Correlation of structure and function in the Ca2+-ATPase of sarcoplasmic reticulum: a Fourier transform infrared spectroscopy (FTIR) study on the effects of dimethyl sulfoxide and urea

The effect of dimethyl sulfoxide (DMSO) on the structure of sarcoplasmic reticulum was analyzed by Fourier transform infrared (FTIR) and fluorescence spectroscopy. Exposure of sarcoplasmic reticulum vesicles to 35% DMSO (v/v) at 2 degrees C for several hours in a D2O medium produced no significant change in the phospholipid and protein Amide I regions of the FTIR spectra, but the intensity of the Amide II band decreased, presumably due to proton/deuterium exchange. At 40% to 60% DMSO concentration a shoulder appeared in the FTIR spectra at 1630 cm-1, that is attributed to the formation of new beta or random coil structures; irreversible loss of ATPase activity accompanied this change. At 70% DMSO concentration the intensity of the main Amide I band at 1639 cm-1 decreased and a new band appeared at 1622 cm-1, together with a shoulder at 1682 cm-1. These changes indicate an abrupt shift in the conformational equilibrium of Ca2+-ATPase from alpha to beta structure or to a new structure characterized by weaker hydrogen bonding. Decrease of ionization of aspartate and glutamate carboxyl groups in the presence of DMSO may also contribute to the change in intensity at 1622 cm-1. The changes were partially reversed upon removal of DMSO. Exposure of sarcoplasmic reticulum vesicles to 1.5 kbar pressure for 1 h at 2 degrees C in an EGTA-containing (low Ca2+) medium causes irreversible loss of ATPase activity, with the appearance of new beta structure, and abolition of the Ca2+-induced fluorescence response of FITC covalently bound to the Ca2+-ATPase; DMSO (35%) stabilized the Ca2+-ATPase against pressure-induced changes in structure and enzymatic activity, while urea (0.8 M) had the opposite effect.     

Infrared spectroscopic signals arising from ligand binding and conformational changes in the catalytic cycle of sarcoplasmic reticulum calcium ATPase.

Fourier transform infrared spectroscopy was used to investigate ligand binding and conformational changes in the Ca2(+)-ATPase of sarcoplasmic reticulum during the catalytic cycle. The ATPase reaction was started in the infrared sample by release of ATP from the inactive, photolabile ATP derivative P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP). Absorption spectroscopy in the visible spectral region using the Ca2(+)-sensitive dye Antipyrylazo III ensured that the infrared samples were able to transport Ca2+ in spite of their low water content, which is required for mid-infrared measurements (1800-950 cm-1). Small, but characteristic and highly reproducible infrared absorbance changes were observed upon ATP release. These infrared absorbance changes exhibit different kinetic properties. Comparison with model compound infrared spectra indicates that they are related to photolysis of caged ATP, hydrolysis of ATP in consequence of ATPase activity and to molecular changes in the active ATPase. The absorbance changes due to alterations in the ATPase were observed mainly in the region of Amide I and Amide II protein absorbance and presumably reflect the molecular processes upon phosphoenzyme formation. Since the absorbance changes were small compared to the overall ATPase absorbance, no major rearrangement of ATPase conformation as the result of catalysis could be detected.     

A time-resolved Fourier transformed infrared difference spectroscopy study of the sarcoplasmic reticulum Ca(2+)-ATPase: kinetics of the high-affinity calcium binding at low temperature.

