Altered property of sarcoplasmic Ca-ATPase from vitamin E-deficient dystrophic rabbit is associated with the protein and not the lipid component

The effects of temperature on reconstituted sarcoplasmic Ca-ATPase preparations from vitamin E-deficient dystrophic and control rabbits were studied. Delipidated Ca-ATPase from vitamin E-deficient sarcoplasmic reticulum (SR) reconstituted with lipid of control SR exhibited properties similar to preparations reconstituted with lipid of vitamin E-deficient SR, namely low Ca-ATPase activity and a linear Arrhenius plot of enzyme activity. On the other hand, delipidated control SR Ca-ATPase reconstituted with lipid of vitamin E-deficient SR showed a reduction in activity but retained the discontinuity in the Arrhenius plot. These results indicated that the altered property of sarcoplasmic Ca-ATPase from vitamin E-deficient dystrophic rabbit was associated with the protein and not the lipid component.     

The physical mechanism of calcium pump regulation in the heart.

The Ca-ATPase in the cardiac sarcoplasmic reticulum membrane is regulated by an amphipathic transmembrane protein, phospholamban. We have used time-resolved phosphorescence anisotropy to detect the microsecond rotational dynamics, and thereby the self-association, of the Ca-ATPase as a function of phospholamban phosphorylation and physiologically relevant calcium levels. The phosphorylation of phospholamban increases the rotational mobility of the Ca-ATPase in the sarcoplasmic reticulum bilayer, due to a decrease in large-scale protein association, with a [Ca2+] dependence parallel to that of enzyme activation. These results support a model in which phospholamban phosphorylation or calcium free the enzyme from a kinetically unfavorable associated state.     

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.     

Two populations of phospholipids exist in sarcoplasmic reticulum and in recombined membranes containing Ca-ATPase

Phosphorus nuclear magnetic resonance spectra of sarcoplasmic reticulum membranes from rabbit muscle and of recombined membranes containing the calcium-dependent adenosinetriphosphatase (Ca-ATPase) of sarcoplasmic reticulum reveal two distinguishable, overlapping resonances. One resonance resembles a normal phospholipid bilayer resonance, and the other is much broader. The broader component is not seen in protein-free phospholipid vesicles. In recombined membranes of the Ca-ATPase, the intensity found in the broad component was proportional to the concentration of protein in the vesicles. The two-component spectra are interpreted to arise from at least two different domains of phospholipids, one of which is motionally restricted by the Ca-ATPase. Phospholipids exchange between these two domains at a rate less than 10(3) s-1. A model for protein-lipid interactions in membranes containing the Ca-ATPase is proposed in which some of the phospholipid head groups of the membrane interact directly with the protein.     

Immunochemical quantification of sarcoplasmic reticulum Ca-ATPase, of calsequestrin and of parvalbumin in rabbit skeletal muscles of defined fiber composition

Antibodies directed against purified Ca-ATPase from sarcoplasmic reticulum, calsequestrin and parvalbumin from rabbit fast-twitch muscle were raised in sheep. The specificity of the antibodies was shown by immunoblot analysis and by enzyme-linked immunoadsorbent assays (ELISAs). IgG against the sarcoplasmic reticulum Ca-ATPase inhibited the catalytic activities of Ca-ATPase from fast-twitch (psoas, tibialis anterior) and slow-twitch (soleus) muscles to the same degree. In non-equilibrium competitive ELISAs the anti(Ca-ATPase) IgG displayed a slightly higher affinity for the Ca-ATPase from fast-twitch muscle than for that from slow-twitch muscle. This suggests a fiber-type-specific polymorphism of the sarcoplasmic reticulum Ca-ATPase. Quantification of Ca-ATPase, calsequestrin and parvalbumin in various rabbit skeletal muscles of histochemically determined fiber composition was achieved by sandwich ELISA. Ca-ATPase was found to be 6-7 times higher in fast than in slow-twitch muscles. A slightly higher concentration was found in fast-twitch muscles with a higher percentage of IIb fibers when compared with fast-twitch muscles with a higher percentage of IIa fibers. Thus Ca-ATPase is distributed as follows, IIb greater than or equal to IIa much greater than I. Calsequestrin was uniformly distributed in fast-twitch muscles independently of their IIa/IIb fiber ratio and displayed 50% lower concentrations in slow than in fast-twitch muscles (IIb = IIa greater than I). Parvalbumin contents were 200-300-fold higher in fast than in slow-twitch muscles. Significantly lower parvalbumin concentrations were found in fast-twitch muscles with a higher percentage of IIa fibers than in fast-twitch muscles with a higher percentage of IIb fibers (IIb greater than IIa much greater than I).     

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.     

