Positions of the sites labeled by N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)carbodiimide on the (Ca2+ + Mg2+)-ATPase

N-Cyclohexyl-N'-(4-dimethylamino-1-naphthyl)carbodiimide (NCD-4) labels (Ca2+ + Mg2+)-ATPase at Ca2+-protectable sites, believed to be at or near the two Ca2+ binding sites on the ATPase, and at nonspecific sites. The labeled ATPase has been reconstituted into lipid bilayers containing phosphatidylethanolamine labeled with fluorescein isothiocyanate. The distance between NCD-4 and fluorescein groups was measured using Forster energy transfer and the NCD-4 labels were found to be approx. 20 A from the lipid/water interface suggesting that the Ca2+ binding sites on the ATPase are also 20 A from the lipid/water interface. Addition of vanadate causes no change in the efficiency of energy transfer, suggesting that the Ca2+ binding sites on the E1 conformation of the ATPase do not move significantly with respect to the lipid/water interface in the E1-E2 transition.     

Modification of the functional properties of sarcoplasmic reticulum Ca-ATPase in hypercholesterolemia

Using alamethicin, permitting the measurement of genuine catalytic enzyme activity, hypercholesterolemia was shown to cause a 10-30% reduction of specific Ca-ATPase activity registered at 37 degrees C and the shift of Arrhenius plot in 20-30 degrees C temperature range. Reconstruction of delipidated Ca-ATPase of sarcoplasmic reticulum membranes by egg lecithin in animals with hypercholesterolemia does not lead to the recovery of Arrhenius plot. The data obtained demonstrate that modification of temperature-dependent Ca-ATPase activity in hypercholesterolemia is associated with the changes in the polypeptide with a catalytic function and is not induced by the changes in phospholipid enzyme surroundings.     

Change in the molecular properties of sarcoplasmic reticulum Ca-ATPase during modification of the membranes with cholesterol

The thiol reagent NBD-chloride (4-chloro-7-nitro-benzo-2-oxo-1,3-diazole) was used to determine the amount and reactivity of SH-groups of Ca-ATPase of rat skeletal muscle sarcoplasmic reticulum during hypercholesterolemia. Modification of membranes with cholesterol brought about a decrease in the total amount and reactivity of SH-groups at the cost of reduction of rapid SH-groups and decrease of the modification constant of these SH-groups. The masking effect of high concentrations of ATP on the reactivity of SH-groups in hypercholesterolemia was noticed. It is inferred that the reduced efficacy of Ca-pump work found under the same experimental conditions before is a consequence of the modification of sarcoplasmic reticulum membranes with cholesterol and change in the molecular conformation of Ca-ATPase.     

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum vesicles was studied in a native and a fluorescein-labeled ATPase preparation (Pick, U., and Karlish, S. J. D. (1980) Biochim. Biophys. Acta 626, 255-261). Vanadate induced a fluorescence enhancement in a fluorescein-labeled enzyme, indicating that it shifts the equilibrium between the two conformational states of the enzyme by forming a stable E2-Mg-vanadate complex (E2 is the low affinity Ca2+ binding conformational state of the sarcoplasmic reticulum Ca-ATPase). Indications for tight binding of vanadate to the enzyme (K1/2 = 10 microM) in the absence of Ca2+ and for a slow dissociation of vanadate from the enzyme in the presence of Ca2+ are presented. The enzyme-vanadate complex was identified by the appearance of a time lag in the onset of Ca2+ uptake and by a slowing of the fluorescence quenching response to Ca2+. Ca2+ prevented the binding of vanadate to the enzyme. Pyrophosphate (Kd = 2 mM) and ATP (Kd = 25 microM) competitively inhibited the binding of vanadate, indicating that vanadate binds to the low affinity ATP binding site. Binding of vanadate inhibited the high affinity Ca2+ binding to the enzyme at 4 degrees C. Vanadate also inhibited the phosphorylation reaction by inorganic phosphate (Ki = 10 microM) but had no effect on the phosphorylation by ATP. It is suggested that vanadate binds to a special region in the low affinity ATP binding site which is exposed only in the E2 conformation of the enzyme in the absence of Ca2+ and which controls the rate of the conformation transition in the dephosphorylated enzyme. The implications of these results to the role of the low affinity ATP binding sites are discussed.     

Anesthetics alter the physical and functional properties of the Ca-ATPase in cardiac sarcoplasmic reticulum.