We have used time-resolved Fourier transformed infrared difference spectroscopy to characterize the amplitude, frequency, and kinetics of the absorbance changes induced in the infrared (IR) spectrum of sarcoplasmic reticulum Ca(2+)-ATPase by calcium binding at the high-affinity transport sites. 1-(2-Nitro-4,5-dimethoxyphenyl)-N,N,N',N'-tetrakis [(oxycarbonyl)methyl]-1,2-ethanediamine (DM-nitrophen) was used as a caged-calcium compound to trigger the release of calcium in the IR samples. Calcium binding to Ca(2+)-ATPase induces the appearance of spectral bands in difference spectra that are all absent in the presence of the inhibitor thapsigargin. Spectral bands above 1700 cm-1 indicate that glutamic and/or aspartic acid side chains are deprotonated upon calcium binding, whereas other bands may be induced by reactions of asparagine, glutamine, and tyrosine residues. Some of the bands appearing in the 1690-1610 cm-1 region arise from modifications of peptide backbone carbonyl groups. The band at 1653 cm-1 is a candidate for a change in an alpha-helix, whereas other bands could arise from modifications of random, turn, or beta-sheet structures or from main-chain carbonyl groups playing the role of calcium ligands. Only a few residues are involved in secondary structure changes. The kinetic evolution of these bands was recorded at low temperature (-9 degrees C). All bands exhibited a monophasic kinetics of rate constant 0.026 s-1, which is compatible with that measured in previous study at the same temperature in a suspension of sarcoplasmic reticulum vesicles by intrinsic fluorescence of Ca(2+)-ATPase.     

Optical probe responses on sarcoplasmic reticulum: oxacarbocyanines as probes of membrane potential.

The relationship between Ca2+ fluxes and the ion diffusion potential was analyzed on sarcoplasmic reticulum membranes using oxacarbocyanine dyes as optical probes for membrane potential. 3.3'-Diethyloxodicarbocyanine responds to ATP-induced Ca2+ uptake by isolated sarcoplasmic reticulum vesicles with a decrease in absorbance at 600 nm. The optical change is reversed during Ca2+ release from sarcoplasmic reticulum induced by KCl or by ADP and inorganic phosphate. The absorbance changes are largely attributable to the binding of accumulated Ca2+ to the membrane. There is no indication that sustained changes in membrane diffusion potential would accompany pump-mediated Ca2+ fluxes. A large change in the absorbance of 3,3'-diethyloxodicarbocyanine was observed on sarcoplasmic reticulum vesicles under the influence of membrane potential generated by valinomycin in the presence of a K+ gradient or by ionophore A23187 in the presence of a Ca2+ gradient. The maximum of the potential-dependent absorbance change is at 575--580 nm. The potentials generated by valinomycin or ionophore A23187 are short-lived due to the high permeability of sarcoplasmic reticulum membranes for cations and anions. There is no correlation between the direction and magnitude of the artifically imposed membrane potential and the rate of Ca2+ uptake or release by isolated sarcoplasmic reticulum vesicles.     

Rapid filtration studies of Ca2+-induced Ca2+ release from skeletal sarcoplasmic reticulum. Role of monovalent ions

We have developed a rapid filtration technique for the measurement of Ca2+ release from isolated sarcoplasmic reticulum vesicles. Using this technique, we have studied the Ca2+-induced Ca2+ release of sarcoplasmic reticulum vesicles from rabbit skeletal muscle passively loaded with 5 mM Ca2+. The effect of known effectors (adenine nucleotides and caffeine) and inhibitors (Mg2+ and ruthenium red) of this release were investigated. In a medium composed of 100 mM KCl buffered at pH 6.8 with 20 mM K/3-(N-morpholino)propanesulfonic acid the Ca2+ release rate was maximal (500 nmol of Ca2+ released.(mg of protein)-1.s-1) at 1 micron external Ca2+ and 5 mM ATP. We also observed a rapid Ca2+ release induced by micromolar Ag+ in the presence of ATP (at 1 nM Ca2+). The Ag+-induced Ca2+ release was totally inhibited by 5 micron ruthenium red. We have also investigated the effect of monovalent ions on the Ca2+ release elicited by Ca2+ or Ag+. We show that the Ca2+ release rate: 1) was dependent upon the presence of K+ or Na+ in the release medium and 2) was influenced by a K+ gradient created across the sarcoplasmic reticulum membrane. These results directly support the idea of the involvement of an influx of K+ (through K+ channels) during the Ca2+ release and allow to reconsider a possible influence of the membrane potential of the sarcoplasmic reticulum on the Ca2+ release.     

