Ca-ATPase activity and protein composition of sarcoplasmic reticulum membranes isolated from skeletal muscles of typical hibernator,the ground squirrel Spermophilus undulatus

Ca-ATPase activity in sarcoplasmic reticulum (SR) membranes isolated from skeletal muscles of the typical hibernator, the ground squirrel Spermophilus undulatus, is about 2-fold lower than that in SR membranes of rats and rabbits and is further decreased 2-fold during hibernation. The use of carbocyanine anionic dye Stains-All has revealed that Ca-binding proteins of SR membranes, histidine-rich Ca-binding protein and sarcalumenin, in ground squirrel, rat, and rabbit SR have different electrophoretic mobility corresponding to apparent molecular masses 165, 155, and 170 kDa and 130, 145, and 160 kDa, respectively; the electrophoretic mobility of calsequestrin (63 kDa) is the same in all preparations. The content of these Ca-binding proteins in SR membranes of the ground squirrels is decreased 3–4 fold and the content of 55, 30, and 22 kDa proteins is significantly increased during hibernation.     

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 corticotropin and hydrocortisone on the Ca2+-ATPase activity of skeletal muscle sarcoplasmic reticulum

The effect of corticotropin (ACTH1-39), synacthen (ACTH1-24) and hydrocortisone-hemisuccinate on the activity of Ca-ATPase of skeletal muscle sarcoplasmic reticulum (SR) and calcium (Ca) accumulation in SR vesicles has been studied. It has been shown that ACTH1-39 (I U per 100 g body weight) increased the activity of Ca-ATPase in skeletal muscle SR of rats, while hydrocortisone (5 mg per 100 g body weight) did not change the activity of Ca-ATPase in skeletal muscle SR. However, both hormones increase the total activity of ATPase. ACTH1-39 and ACTH1-24 (0.05-0.0005 U/ml) and hydrocortisone (2.8 X 10(-7)-2.8 X 10(-9) mol/l) increased in vitro Ca-ATPase isolated from rabbit skeletal muscle SR and accumulation of Ca is SR vesicles. At the same time, hydrocortisone reduced calcium/phosphorus ratio, while ACTH1-39 and ACTH1-24 increased it, i.e. hydrocortisone facilitated Ca accumulation in SR requiring more ATP energy, whereas ACTH facilitated Ca accumulation in SR requiring less ATP energy.     

Comparative characteristics of sarcoplasmic reticulum preparations from skeletal muscles of the ground squirrel Spermophilus undulatus, rats, and rabbits

A comparison of sarcoplasmic reticulum (SR) preparations from skeletal muscles of ground squirrels Spermophilus undulatus, rats, and rabbits established that on the basis of protein yield and phospholipid/protein ratio these preparations are practically the same. Nevertheless, the specific activity of Ca-ATPase, the main protein component of SR membranes, in SR preparations of the ground squirrel skeletal muscles is only about half of the activity in SR preparations of rats and rabbits. Significant differences in protein composition of the preparations were detected: ground squirrel SR differed by an unusually high content of a 205 kD protein (probably myosin) and a number of low-molecular-weight SR protein components, and the SR preparations of rabbits are characterized by a high content of the Ca-binding proteins calsequestrin and sarcalumenin. Use of the anionic carbocyanine dye Stains-All established that all preparations contained only three proteins which are stained dark blue by this dye: calsequestrin, sarcalumenin, and a histidine-rich Ca-binding protein. The electrophoretic mobility of calsequestrin was identical in all preparations (molecular mass 63 kD), whereas sarcalumenin and histidine-rich Ca-binding protein are probably present in different isoforms with molecular masses of 130, 145, and 160 and 165, 155, and 170 kD, respectively, in SR preparations of ground squirrels, rats, and rabbits. Analysis of the fluorescence parameters of the fluorescent probes 8-anilino-1-naphthalene sulfonic acid and pyrene bound to SR membranes showed that the properties of the lipid bilayer in the SR membranes of the preparations differed considerably. It is suggested that the differences in protein composition and/or structural state of the ground squirrel SR membrane lipid bilayer could be the reason for the low Ca-ATPase activity in these preparations.     

Characteristics of the interaction of melittin with sarcoplasmic reticulum membranes.