We have studied the effects of the local anesthetic lidocaine, and the general anesthetic halothane, on the function and oligomeric state of the CA-ATPase in cardiac sarcoplasmic reticulum (SR). Oligomeric changes were detected by time-resolved phosphorescence anisotropy (TPA). Lidocaine inhibited and aggregated the Ca-ATPase in cardiac SR. Micromolar calcium or 0.5 M lithium chloride protected against lidocaine-induced inhibition, indicating that electrostatic interactions are essential to lidocaine inhibition of the Ca-ATPase. The phospholamban (PLB) antibody 2D12, which mimics PLB phosphorylation, had no effect on lidocaine inhibition of the Ca-ATPase in cardiac SR. Inhibition and aggregation of the Ca-ATPase in cardiac SR occurred at lower concentrations of lidocaine than necessary to inhibit and aggregate the Ca-ATPase in skeletal SR, suggesting that the cardiac isoform of the enzyme has a higher affinity for lidocaine. Halothane inhibited and aggregated the Ca-ATPase in cardiac SR. Both inhibition and aggregation of the Ca-ATPase by halothane were much greater in the presence of PLB antibody or when PLB was phosphorylated, indicating a protective effect of PLB on halothane-induced inhibition and aggregation. The effects of halothane on cardiac SR are opposite from the effects of halothane observed in skeletal SR, where halothane activates and dissociates the Ca-ATPase. These results underscore the crucial role of protein-protein interactions on Ca-ATPase regulation and anesthetic perturbation of cardiac SR.     

Kinetics of proton binding to the sarcoplasmic reticulum Ca-ATPase in the E1 state

A new caged proton, 2-methoxy-5-nitrophenyl sulfate, was synthesized and used in time-resolved pH jump experiments to study proton binding in the sarcoplasmic reticulum Ca-ATPase. The major advantage of this compound is that it does not produce significant artifacts in experiments in which the fluorescent styryl dye 2BITC is used to monitor ion movements in the Ca pump. Two rate-limiting processes were resolved and their dependence on pH, Ca(2+) concentration, and temperature investigated. The faster process showed a relaxation time between 4 and 8 ms independent on pH and Ca(2+) concentration, and the time constant of the slower process varied between 31 ms (0 Ca(2+)) and 100 ms (100 microM Ca(2+)). A consistent mechanism to explain the results was derived in agreement with previous studies and the generally accepted Post-Albers scheme of the pump cycle. This mechanism requires that under physiological conditions the ion-binding sites are always occupied and two protons and a Ca(2+) ion replace each other. In the absence of ATP at low pH a nonphysiological state can be induced in which up to four protons bind to the Ca pump in the E(1) conformation. So far it could not be verified whether these additional protons bind to amino acid side chains or are coordinated as hydronium ions.     

Ca-ATPase self-fluorescence in the sarcoplasmic reticulum of the rabbit skeletal muscle

The quenching of the intrinsic protein fluorescence of sarcoplasmic reticulum Ca-ATPase from the rabbit skeletal muscles by hydrophylic (NaI, CsCl) or hydrophobic (pyrene, fluorescamine) substances has been studied. CsCl (up to 1 M) has been shown not to affect the intrinsic protein fluorescence while NaI (250 mM) quenches it at 15%, pyrene (8 mkM) decreases the intrinsic fluorescence of Ca-ATPase at 35% and fluorescamine (up to 40 mkM)--at 80%. Possible mechanisms of the interaction of the quenchers with the intrinsic fluorescence of sarcoplasmic reticulum Ca-ATPase are being discussed.     

Competitive inhibition of Ca-ATPase in the sarcoplasmic reticulum by sodium fluoride

Effect of NaF and AlCl3 the activity of the sarcoplasmic reticulum Ca-ATP-ase has been investigated. NaF (mM) completely inhibits the Ca-ATP-ase activity in presence of 0.02% tween-20. The inhibition is time- and NaF-concentration-dependent and increases as affected by AlCl3 (microM). The potentiated action of AlCl3 depends on the NaF concentration. AlCl3 without NaF does not change the Ca-ATP-ase activity. NaF inhibits the Ca-ATP-ase competitively with respect to ATP, but NaF plus AlCl3 make the inhibition combined. The affinity of the Ca-ATP-ase to the NaF + AlCl3 complex, but not to NaF decreases by 5 mM with an increase of the Pi concentration. NaF probably interacts with the ATP-binding site and the NaF + AlCl3 complex interacts with the phosphate-binding site of the ATP-ase.     