Infrared spectroscopic characterization of the structural changes connected with the E1----E2 transition in the Ca2+-ATPase of sarcoplasmic reticulum

The Ca2+-transporting ATPase (EC 3.6.1.38) of sarcoplasmic reticulum alternates between several conformational states during ATP-dependent Ca2+ transport. The E1 conformation is stabilized by 0.1 mM Ca2+ and the E2 conformation by vanadate in a Ca2+-free medium. Fourier transform infrared spectroscopy reveals significant differences between the two states that indicate differences in the protein secondary structure. The two states and the corresponding spectra can be interconverted reversibly by changing the Ca2+ concentration of the medium. The infrared spectral changes indicate the appearance of a new alpha-helical substructure connected with the E1----E2 conversion accompanied by small changes in beta-turns, while the beta-sheet content remains essentially unchanged. There are also differences between the E1 and E2 states in the C = O stretching vibrations of the ester carbonyl groups of phospholipids in intact sarcoplasmic reticulum that are not observed under identical conditions in isolated sarcoplasmic reticulum lipid dispersions. These observations imply an effect of proteins on the structure of the interfacial regions of the phospholipids that is dependent on the conformational state of the Ca2+-ATPase. The CH2- and CH3-stretching frequencies of the membrane lipids are not affected significantly by the E1----E2 transition. The Fourier transform infrared spectra of sarcoplasmic reticulum vesicles in the presence of 20 mM Ca2+ suggest the stabilization of a protein conformation similar to the E2 state except for differences in the behavior of COO- and phospholipid ester C = O groups that may reflect charge effects of the bound Ca2+.     

Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum

The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.     

The effect of dicyclohexylcarbodiimide and cyclopiazonic acid on the difference FTIR spectra of sarcoplasmic reticulum induced by photolysis of caged-ATP and caged-Ca2+.

The photochemical release of Ca2+ from caged-Ca2+ in the absence of ATP, and the release of ATP from caged-ATP in the presence of Ca2+ induce characteristic difference FTIR spectra on rabbit sarcoplasmic reticulum that are related to the formation of Ca2-E1 and E approximately P intermediates of the Ca(2+)-ATPase, respectively. Dicyclohexylcarbodiimide (10 nmol/mg protein) abolished both the Ca(2+)-and ATP-induced difference FTIR spectra parallel with inhibition of ATPase activity. Cyclopiazonic acid (50 nmol/mg protein) inhibited the Ca(2+)-induced difference spectrum measured in the absence of ATP, but had no significant effect on the ATP-induced difference spectrum measured in the presence of 1 mM Ca2+. The dog kidney Na+,K(+)-ATPase did not give significant difference spectrum after photolysis of caged-ATP in Ca(2+)-free media containing 90 mM Na+ and 10 mM K+, with or without ouabain. We propose that both the Ca2+ and the ATP-induced difference FTIR spectra of the Ca(2+)-ATPase reflect the occupancy of the high-affinity Ca2+ transport site of the enzyme.     

Enhanced Ca2+-induced calcium release by isolated sarcoplasmic reticulum vesicles from malignant hyperthermia susceptible pig muscle