Ascorbic acid-Fe2+ treatment mimics effect of vitamin E deficiency on sarcoplasmic Ca-ATPase of rabbit muscle

After 90 min treatment with ascorbic acid and FeSO4 at 4 degrees C, the activity of rabbit sarcoplasmic reticulum Ca-ATPase was reduced to 22% and the Arrhenius plot of enzyme activity showed an absence of a discontinuity. The presence of vitamin E restored enzyme activity (60%) and the discontinuity in the Arrhenius plot. Ca-ATPase reconstituted with delipidated protein from ascorbic acid-Fe-treated preparation and normal lipid exhibited properties similar to the intact treated enzyme, whereas that reconstituted with delipidated normal protein and lipid from treated preparation exhibited reduced activity but retained the Arrhenius discontinuity. These properties are similar to those observed for sarcoplasmic reticulum Ca-ATPase from the vitamin E-deficient muscular dystrophic rabbit.     

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.     

Energy-dependent transport of Ca2+ and lipid peroxidation in the sarcoplasmic reticular membranes of rats during hypokinesia

Thirty-day hypokinesia in Wistar rats did not affect the rate of Ca2+ transport and the activity of Ca-ATPase in light and heavy fractions of sarcoplasmic reticulum of primarily white muscles. As hypokinesia was raised up to 90 days, these indicators increased. The intensity of lipid peroxidation in sarcoplasmic reticulum was lower in hypokinetic animals.     

Effect of urea on conformational changes in the active site of native and reconstituted Ca-ATPase of the sarcoplasmic reticulum]

The effect of urea (1-3 M) on conformational changes in the active center of native and reconstituted Ca-ATPase of sarcoplasmic reticulum modified by fluorescein-5-isothiocyanate (FITC) was studied using the method of fluorescence titration by neodymium (Nd3+) ions. Based on the analysis of curves of fluorescence quenching of FITC-labeled Ca-ATPase by Nd3+ ions, the parameters characterizing the structural changes in the Mg-ATP binding center were determined. It was assumed that FITC and Nd3+ ion bind to different polypeptide fragments moving relative to each other, which provides the conformational lability of the nucleotide binding site at some stages of the catalytic cycle. A comparison of structural changes caused by urea at the active site of native and reconstituted Ca-ATPase of sarcoplasmic reticulum indicates that the Nd3+ binding center is localized in the region of contacts of monomers in the oligomer.     

Ability of nucleoside triphosphates to provide for Ca 2+ transport by sarcoplasmic reticulum fragments

The Ca2+ transport by sarcoplasmic reticulum fragments was studied. ATP, CTP, ITP, GTP and UTP provided the same Ca-pump efficiency. When the NTP was exhausted, Ca2+ actively accumulated from the sarcoplasmic reticulum vesicles outflow, and with the higher rate of ATP was a substrate. The Ca-ATPase conformational transitions induced by ATP are discussed for their role in the provision of energy.     

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.     

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.     

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.     

Spin labels in the analysis of Ca-ATPase in the sarcoplasmic reticulum of rabbit skeletal muscles in hypercholesteremia

The method of electron paramagnetic resonance with spin-labeled maleimide was used to study variation of the structure of Ca-ATPase of the sarcoplasmic reticulum (SR) in rabbit skeletal muscles under long-term hypercholesterolemia (HC). The rate of the maleimide spin label binding with Ca-ATPase of the SR was decreased in HC, which correlated with a lesser access of spin-labeled thiol groups for potassium ferricyanide and sodium ascorbate. HC led to a considerable reduction in the lability and to enhancement of hydrophobia of the spin-labeled fragment of the enzyme. It is concluded that the disordered function of the SR Ca-pump is a consequence of structural changes in the Ca-ATPase molecule in HC.     

Thyroid hormone differentially affects mRNA levels of Ca-ATPase isozymes of sarcoplasmic reticulum in fast and slow skeletal muscle

mRNA levels for the type I and type II isoforms of sarcoplasmic reticulum (SR) Ca-ATPase were determined in soleus (SOL) and extensor digitorum longus (EDL) muscle of euthyroid (normal), hypothyroid, and hyperthyroid rats. Total Ca-ATPase mRNA content of hyperthyroid muscle was 1.5-fold (EDL) and 6-fold (SOL) higher compared to hypothyroid muscle, with corresponding increases in total SR Ca-ATPase activity. EDL contained only type II Ca-ATPase mRNA. In SOL type I mRNA was the major form in hypothyroidism (98%), but the type II mRNA content was stimulated 150-fold by T3, accounting for 50% of the Ca-ATPase mRNA in hyperthyroidism.     

The effect of ionizing radiation on the structure of the hydrophobic fragment of Ca-ATPase in skeletal muscle sarcoplasmic reticulum

Early (1 and 24 h) after X-irradiation with a dose of 0.21 C/kg changes occurred in the acceptability of the polypeptide chain parts of sarcoplasmic reticulum Ca-ATPase for the effect of trypsin. The analysis of the results of studying the structural and functional properties of a hydrophobic fragment of this enzyme in the control and after irradiation permitted to define the part of the Ca-ATPase polypeptide chain that provided ion selectivity of the fragment.     

Radiation inactivation analysis of sarcoplasmic reticulum Ca-ATPase in membrane-bound form and in detergent-solubilized monomeric states

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.