Addition of an amphipathic bee venom peptide, melittin, to sarcoplasmic reticulum (SR) vesicles isolated from rabbit skeletal muscles resulted in a fast (<1 min) blue shift in the fluorescence maximum of the melittin--SR membrane complex. Over the following 45 min the position of the fluorescence maximum did not change, but the fluorescence intensity of the melittin--SR membrane complex decreased by approximately 35% with rate constant 0.14 min-1. Melittin rapidly quenched the isotropic signal in the EPR spectrum of spin-labeled stearic acid added to SR membranes. Further changes in the spectral parameters of the spin probe bound to SR membranes in the presence of melittin indicated an increase of the viscosity of the probe microenvironment (empiric parameter T/eta was decreased by approximately 35% with rate constant 0.11 min-1). The surface potential of SR membranes measured using a pH-sensitive dye, neutral red, decreased after melittin addition from -60 to -30 mV. It was demonstrated with the use of a cross-linking agent, cupric o-phenanthroline, that melittin induced slow aggregation of Ca-ATPase protein in SR membranes; the content of enzyme in the monomeric form decreased with rate constant 0.14 min-1. It is concluded that melittin binds rapidly to SR membranes, inducing slow changes in Ca-ATPase conformation and oligomeric state as well as structural transitions in the lipid bilayer of SR membranes.     

Comparable levels of Ca-ATPase inhibition by phospholamban in slow-twitch skeletal and cardiac sarcoplasmic reticulum

Alterations in expression levels of phospholamban (PLB) relative to the sarcoplasmic reticulum (SR) Ca-ATPase have been suggested to underlie defects of calcium regulation in the failing heart and other cardiac pathologies. To understand how variation in PLB expression relative to that of the Ca-ATPase can modulate calcium transport, we have investigated the inhibition of the Ca-ATPase by PLB in native SR membranes from slow-twitch skeletal and cardiac muscle and in reconstituted proteoliposomes. Quantitative immunoblotting in combination with affinity-purified protein standards was used to measure protein concentrations of PLB and of the Ca-ATPase. Functional inhibition of the Ca-ATPase was determined from both the calcium concentrations for half-maximal activation (Ca(1/2)) and the shift in the calcium concentrations following release of PLB inhibition (i.e., (Delta)Ca(1/2)) by incubation with monoclonal antibodies against PLB, which are equivalent to phosphorylation of PLB by cAMP-dependent protein kinase. We report that equivalent levels of PLB inhibition and antibody-induced activation ((Delta)Ca(1/2) = 0.25 +/- 0.02 microM) are observed in SR membranes from slow-twitch skeletal and cardiac muscle, where molar stoichiometries of PLB expressed per Ca-ATPase vary, respectively, from 0.9 +/- 0.1 to 4.1 +/- 0.8. Similar levels of inhibition to those observed in isolated SR vesicles were observed using reconstituted proteoliposomes following co-reconstitution of affinity-purified Ca-ATPase with PLB. These results indicate that total expression levels of one PLB per Ca-ATPase result in full inhibition of the Ca-ATPase and, based on the measured K(D) (140 +/- 30 microM), suggests one PLB complexed with two Ca-ATPase molecules is sufficient for full inhibition of activity. Therefore, the excess PLB expressed in the heart over that required for inhibition suggests a capability for graded responses of the Ca-ATPase activity to endogenous kinases and phosphatases that modulate the level of phosphorylation necessary to relieve inhibition of the Ca-ATPase by PLB.     

Several properties of the Ca-pump of rabbit skeletal muscle sarcoplasmic reticulum in hypercholesteremia]

Sarcoplasmic reticulum (SR) fragments from the skeletal muscles of rabbit with marked atherosclerosis possessed decreased Ca2+-accumulating capacity. Lowering of transport efficiency, namely reduction of the Ca/ATP ratio from 1.9--normal value--to 0.9 during the experiment at 26 degrees C was accompanied by activation of Ca-ATPase and simultaneously of the rate of Ca2+ outflux from the SR. Arrhenius plots of Ca-ATPase temperature dependence characterized under normal conditions by a break at 20--21 degrees C was linearized under hypercholesterolemia. At the same time there was a rise (from 0.03 under normal conditions to 0.15 in atherosclerosis) of cholesterol/protein ratio in the SR membrane preparations. Activation energy for Ca-ATPase crude membranes under normal conditions was equal to 15.6 and 28.7 kcal/mol above and below the break point respectively; this value for Ca-ATPase of membranes with increased cholesterol level was 19 kcal/mol for all the temperatures investigated.     