To further define the possible involvement of sarcoplasmic reticulum calcium accumulation and release in the skeletal muscle disorder malignant hyperthermia (MH), we have examined various properties of sarcoplasmic reticulum fractions isolated from normal and MH-susceptible pig muscle. A sarcoplasmic reticulum preparation enriched in vesicles derived from the terminal cisternae, was further fractionated on discontinuous sucrose density gradients (Meissner, G. (1984) J. Biol. Chem. 259, 2365-2374). The resultant MH-susceptible and normal sarcoplasmic reticulum fractions, designated F0-F4, did not differ in yield, cholesterol and phospholipid content, or nitrendipine binding capacity. Calcium accumulation (0.27 mumol Ca/mg per min at 22 degrees C), Ca2+-ATPase activity (0.98 mumol Pi/mg per min at 22 degrees C), and calsequestrin content were also similar for MH-susceptible and normal sarcoplasmic reticulum fraction F3. To examine sarcoplasmic reticulum calcium release, fraction F3 vesicles were passively loaded with 45Ca (approx. 40 nmol Ca/mg), and rapidly diluted into a medium of defined Ca2+ concentration. Upon dilution into 1 microM Ca2+, the extent of Ca2+-dependent calcium release measured after 5 s was significantly greater for MH-susceptible than for normal sarcoplasmic reticulum, 65.9 +/- 2.8% vs. 47.7 +/- 3.9% of the loaded calcium, respectively. The C1/2 for Ca2+ stimulation of this calcium release (5 s value) from MH-susceptible sarcoplasmic reticulum also appeared to be shifted towards a higher Ca2+-sensitivity when compared to normal sarcoplasmic reticulum. Dantrolene had no effect on calcium release from fraction F3, however, halothane (0.1-0.5 mM) increased the extent of calcium release (5 s) similarly in both MH-susceptible and normal sarcoplasmic reticulum. Furthermore, Mg2+ was less effective at inhibiting, while ATP and caffeine were more effective in stimulating, this Ca2+-dependent release of calcium from MH-susceptible, when compared to normal sarcoplasmic reticulum. Our results demonstrate that while sarcoplasmic reticulum calcium-accumulation appears unaffected in MH, aspect(s) of the sarcoplasmic reticulum Ca2+-induced calcium release mechanism are altered. Although the role of the Ca2+-induced calcium release mechanism of sarcoplasmic reticulum in situ is not yet clear, our results suggest that an abnormality in the regulation of sarcoplasmic reticulum calcium release may play an important role in the MH syndrome.     

Effect of Mg ions and spermine on ATP-dependent Ca2+ transport in myometrial intracellular structures. I. Comparative study of Ca2+ accumulation in mitochondria and sarcoplasmic reticulum

In experiments, which were carried out with the use of a radioactive label (45Ca2+) on the suspension of rat uterus myocytes treated by digitonin solution (0.1 mg/ml), influence of Mg ions and spermine on Mg2+, ATP-dependent Ca2+ transport in mitochondria and sarcoplasmic reticulum was investigated. Ca2+ accumulation in mitochondria (1324 +/- 174 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to thapsigargin (100 nM) and was blocked by ruthenium red (10 microM). Oxalate-stimulated Ca2+ accumulation in sarcoplasmic reticulum (136 +/- 17 pmol Ca2+/10(6) cells for 1 min - the control) was tested as such which was not sensitive to ruthenium red and was blocked by thapsigargin. It has been shown, that initial speed and level of energy-dependent Ca2+ accumulation in mitochondria considerably exceeded the values of these parameters for sarcoplasmic reticulum Ca2+-accumulation system. Ca2+ accumulation kinetic in mitochondria was characterized by a steady-state phase (for 5-10 min. of incubation) while accumulation kinetic of this cation in sarcoplasmic reticulum corresponded to zero order reaction. Increase of Mg2+ concentration up to 5 mM led to activation of Ca2+-accumulation systems in mitochondria and sarcoplasmic reticulum (values of activation constants K(Mg) for Mg2+ were 2.8 and 0.6 mM, accordingly). Concentration dependence of spermine action on Ca2+ accumulation in mitochondria was described by a dome-shaped curve with a maximum at 1 mM spermine. In case of sarcoplasmic reticulum Ca2+ pump only the inhibition phase was tested at spermine concentration above 1 mM. However values of inhibition constants for both transporting systems were practically identical--5.2 +/- 0.6 and 5.7 +/- 0.7 mM, accordingly. Hence, Mg ions carry out the important role in regulation of energy-dependent Ca2+ transporting systems both in uterus smooth muscle mitochondria and sarcoplasmic reticulum. Spermine acts first of all on mitochondrial calcium uniporter.     

Comparison of the (Ca2+ + Mg2+)-ATPase proteins from normal and dystrophic chicken sarcoplasmic reticulum.