Functional role of the oligomeric organization of Ca-ATPase of the sarcoplasmic reticulum

The analysis of the present-day concepts on the possible physiological role of the oligomeric organization of Ca-ATPase sarcoplasmic reticulum is given. According to the proposed conception the main functional role of the protein-protein interactions is connected with the possibilities of regulation Ca2+ outflux from the lumens reticulum in the region of interprotein contacts.     

Excimer formation of ATPase from sarcoplasmic reticulum labeled with N-(3-pyrene)maleinimide

Sarcoplasmic reticulum ATPase from fast skeletal muscle was labeled in native vesicles with N-(3-pyrene)maleinimide. At labeling ratios larger than 1 mol pyrenemaleinimide/2.5 mol ATPase significant amounts of excimers are detected. Excimer concentration decreases at low, non-solubilizing amounts of detergents (0.2 mg X mg protein-1) and completely disappears after solubilization of the membranes. These results exclude that excimers are formed due to 'double-labeling' of one ATPase molecule. It is concluded that the ATPase exists as an oligomer within the membrane of native vesicles.     

Decreased conformational stability of the sarcoplasmic reticulum Ca-ATPase in aged skeletal muscle

Sarcoplasmic reticulum (SR) membranes purified from young adult (4–6 months) and aged (26–28 months) Fischer 344 male rat skeletal muscle were compared with respect to the functional and structural properties of the Ca-ATPase and its associated lipids. While we find no age-related alterations in (1) expression levels of Ca-ATPase protein, and (2) calcium transport and ATPase activities, the Ca-ATPase isolated from aged muscle exhibits more rapid inactivation during mild (37°C) heat treatment relative to that from young muscle. Saturation-transfer EPR measurements of maleimide spin-labeled Ca-ATPase and parallel measurements of fatty acyl chain dynamics demonstrate that, accompanying heat inactivation, the Ca-ATPase from aged skeletal muscle more readily undergoes self-association to form inactive oligomeric species without initial age-related differences in association state of the protein. Neither age nor heat inactivation results in differences in acyl chain dynamics of the bilayer including those lipids at the lipid-protein interface. Initial rates of tryptic digestion associated with the Ca-ATPase in SR isolated from aged muscle are 16( ± 2)% higher relative to that from young muscle, indicating more solvent exposure of a portion of the cytoplasmic domain. During heat inactivation these structural differences are amplified as a result of immediate and rapid further unfolding of the Ca-ATPase isolated from aged muscle relative to the delayed unfolding of the Ca-ATPase isolated from young muscle. Thus age-related alterations in the solvent exposure of cytoplasmic peptides of the Ca-ATPase are likely to be critical to the loss of conformational and functional stability.     

Common structural domains in the sarcoplasmic reticulum Ca-ATPase and the transverse tubule Mg-ATPase

Transverse tubule (TT) membranes isolated from chicken skeletal muscle possess a very active magnesium-stimulated ATPase (Mg-ATPase) activity. The Mg-ATPase has been tentatively identified as a 102-kD concanavalin A (Con A)-binding glycoprotein comprising 80% of the integral membrane protein (Okamoto, V.R., 1985, Arch. Biochem. Biophys., 237:43-54). To firmly identify the Mg-ATPase as the 102-kD TT component and to characterize the structural relationship between this protein and the closely related sarcoplasmic reticulum (SR) Ca-ATPase, polyclonal antibodies were raised against the purified SR Ca-ATPase and the TT 102-kD glycoprotein, and the immunological relationship between the two ATPases was studied by means of Western immunoblots and enzyme-linked immunosorbent assays (ELISA). Anti-chicken and anti-rabbit SR Ca-ATPase antibodies were not able to distinguish between the TT 102-kD glycoprotein and the SR Ca-ATPase. The SR Ca-ATPase and the putative 102-kD TT Mg-ATPase also possess common structural elements, as indicated by amino acid compositional and peptide mapping analyses. The two 102-kD proteins exhibit similar amino acid compositions, especially with regard to the population of charged amino acid residues. Furthermore, one-dimensional peptide maps of the two proteins, and immunoblots thereof, show striking similarities indicating that the two proteins share many common epitopes and peptide domains. Polyclonal antibodies raised against the purified TT 102-kD glycoprotein were localized by indirect immunofluorescence exclusively in the TT-rich I bands of the muscle cell. The antibodies substantially inhibit the Mg-ATPase activity of isolated TT vesicles, and Con A pretreatment could prevent antibody inhibition of TT Mg-ATPase activity. Further, the binding of antibodies to intact TT vesicles could be reduced by prior treatment with Con A. We conclude that the TT 102-kD glycoprotein is the TT Mg-ATPase and that a high degree of structural homology exists between this protein and the SR Ca-ATPase.     

