmic reticulum when added after exogenous Ca²⁺ was accumulated by the vesicles, and also inhibited Ca²⁺ uptake when added before Ca²⁺. This inhibitory effect on the calcium transport by arachidonic, oleic and linoleic acid was prevented by bovine serum albumin through its ability to bind with the fatty acid. The order of effectiveness of the fatty acids in inhibiting calcium transport by isolated sarcoplasmic reticulum was $\text{arachidonic acid> oleic acid >}$ linoleic acid. Similar inhibition of calcium uptake and induction of calcium release by arachidonic acid was observed in muscle homogenate sarcoplasmic reticulum preparations. Both arachidonic and oleic acid stimulated the (Ca²⁺ + Mg²⁺)-ATPase activity of sarcoplasmic reticulum at low concentrations, but inhibited the (Ca²⁺ + Mg²⁺)-ATPase activity at high concentrations. The maximal (Ca²⁺ + Mg²⁺-ATPase activity observed with arachidonic acid was twice that obtained with oleic acid, but the concentration of arachidonic acid required was 3–4-times greater than that of oleic acid. The concentration of arachidonic acid required to give maximum stimulation of the (Ca²⁺ + Mg²⁺)-ATPase activity was 3.6-times greater than that needed for complete inhibition of calcium accumulation by the sacroplasmic reticulum. With oleic acid, however, the concentration required to give maximum stimulation of the (Ca²⁺ + Mg²⁺)-ATPase activity inhibited the sarcoplasmic reticulum Ca²⁺ accumulation by 72%. The present data support our hypothesis that, in porcine malignant hyperthermia, unsaturated fatty acids from mitochondrial membranes released by endogenous phospholipase A₂ would induce the sarcoplasmic reticulum to release calcium (Cheah K.S. and Cheah, A.M. (1981) Biochim. Biophys. Acta 634, 70–84).     

In vitro effects of toxaphene on mitochondrial calcium ATPase and calcium uptake in selected rat tissues

$\text{In vitro}$ effects of toxaphene on Ca²⁺-ATPase activity and ⁴⁵Ca²⁺-uptake were studied in mitochondrial fractions of heart, kidney and liver tissues of rat. Mitochondrial fractions were prepared by the conventional centrifugation method. Ca²⁺-ATPase activity was determined by measuring the inorganic phosphate liberated during ATP hydrolysis. Toxaphene inhibited Ca²⁺-ATPase in a concentration dependent manner in all the three tissues. Substrate activation kinetics, with heart, kidney and liver tissue fractions, revealed that toxaphene inhibited Ca²⁺-ATPase activity non-competetively by decreasing the maximum velocity of the enzyme without affecting the enzyme-substrate affinity. Toxaphene also inhibited mitochondrial ⁴⁵Ca²⁺-uptake in the three selected tissues in a concentration dependent manner. These results indicate that toxaphene is an inhibitor of mitochondrial Ca²⁺-ATPase and calcium transport in heart, kidney and liver tissues of rat.     

Involvement of Ca2+-stimulated adenosine 5′-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei

The regulatory role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ transport system of rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. The release of Ca²⁺ from the Ca²⁺-loaded nuclei was evoked progressively after Ca²⁺ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca²⁺ from the Ca²⁺-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca²⁺ uptake and induced a promotion of Ca²⁺ release from the Ca²⁺-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca²⁺-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca²⁺ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca²⁺ channels, did not prevent the NAD⁺ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca²⁺ release, suggesting that Ca²⁺ channels do not involve on Ca²⁺ release from the nuclei. These results indicates that an inhibition of nuclear Ca²⁺-ATPase activity causes the decrease in nuclear Ca²⁺ uptake and the release of Ca²⁺ from the Ca²⁺-loaded nuclei. The present finding suggests that Ca²⁺-ATPase plays a critical role in the regulatory mechanism of Ca²⁺ uptake and release in rat liver nuclei.     

Relationship between plasma membrane Ca2+-ATPase activity and acrosome reaction in guinea pig sperm

The results obtained by biochemical measurement demonstrated for the first time that significant decrease of the plasma membrane Ca²⁺-ATPase activity occurred during capacitation and acrosome reaction of guinea pig sperm. Ethaorynic acid, one kind of Ca²⁺-ATPase antagonists, inhibited the plasma membrane Ca²⁺-ATPase activity, but calmodulin (50μg/mL) and trifluoperazine (200- 500μmol/L) did not, suggesting that calmodulin is not involved in ATP-driven Ca²⁺ efflux from sperm. However, calmodulin is involved in the control of Ca²⁺ influx. TFP, one kind of calmodulin antagonists, accelerated the acrosome reaction and Ca²⁺ uptake into sperm cells significantly. Ca²⁺-ATPase antagonists, quercetin, sodium orthovandate, furosemide and ethacrynic acid promoted the acrosome reaction, but inhibited Ca2+ uptake, which cannot be explained by their inhibitory effects on the plasma membrane Ca²⁺-ATPase activity. It is speculated that this phenomenon might be caused by simultaneous inhibitions of the activities of Ca²⁺-ATPase present in the plasma membrane, the outer acrosome membrane and the outer mitochondrion membrane resulting in Ca²⁺ accumulation in the cytoplasm, which in turn blocks further Ca2+ entry through some negative feedback mechanism(s). The inhibitory effect of Ca²⁺-ATPase antagonist on glycolytic activity may also be the reason for Ca²⁺ accumulation in cytoplasm and inhibition of Ca²⁺ uptake.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: II. Mechanism of impairment in ATP-dependent Ca2+ transport