The involvement of membrane protein in dystrophic chicken fragmented sarcoplasmic reticulum alterations has been examined. A purified preparation of the (Ca2+ + Mg2+)-ATPase protein from dystrophic fragmented sarcoplasmic reticulum was found to have a reduced calcium-sensitive ATPase activity and phosphoenzyme level, in agreement with alterations found in dystrophic chicken fragmented sarcoplasmic reticulum. An amino acid analysis of the ATPase preparations showed no difference in the normal and dystrophic (Ca2+ + Mg2+)-ATPase. The (Ca2+ + Mg2+)-ATPase was investigated further by isoelectric focusing and proteolytic digestion of the fragmented sarcoplasmic reticulum. Neither of these methods indicated any alteration in the composition of the dystrophic (Ca2+ + Mg2+)-ATPase. We have concluded that the alterations observed in dystrophic fragmented sarcoplasmic reticulum are not due to increased amounts of non-(Ca2+ + Mg2+)-ATPase protein, and that the normal and dystrophic (Ca2+ + Mg2+)-ATPase protein are not detectably different.     

Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. II. Releases involving a Ca2+-induced Ca2+ release channel

Calcium ions that have been preloaded into isolated sarcoplasmic reticulum subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances. We report here that not only caffeine, but also Ca2+ ions, thymol, quercetin, menthol, halothane, chloroform, 1-ethyl-2-methylbenzimidazole, ryanodine, tetraphenylboron, ketoconazole, miconazole, clotrimazole, W-7, doxorubicin, 5,5'-dithiobis-(2-nitrobenzoic acid), p-chloromercuribenzoic acid, and low concentrations of Ag+ induce Ca2+ release from such triadic sarcoplasmic reticulum. All these drugs induce increased undirectional Ca2+ efflux. We believe all these drug-induced Ca2+ releases are mediated by Ca2+ efflux through the same ion channel since these releases are all greatly attenuated when light sarcoplasmic reticulum is substituted for triads and are even more pronounced when transverse tubule-free terminal cisternae are substituted for triads, and all these forms of drug-induced Ca2+ release are inhibited by submicromolar concentrations of ruthenium red, and by submillimolar concentrations of tetracaine, 9-aminoacridine, and Ba2+, yet they are not affected by nifedipine even at a concentration of 50 microM.     

ATP-Induced phosphorylation of the sarcoplasmic reticulum Ca2+ ATPase: molecular interpretation of infrared difference spectra.

Time-resolved infrared difference spectra of the ATP-induced phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase have been recorded in H2O and 2H2O at pH 7.0 and 1 degrees C. The reaction was induced by ATP release from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and from [gamma-18O3]caged ATP. A band at 1546 cm-1, not observed with the deuterated enzyme, can be assigned to the amide II mode of the protein backbone and indicates that a conformational change associated with ATPase phosphorylation takes place after ATP binding. This is also indicated between 1700 and 1610 cm-1, where bandshifts of up to 10 cm-1 observed upon protein deuteration suggest that amide I modes of the protein backbone dominate the difference spectrum. From the band positions it is deduced that alpha-helical, beta-sheet, and probably beta-turn structures are affected in the phosphorylation reaction. Model spectra of acetyl phosphate, acetate, ATP, and ADP suggest the tentative assignment of some of the bands of the phosphorylation spectrum to the molecular groups of ATP and Asp351, which participate directly in the phosphate transfer reaction: a positive band at 1719 cm-1 to the C==O group of aspartyl phosphate, a negative band at 1239 cm-1 to the nuas(PO2-) modes of the bound ATP molecule, and a positive band at 1131 cm-1 to the nuas(PO32-) mode of the phosphoenzyme phosphate group, the latter assignment being supported by the band's sensitivity toward isotopic substitution in the gamma-phosphate of ATP. Band positions and shapes of these bands indicate that the alpha- and/or beta-phosphate(s) of the bound ATP molecule become partly dehydrated when ATP binds to the ATPase, that the phosphoenzyme phosphate group is unprotonated at pH 7.0, and that the C==O group of aspartyl phosphate does not interact with bulk water. The Ca2+ binding sites seem to be largely undisturbed by the phosphorylation reaction, and a functional role of the side chains of Asn, Gln, and Arg residues was not detected.     