Effect of phenothiazines on Ca-ATPase in the sarcoplasmic reticulum of rabbit skeletal muscles

The phenothiazines trifluoroperazine , chlorpromazine and etmozine inhibit Ca-ATPase of the sarcoplasmic reticulum of rabbit skeletal muscles. The inhibitory action decreases in the order of trifluoroperazine greater than chlorpromazine greater than etmozine . The data are provided, indicating that the inhibitory effects of the phenothiazines on Ca-ATPase of the reticulum of the skeletal muscles are not mediated via calmodulin.     

Fluorescent staining of microfilaments with heavy meromyosin labeled with N-(7-dimethylamino-4-methylcoumarinyl) maleimide

For fluorescent staining of microfilaments in cells, heavy meromyosin (HMM) or subfragment-1 (S-1) was labeled with a novel thiol-directed fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide (DACM), instead of the usual dyes, such as fluorescein-isothiocyanate (FITC). DACM-labeled HMM or S-1 gave characteristic fluorescence patterns to a variety of cell types similar to those reported with the use of FITC-labeled HMM or S-1 or with immunofluorescence techniques using anti-actin antibody. The fluorescence of DACM was fairly photoresistant as compared with FITC, so that HMM or S-1 required only 1 mol of the dye per myosin head. Consequently, F-actin need not be used to preserve the actin binding activity of the myosin fragments when labeling with the dye.     

Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.

The role of ATP-dependent calcium uptake into intracellular storage compartments is an essential feature of hormonally induced calcium signaling. Thapsigargin, a non-phorboid tumor promoter, increasingly is being used to manipulate calcium stores because it induces a hormone-like elevation of cytosolic calcium. It has been suggested that thapsigargin acts through inhibition of the endoplasmic reticulum calcium pump. We have directly tested the specificity of thapsigargin on all of the known intracellular-type calcium pumps (referred to as the sarcoplasmic or endoplasmic reticulum Ca-ATPase family (SERCA]. Full-length cDNA clones encoding SERCA1, SERCA2a, SERCA2b, and SERCA3 enzymes were expressed in COS cells, and both calcium uptake and calcium-dependent ATPase activity were assayed in microsomes isolated from them. Thapsigargin inhibited all of the SERCA isozymes with equal potency. Furthermore, similar doses of thapsigargin abolished the calcium uptake and ATPase activity of sarcoplasmic reticulum isolated from fast twitch and cardiac muscle but had no influence on either the plasma membrane Ca-ATPase or Na,K-ATPase. The interaction of thapsigargin with the SERCA isoforms is rapid, stoichiometric, and essentially irreversible. These properties demonstrate that thapsigargin interacts with a recognition site found in, and only in, all members of the endoplasmic and sarcoplasmic reticulum calcium pump family.     

The modulation of Ca-ATPase activity and protein-lipid interactions in the sarcoplasmic reticulum by ATP

The time-course of ATP hydrolysis by Ca-ATPase of purified sarcoplasmic reticulum is biphasic with an initial rate over 1 to 2 min exceeding the subsequent rate. Hydrolysis of GTP and p-nitrophenylphosphate (pNPP) occurs at a slower but constant rate. Arrhenius plots of GTP, p-nitrophenylphosphate and initial rates of ATP hydrolysis all exhibit a discontinuity at about 20-24 degrees C; no breaks are observed in plots of the slower phase of ATP hydrolysis. The effect of substrate hydrolysis on the disposition of the enzyme in the membrane was examined by monitoring the quenching of tryptophan fluorescence by pyrene present in the hydrophobic domain of the membrane. The presence of ATP, but not GTP, prevents a temperature-dependent decrease in fluorescence quenching suggesting that ATP binding causes a change in the protein domain in contact with the membrane lipids.     