Identification of the multifunctional calmodulin-dependent protein kinase in the cytosol, sarcoplasmic reticulum, and sarcolemma of rabbit skeletal muscle

A multifunctional calmodulin-dependent protein kinase (calmodulin kinase) was purified from the cytosol of rabbit skeletal muscle as a subunit of 58 kDa. A 58-kDa protein in sarcoplasmic reticulum (SR) and sarcolemma (SL) of rabbit skeletal muscle was endogenously phosphorylated in a calmodulin-dependent manner. The 58-kDa protein in SR and SL was considered to be identical to the subunit of cytosol calmodulin kinase on the basis of immunoreactivity, calmodulin binding, and autophosphorylation studies and on the patterns of protease-treated phosphopeptides. Calmodulin kinase showed broad substrate specificity and phosphorylated troponins I and T.     

Effect of temperature on fluorescence of labels and probes of various localizations incorporated into sarcoplasmic reticulum samples

Studies were carried out of temperature relationship of dansylchloride, N-3-pyrenylmaleinimide fluorescence, SR membranes, self-luminescence caused by Ca-ATPase tryptophane - provided fluorescence and of pyrene excimerization in membrane preparations of sarcoplasmic reticulum (SR) of rabbit skeletal muscles. Temperature relationship of fluorescence intensity of dansylchloride and N-3-pyrenylmaleimide in Arrhenius coordinates has bends at 15 and 35 degrees. Selffluorescence of protein samples linearly depends on temperature. Temperature relationship of the ratio between the intensities of exsimeric and monomeric forms of pyrene Fa/Fm in Arrhenius coordinates has the bend at 20-22 degrees. Hence only the latter relationship coincides with the shape of Arrhenius graph for enzymatic activity of SR Ca-ATPase.     

Molecular dynamics in mouse atrial tumor sarcoplasmic reticulum.

We have determined directly the effects of the inhibitory peptide phospholamban (PLB) on the rotational dynamics of the calcium pump (Ca-ATPase) of cardiac sarcoplasmic reticulum (SR). This was accomplished by comparing mouse ventricular SR, which has PLB levels similar to those found in other mammals, with mouse atrial SR, which is effectively devoid of PLB and thus has much higher (unregulated) calcium pump activity. To obtain sufficient quantities of atrial SR, we isolated the membranes from atrial tumor cells. We used time-resolved phosphorescence anisotropy of an erythrosin isothiocyanate label attached selectively and rigidly to the Ca-ATPase, to detect the microsecond rotational motion of the Ca-ATPase in the two preparations. The time-resolved phosphorescence anisotropy decays of both preparations at 25 degrees C were multi-exponential, because of the presence of different oligomeric species. The rotational correlation times for the different oligomers were similar for the two preparations, but the total decay amplitude was substantially greater for atrial tumor SR, indicating that a smaller fraction of the Ca-ATPase molecules exists as large aggregates. Phosphorylation of PLB in ventricular SR decreased the population of large-scale Ca-ATPase aggregates to a level similar to that of atrial tumor SR. Lipid chain mobility (fluidity), detected by electron paramagnetic resonance of stearic acid spin labels, was very similar in the two preparations, indicating that the higher protein mobility in atrial tumor SR is not due to higher lipid fluidity. We conclude that PLB inhibits by inducing Ca-ATPase lateral aggregation, which can be relieved either by phosphorylating or removing PLB.     

Protective effect of alpha-tocopherol on the Ca2+-transport system of the sarcoplasmic reticulum membranes in hypercholesterolemia

The effect of antioxidant--alpha-tocopherol--on Ca2+-transporting system in sarcoplasmic reticulum (SR) of the rabbit skeletal muscles was studied in hypercholesterolemia (HC). alpha-tocopherol administration to animals with HC produced a break on the curve of temperature dependence of Ca-ATPase activity at about 20 degrees C, that disappeared in HC, increased the rate of "rapid" SH-group binding by thiol reagents, and normalized the level of unsaturated fatty acids in SR membranes without altering phospholipid content. It is suggested that the damage of Ca-ATPase in HC is mainly due to activation of lipid peroxidation.     

Effect of cholesterol on the cooperativeness of the Ca-ATPase of the sarcoplasmic reticulum in rabbit skeletal muscle

Cooperative properties of Ca-ATPase of the sarcoplasmic reticulum (SR) of rabbit skeletal muscles were examined in health and hypercholesterolemia. As the concentration of ATP was raised (from 50-100 microM to 5 mM) the Hill ratio (Nh) for ATP increased from 0.4 to 3.2. It is assumed that increased cooperative interaction between Ca-ATPase polymers led to a rise in the efficacy of Ca-pump work. Under the conditions described the Nh for UTP increased from 0.43 to 1.0. During hypercholesterolemia (1 g/kg cholesterol for 1, 3 and 6 months), the maximal values of the Nh for ATP did not exceed 2.0, whereas those for UTP 1.0.     