Mechanism of the stimulation of Ca2+-dependent ATPase of skeletal muscle sarcoplasmic reticulum by protein kinase

Sarcoplasmic reticulum isolated from moderately fast rabbit skeletal muscle contains intrinsic adenosine 3',5'-monophosphate (cAMP)-independent protein kinase activity and a substrate of 100 000 Mr. Phosphorylation of skeletal sarcoplasmic reticulum by either endogenous membrane bound or exogenous cAMP-dependent protein kinase results in stimulation of the initial rates of Ca2+ transport and Ca2+-ATPase activity. To determine the molecular mechanism by which protein kinase-dependent phosphorylation regulates the calcium pump in skeletal sarcoplasmic reticulum, we examined the effects of protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Skeletal sarcoplasmic reticulum vesicles were preincubated with cAMP and cAMP-dependent protein kinase in the presence (phosphorylated sarcoplasmic reticulum) and absence (control sarcoplasmic reticulum) of adenosine 5'-triphosphate (ATP). Control and phosphorylated sarcoplasmic reticulum were subsequently assayed for formation (5-100 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase. Protein kinase mediated phosphorylation of skeletal sarcoplasmic reticulum resulted in pronounced stimulation of initial rates and levels of E approximately P in sarcoplasmic reticulum preincubated with either ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) prior to assay (Ca2+-free sarcoplasmic reticulum), or with calcium/EGTA buffer (Ca2+-bound sarcoplasmic reticulum). These effects were evident within a wide range of ionized Ca2+. Phosphorylation of skeletal sarcoplasmic reticulum by protein kinase also increased the initial rate of E approximately P decomposition. These findings suggest that protein kinase-dependent phosphorylation of skeletal sarcoplasmic reticulum regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the active calcium transport observed at steady state.     

Inhibitors of Ca2+ release from the isolated sarcoplasmic reticulum. II. The effects of dantrolene on Ca2+ release induced by caffeine, Ca2+ and depolarization

The effects of dantrolene, which is a known muscle relaxant, on Ca2+ release from the isolated sarcoplasmic reticulum induced by several different methods [1) addition of caffeine, (2) Ca2+ jump, and (3) membrane-depolarization produced by choline chloride replacement of potassium gluconate) were investigated. Dantrolene inhibited caffeine-induced Ca2+ release with C1/2 = 2.5 microM, whereas there was no effect on Ca2+ release induced by a Ca2+ jump. The amount of Ca2+ released by depolarization was reduced if Ca2+ release was triggered in an earlier phase of the steady state of Ca2+ uptake (time elapsed between the addition of ATP and the triggering of Ca2+ release, tATP less than 4 min); while, if triggered in a latter phase (tATP greater than 4 min) dantrolene enhanced depolarization-induced Ca2+ release. C1/2 for the inhibition and that for enhancement of depolarization-induced Ca2+ release were 1.0 and 0.3 microM, respectively. These results suggest that dantrolene affects several different steps of the mechanism by which Ca2+ release is triggered. The sarcoplasmic reticulum and T-tubule membrane fractions had 7.9 nmol dantrolene-binding sites/mg (Kassoc = 1.0 X 10(5) M-1) and 21.0 nmol/mg (Kassoc = 1.1 X 10(5) M-1), respectively. The time-course of dantrolene binding to sarcoplasmic reticulum was monophasic, while that to T-tubules was biphasic.     

Localization of Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in adult rat papillary muscle

Localization of the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in rat papillary muscle was determined by indirect immunofluorescence and immunoferritin labeling of cryostat and ultracryotomy sections, respectively. The Ca2+ + Mg2+-ATPase was found to be rather uniformly distributed in the free sarcoplasmic reticulum membrane but to be absent from both peripheral and interior junctional sarcoplasmic reticulum membrane, transverse tubules, sarcolemma, and mitochondria. This suggests that the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum is antigenically unrelated to the Ca2+ + Mg2+-ATPase of the sarcolemma. These results are in agreement with the idea that the sites of interior and peripheral coupling between sarcoplasmic reticulum membrane and transverse tubules and between sarcoplasmic reticulum and sarcolemmal membranes play the same functional role in the excitation-contraction coupling in cardiac muscle.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca2+ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37 degrees C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 microM CaCl2, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca2+ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca2+ uptake, a second phase of Ca2+ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca2+ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca2+ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca2+ influx of sarcoplasmic reticulum near steady state of Ca2+ uptake was measured by pulse labeling with 45Ca2+. The Ca2+ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca2+ uptake in normal medium, Ca2+ influx was balanced by Ca2+ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca2+ exchange rate at the first plateau of Ca2+ uptake was about half of that in normal medium. When the second phase of Ca2+ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca2+ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 micrograms/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca2+ uptake was also observed. These data suggest that the Ca2+ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca2+ efflux, which subsequently stimulates Ca2+ exchange.     