Rotational dynamics of the Ca-ATPase in sarcoplasmic reticulum studied by time-resolved phosphorescence anisotropy

We have investigated the microsecond rotational motions of the Ca-ATPase in rabbit skeletal sarcoplasmic reticulum (SR), by measuring the time-resolved phosphorescence anisotropy of erythrosin 5-isothiocyanate (ERITC) covalently and specifically attached to the enzyme. Over a wide range of solvent conditions and temperatures, the phosphorescence anisotropy decay was best fit by a sum of three exponentials plus a constant term. At 4 degrees C, the rotational correlation times were phi 1 = 13 +/- 3 microseconds, phi 2 = 77 +/- 11 microseconds, and phi 3 = 314 +/- 23 microseconds. Increasing the solution viscosity with glycerol caused very little effect on the correlation times, while decreasing the lipid viscosity with diethyl ether decreased the correlation times substantially, indicating that the decay corresponds to rotation of the protein within the membrane, not to vesicle tumbling. The normalized residual anisotropy (A infinity) is insensitive to viscosity and temperature changes, supporting the model of uniaxial rotation of the protein about the membrane normal. The value of A infinity (0.20 +/- .02) indicates that each of the three decay components can be analyzed as a separate rotational species, with the preexponential factor Ai equal to 1.25X the mole fraction. An empirically accurate measurement of the membrane lipid viscosity was obtained, permitting a theoretical analysis of the correlation times in terms of the sizes of the rotating species. At 4 degrees C, the dominant correlation time (phi 3) is too large for a Ca-ATPase monomer, strongly suggesting that the enzyme is primarily aggregated (oligomeric).(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of phenothiazines on the sarcoplasmic reticulum Ca-ATPase from skeletal and cardiac muscles

Phenothiazines--trifluoperazine, chloropromazine and ethmozine-- inhibit the sarcoplasmic reticulum Ca-ATPase from skeletal and cardiac muscles of the rabbit. The inhibition constants for both preparations are of the same order of magnitude. The experimental data suggest that the effect of phenothiazine on the sarcoplasmic reticulum Ca-ATPase is not mediated by CaM, but is directed toward the enzyme molecule.     

Endoplasmic reticulum of rat liver contains two proteins closely related to skeletal sarcoplasmic reticulum Ca-ATPase and calsequestrin

Rat liver endoplasmic reticulum (ER) membranes were investigated for the presence of proteins having structural relationships with sarcoplasmic reticulum (SR) proteins. Western immunoblots of ER proteins probed with polyclonal antibodies raised against the 100-kDa SR Ca-ATPase of rabbit skeletal muscle identified a single reactive protein of 100 kDa. Also, the antibody inhibited up to 50% the Ca-ATPase activity of isolated ER membranes. Antisera raised against the major intraluminal calcium binding protein of rabbit skeletal muscle SR, calsequestrin (CS), cross-reacted with an ER peptide of about 63 kDa, by the blotting technique. Stains-All treatment of slab gels showed that the cross-reactive peptide stained metachromatically blue, similarly to SR CS. Two-dimensional electrophoresis (Michalak, M., Campbell, K. P., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 1317-1326) of ER proteins showed that the CS-like component of liver ER, similarly to skeletal CS, fell off the diagonal line, as expected from the characteristic pH dependence of the rate of mobility of mammalian CS. In addition, the CS-like component of liver ER was released from the vesicles by alkaline treatment and was found to be able to bind calcium, by a 45Ca overlay technique. From these findings, we conclude that a 100-kDa membrane protein of liver ER is the Ca-ATPase, and that the peripheral protein in the 63-kDa range is closely structurally and functionally related to skeletal CS.     

Nitric oxide-dependent modification of the sarcoplasmic reticulum Ca-ATPase: localization of cysteine target sites

Skeletal muscle contraction and relaxation is modulated through the reaction of sarcoplasmic reticulum (SR) protein thiols with reactive oxygen and nitrogen species. Here, we have utilized high-performance liquid chromatography-electrospray mass spectrometry and a specific thiol-labeling procedure to identify and quantify cysteine residues of the SR Ca-ATPase that are modified by exposure to nitric oxide (NO). NO and/or NO-derived species inactivate the SR Ca-ATPase and modify a broad spectrum of cysteine residues with highest reactivities towards Cys364, Cys670, and Cys471. The selectivity of NO and NO-derived species towards the SR Ca-ATPase thiols is different from that of peroxynitrite. The efficiency of NO at thiol modification is significantly higher compared with that of peroxynitrite. Hence, NO has the potential to modulate muscle contraction through chemical reaction with the SR Ca-ATPase in vivo.