Role of lipid peroxidation in changes in the structure of Ca-ATPase in skeletal muscle sarcoplasmic reticulum during hypercholesterolemia

Using the method of spin labels it was shown that in hypercholesterolemia (HCh), the following parameters decreased: the velocity of maleimide spin label binding to sarcoplasmic reticulum (SR) Ca-ATPase of rabbit skeletal muscles, the accessibility of spin-labeled thiol groups of the enzyme to potassium ferricyanide and sodium ascorbate, and the mobility of the Ca-ATPase molecule fragment to which the spin label was attached. In addition, intensification of lipid peroxidation was demonstrated in SR membranes. Supplementation of the high-cholesterol diet with alpha-tocopherol resulted in the decreased rates of lipid peroxidation in SR membranes and increased values of the above parameters relative to the values found under HCh. It is concluded that the effect of alpha-tocopherol in vivo on the structure of the Ca-ATPase proteolipid complex in HCh is due mainly to antioxidant properties of the diet-supplementing substance.     

Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers

The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca(2+)-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped viruses indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.     

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.     

Purification, characterization, and molecular cloning of a 60-kDa phosphoprotein in rabbit skeletal sarcoplasmic reticulum which is an isoform of phosphoglucomutase.

A 60-kDa substrate of calmodulin-dependent protein kinase in rabbit "heavy" skeletal sarcoplasmic reticulum (SR) was characterized by purification and cDNA cloning. Purification was achieved by column chromatography using DEAE-Sephacel, heparin-agarose, and hydroxylapatite in 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS). Analyses of amino acid sequence and composition indicated that the CHAPS-soluble 60-kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding two isoforms of PGM were isolated from rabbit skeletal muscles. The translated amino acid sequences show that the isoforms, PGM1 and PGM2, differ in the N-terminal 77 amino acids and that PGM2 is identical to the 60-kDa protein in the SR. Northern blot analysis showed that the size of the mRNA encoding PGM2 is 2.4 kilobases. The PGM enzyme activity was markedly inhibited in SR membranes, while perturbation of the membranes with CHAPS or guanidine-HCl recovered the enzyme activity. KCl (0.15-1 M) led to a partial recovery of the enzyme activity suggesting that the charge interaction is not the primary force for PGM-SR interaction. PGM is localized in the heavy fraction of SR, where calsequestrin and Ca2+ release channel are enriched. Our results demonstrate that an isoform of PGM localized in junctional skeletal SR is the 60-kDa substrate of calmodulin-dependent protein kinase.     

Mesencephalic astrocyte-derived neurotrophic factor protects the heart from ischemic damage and is selectively secreted upon sarco/endoplasmic reticulum calcium depletion

The endoplasmic reticulum (ER) stress protein mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to protect cells from stress-induced cell death before and after its secretion; however, the conditions under which it is secreted are not known. Accordingly, we examined the mechanism of MANF release from cultured ventricular myocytes and HeLa cells, both of which secrete proteins via the constitutive pathway. Although the secretion of proteins via the constitutive pathway is not known to increase upon changes in intracellular calcium, MANF secretion was increased within 30 min of treating cells with compounds that deplete sarcoplasmic reticulum (SR)/ER calcium. In contrast, secretion of atrial natriuretic factor from ventricular myocytes was not increased by SR/ER calcium depletion, suggesting that not all secreted proteins exhibit the same characteristics as MANF. We postulated that SR/ER calcium depletion triggered MANF secretion by decreasing its retention. Consistent with this were co-immunoprecipitation and live cell, zero distance, photo affinity cross-linking, demonstrating that, in part, MANF was retained in the SR/ER via its calcium-dependent interaction with the SR/ER-resident protein, GRP78 (glucose-regulated protein 78 kDa). This unusual mechanism of regulating secretion from the constitutive secretory pathway provides a potentially missing link in the mechanism by which extracellular MANF protects cells from stresses that deplete SR/ER calcium. Consistent with this was our finding that administration of recombinant MANF to mice decreased tissue damage in an in vivo model of myocardial infarction, a condition during which ER calcium is known to be dysregulated, and MANF expression is induced.     