The role of the phosphorylation and dephosphorylation of sarcolemma and that of the alteration of membrane lipids in the endotoxin-induced impairment of the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results indicate that the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma was decreased by 30–35% 4h after endotoxin administration. Phosphorylation of sarcolemma by the catalytic subunit of the cAMP-dependent protein kinase or calmodulin stimulated ATP-dependent Ca²⁺ transport in both groups, however, the phosphorylation-stimulated activities remained significantly lower in endotoxic animals. Dephosphorylation of sarcolemma decreased ATP-dependent Ca²⁺ transport in both groups, yet, the time required to reach maximal dephosphorylation was reduced from 120 to 90 min 4 h post-endotoxin. Analysis of sarcolemmal membranes reveals that phosphatidylcholine and phosphatidylethanolamine contents were decreased while their respective lysophosphatide levels were increased significantly after endotoxin injection. Digestion of control heart sarcolemma with phospholipase A₂ inhibited Ca²⁺ transport and the inhibition was reversible by phosphatidylcholine. The inhibition caused by the in vivo administration of endotoxin was completely reversible by the addition of phosphatidylcholine. Based on these data, it is concluded that endotoxin administration impairs ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment may be due to i) a defective phosphorylation of sarcolemma; ii) a reduced number of Ca²⁺ pumps; iii) an accelerated dephosphorylation of sarcolemma; and iv) an alteration in membrane phospholipid profile in response to phospholipase A activation.     

Characterizing phospholamban to sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking

Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys²⁷ of PLB was cross-linked to the Ca²⁺ pump at the cytoplasmic extension of M4 (at or near Lys³²⁸) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 Å). Cross-linking was augmented by ATP but abolished by Ca²⁺ or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca²⁺ affinity conformation of the Ca²⁺-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the anti-PLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a and 2D12 stimulation of Ca²⁺-ATPase activity and Ca²⁺ transport. The results suggest that the inhibitory effect of PLB on Ca²⁺-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca²⁺ to the Ca²⁺ pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca²⁺ pump are largely unchanged in failing myocardium.     

Biochemical characteristics of the Ca2+ pumping ATPase in the peribacteroid membrane from broad bean root nodules

Ca²⁺-ATPase in the peribacteroid membrane (PBM) of symbiosomes isolated from Vicia faba root nodules was characterized in terms of its hydrolytic and transport activities. Both activities were found to be pH-dependent and exhibit pH optimum at pH 7.0. Translocation of Ca²⁺ through the PBM by the Ca²⁺-ATPase was shown to be fueled by ATP and other nucleotide triphosphates in the following order: ATP?>?ITP???GTP???UTP???CTP, the K _m of the enzyme for MgATP being about 100 μM. Ca-dependent ITP-hydrolytic activity of symbiosomes was investigated in the presence of the Ca-EGTA buffer system and showed the affinity of PBM Ca²⁺-ATPase for Ca²⁺ of about 0.1 μM. The transport activity of Ca²⁺-ATPase was inhibited by erythrosin B as well as orthovanadate, but markedly stimulated by calmodulin from bovine brain. These results allowed us to conclude that this enzyme belongs to IIB-type Ca²⁺-ATPases which are present in other plant membranes.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Calcium transport mechanism in crayfish gastrolith epithelium correlated with the molting cycle

Summary Periodical changes in Ca²⁺-ATPase and Mg²⁺-ATPase activity were observed cytochemically in the crayfish gastrolith epithelium during the molting cycle in relation to the calcium transport mechanism. The ATPase activity was demonstrated by a new one-step lead citrate method. The reaction products were mainly restricted to the matrix of type II cell mitochondria. The Ca²⁺-ATPase activity was intensely observed in two calcium moving stages, the small gastrolith period which indicates the beginning of gastrolith formation, and the aftermolt, when the calcified gastrolith has been dissolved in the stomach and then reabsorbed from the stomach epithelium into the newly formed soft exoskeleton through the blood. Although the intensity of reaction products of Mg²⁺-ATPase varied in each stage, the enzymatic activity was observed throughout all molting stages. Reaction products were observed in all mitochondria, basement membranes, apical cytoplasmic membranes, and in some lysosomes. In conclusion, periodical changes in the two types of ATPase activity were seen in the mitochondria of gastrolith epithelium during the molting cycle, but Ca²⁺-ATPase activity seemed to be more prominently synchronized to the calcium movement in the gastrolith epithelium than Mg²⁺-ATPase activity. These results provide the strong evidence that Ca²⁺-ATPase may act strongly in the calcium transport system of crayfish molting.     