Cooperative effects of Ca2+ and Sr2+ on sarcoplasmic reticulum adenosine triphosphatase

The intrinsic fluorescence of purified Ca-ATPase from skeletal sarcoplasmic reticulum was measured in the presence of various concentrations of Ca2+, Sr2+, and Ba2+. Ca2+ and Sr2+ induce positive cooperative fluorescence enhancement, whereas Ba2+ does not change the fluorescence of ATPase. ATP does not seem to modify the kinetic parameters of Ca2+ and Sr2+ binding to ATPase. Nevertheless, p-nitrophenylphosphate hydrolysis, activated by Ca2+ or Sr2+ at various pHs, changes the affinity and the cooperative behavior for both cations and two components appear in the Hill plots. For Ca2+, nH of 1.6 to 3.5 were obtained, and 1.06 to 1.83 for Sr2+; nH changes of the second component seem to be pH dependent. Differences in the ratio between rates of Ca2+ transport and substrate hydrolysis by sarcoplasmic reticulum were found, i.e., two for ATP and one for p-nitrophenylphosphate. For Sr2+ this ratio was one for either ATP or p-nitrophenylphosphate.     

(Ca2+ + Mg2+)-ATPase activity associated with the maintenance of a Ca2+ gradient by sarcoplasmic reticulum at submicromolar external [Ca2+]. The effect of hypothyroidism

The formation and maintenance of Ca2+-filling levels by sarcoplasmic reticulum vesicles from euthyroid (control) and hypothyroid skeletal muscle were investigated using the Ca2+-indicator quin-2, at [Ca2+] in the medium [( Cao2+]) of 0.05-0.3 microM. Rapid ATP-dependent Ca2+ uptake resulted in a steady-state Ca2+-filling level, Cai2+, within one minute. This Ca2+ gradient was maintained for at least three minutes, during which less than 20% of the ATP was consumed. Cai2+ was maximal (120 nmol/mg) for [Cao2+] greater than 0.3 microM and decreased to 40 nmol/mg at [Cao2+] of 0.05 microM. Preparations from both experimental groups showed qualitatively and quantitatively the same relationship between Cai2+ and [Cao2+] at steady state, despite a significantly lower Ca2+-pump content of hypothyroid sarcoplasmic reticulum, which resulted in a 25% lower maximal (Ca2+ + Mg2+)-ATPase activity. Maintenance of the steady state, at all levels of Cai2+, was associated with net ATP consumption by the Ca2+ pump and cycling of Ca2+, which processes were 30% slower in the hypothyroid group as compared to the control group. Determination of the passive efflux of Ca2+, as well as the fraction of leaky or unsealed sarcoplasmic reticulum fragments, excluded either of these possibilities as an explanation for the relatively high (Ca2+ + Mg2+)-ATPase rates at steady state. On the basis of these and previously reported results, it is concluded that the maintenance of a Ca2+ gradient by sarcoplasmic reticulum under physiological conditions with respect to external [Ca2+] and the concentrations of ATP, ADP and Pi, is associated with the cycling of Ca2+ coupled to net ATP hydrolysis. Using the obtained data it is calculated that the sarcoplasmic reticulum may account for 20% of the resting metabolic rate in skeletal muscle. Consequently, together with the previously reported lower sarcoplasmic reticulum content of skeletal muscle in hypothyroidism, we calculate that about one third of the decrease in basal metabolic rate in this thyroid state can be related to the alterations of the sarcoplasmic reticulum.     

Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.