Polyamine transport regulation by calcium and calmodulin: Role of Ca2+-ATPase

The study was conducted on human leukemia (K 562) cells to characterize the mechanisms implicated in the regulation of the polyamine spermicine (Spd) transport process. The antagonists of calmodulin, trifluoperazine (TFP), W-7 (N-[6-aminohexyl]-5-chloro-1-naphthelenesulfonamide), or mellitin inhiblted significantly polyamine Spd uptake in these cells. The translocation of calmodulin towards plasma membrane and a concomitant decrease in its contents in cytosol were directly correlated with the time course increases similar to that of Spd uptake, indicating that calmodulin is recruited towards plasma membrane during the Spd transport process. Diminution of free intracellular calcium, (Ca²⁺)i, by preincubating the cells in BAPTA (bis[2-amino-5-methylphenoxyl]-ethane-N,N′,N′,-tetraacetate) buffer inhibited Spd transport significantly. Addition of lanthanum (LAN), a molecule known to inhibit Ca² efflux via Ca²⁺-ATPase, curtailed Spd uptake by these cells. LAN inhibited Vmax, but not the Km, of Spd uptake, indicating that the former does not directly interact with the polyamine transporter; rather it regulates the transport process, probably via its action on Ca²⁺-ATPase. Calmodulin-stimulated uptake of ⁴⁵Ca²⁺ by inside-out vesicles of K 562 cells, a measure of Ca²⁺-ATPase activity. Furthermore, addition of LAN inhibited both basal and calmodulin-stimulated activity of Ca²⁺-ATPase. Thapsigargin (THAP), a molecule known to elevate (Ca²⁺) i due to its action on the endoplasmic reticulum, increased Spd transport whereas addition of LAN inhibited THAP-stimulated Spd transport activity. THAP increased free (Ca²⁺)i in these cells, and a pre-addition of LAN to these cells curtailed the THAP-stimulated increases of (Ca²⁺)i concentrations. Addition of Spd brought about elevations in (Ca²⁺)i contents. Caffeine also increased (Ca²⁺)i in these cells; however, it failed to stimulate significantly the Spd uptake process, indicating that (Ca²⁺)i which is involved in the regulation of polyamine transport pathways does not belong to the calcium-induced calcium-release (CICR) pool. Replacement of Ca²⁺ from the incubation medium (i.e., 0% Ca²⁺) resulted in higher uptake activity as compared to that in 100% Ca²⁺ medium, demonstrating that in 100% Ca²⁺ medium the calcium efflux process is quickly compensated by calcium refilling/influx from the extracellular medium, while in 0% Ca²⁺ medium there is perpetual efflux of (Ca²⁺)i which contributes to higher Spd uptake process. The results of this study suggest that an increase in free (Ca²⁺)i and its release from the cells via Ca²⁺ ATPase, and concomitant activation of calmodulin, which controls Ca²⁺-pump activity, are involved in the regulation of the Spd uptake process in human leukemia cells. © 1993 Wiley-Liss, Inc.     

The Mechanics of Calcium Transport

With the recent atomic models for the sarcoplasmic reticulum Ca²⁺-ATPase in the Ca²⁺-bound state, the Ca²⁺-free, thapsigargin-inhibited state, and the Ca²⁺-free, vanadate-inhibited state, we are that much closer to understanding and animating the Ca²⁺-transport cycle. These snapshots of the Ca²⁺-transport cycle reveal an impressive breadth and complexity of conformational change. The cytoplasmic domains undergo rigid-body movements that couple the energy of ATP to the transport of Ca²⁺ across the membrane. Large-scale rearrangements in the transmembrane domain suggest that the Ca²⁺-binding sites may alternately cease to exist and reform during the transport cycle. Of the three cytoplasmic domains, the actuator (A) domain undergoes the largest movement, namely a 110° rotation normal to the membrane. This domain is linked to transmembrane segments M1–M3, which undergo large rearrangements in the membrane domain. Together, these movements are a main event in Ca²⁺ transport, yet their significance is poorly understood. Nonetheless, inhibition or modulation of Ca²⁺-ATPase activity appears to target these conformational changes. Thapsigargin is a high-affinity inhibitor that binds to the M3 helix near Phe²⁵⁶, and phospholamban is a modulator of Ca²⁺-ATPase activity that has been cross-linked to M2 and M4. The purpose of this review is to postulate roles for the A domain and M1–M3 in Ca²⁺ transport and inhibition.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport

Effects of endotoxin administration on the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca²⁺ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V_max for ATP and for Ca²⁺ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca²⁺ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca²⁺ transport, but the Mg²⁺-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca²⁺. Additional experiments show that the Ca²⁺ sensitivity of the Ca²⁺-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca²⁺ pumping without affecting Ca²⁺-ATPase activity. Since sarcolemmal ATP-dependent Ca²⁺ transport plays an important role in the regulation of cytosolic Ca²⁺ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca²⁺ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.