Evidence that platelet and skeletal sarcoplasmic reticulum Ca2+-ATPase are structurally distinct

Proteolytic digestion and indirect immunostaining were used to compare the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins. When the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins were digested in the native state with trypsin, the platelet Ca2+-ATPase, which had an apparent undigested molecular mass of 103 kDa, yielded 78-kDa and 25-kDa fragments. Calcium transport activity depended on the integrity of the 103-kDa protein, while the digested protein had residual ATPase activity. Tryptic digestion of the sarcoplasmic reticulum pump protein, which also had an undigested molecular mass of 103 kDa, yielded products with apparent molecular masses of 55 kDa, 36 kDa, and 26 kDa. Distinct patterns were also observed when the platelet and sarcoplasmic reticulum calcium pump proteins were digested with chymotrypsin and Staphylococcus aureus protease in the presence of sodium dodecyl sulfate. Chymotrypsin digestion of the platelet protein resulted in the appearance of products with apparent molecular masses of 70 kDa, 39 kDa, and 31 kDa, while a similar digestion of the sarcoplasmic reticulum calcium pump protein yielded 54-kDa, 52.5-kDa, 46-kDa, 41-kDa, and 36-kDa fragments. Exposure of the sarcoplasmic reticulum and platelet Ca2+-ATPase proteins to S. aureus protease also yielded dissimilar fragmentation patterns. These results indicate that the Ca2+-ATPases from platelets and sarcoplasmic reticulum are distinct proteins.     

Inhibition of dicarboxylic anion transport by fluorescein isothiocyanate in skeletal sarcoplasmic reticulum.

It has been demonstrated previously that dicarboxylic anions are cotransported during ATP-dependent Ca2+ transport by skeletal muscle sarcoplasmic reticulum (SR) membranes, and that anion cotransport stimulates Ca2+ transport. In the current study, we present evidence that dicarboxylic anion cotransport and Ca2+ transport are kinetically distinct in SR, but both functions are mediated by the CaATPase protein. Preincubation of SR with 40 microM fluorescein isothiocyanate (FITC) (pH 7.0) inhibited essentially all of the Ca2+ ATPase activity, as well as active oxalate-supported and oxalate-independent 45Ca2+ accumulation. The addition of 1 mM beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) to the preincubation media fully protected the dicarboxylic anion-independent Ca2+ ATPase activity and the oxalate-independent active 45Ca2+ accumulation from the inhibitory effects of FITC; however, the ATP-associated [14C]oxalate accumulation, the oxalate-dependent 45Ca2+ accumulation, and the oxalate- and maleate-dependent stimulation of Ca2+ ATPase activity were not protected by AMP-PCP. Thus, the dicarboxylic anion accumulation and the stimulation of Ca2+ uptake by dicarboxylic anions could be functionally separated from the ATP-dependent, anion-independent Ca2+ translocation. FITC bound exclusively to the 100-kDa (CaATPase) and 92-kDa (phosphorylase) proteins in the SR membranes and to purified CaATPase in sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 1 mM AMP-PCP inhibited 50-55% of the FITC fluorescence on the 100-kDa protein, but did not significantly alter fluorescence on the 92-kDa protein. Two-dimensional gel analysis demonstrated a single 100-kDa protein in longitudinal SR membranes. FITC appears to inhibit ATP-dependent Ca2+ transport, and dicarboxylic anion translocation through interaction at separate domains of the CaATPase protein.     

Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: electron paramagnetic resonance.

We have performed electron paramagnetic resonance (EPR) experiments on nitroxide spin labels incorporated into rabbit skeletal sarcoplasmic reticulum (SR), in order to investigate the physical and functional interactions between melittin, a small basic membrane-binding peptide, and the Ca-ATPase of SR. Melittin binding to SR substantially inhibits Ca(2+)-dependent ATPase activity at 25 degrees C, with half-maximal inhibition at 9 mol of melittin bound per mole of Ca-ATPase. Saturation transfer EPR (ST-EPR) of maleimide spin-labeled Ca-ATPase showed that melittin decreases the submillisecond rotational mobility of the enzyme, with a 4-fold increase in the effective rotational correlation time (tau r) at a melittin/Ca-ATPase mole ratio of 10:1. This decreased rotational motion is consistent with melittin-induced aggregation of the Ca-ATPase. Conventional EPR was used to measure the submicrosecond rotational dynamics of spin-labeled stearic acid probes incorporated into SR. Melittin binding to SR at a melittin/Ca-ATPase mole ratio of 10:1 decreases lipid hydrocarbon chain mobility (fluidity) 25% near the surface of the membrane, but only 5% near the center of the bilayer. This gradient effect of melittin on SR fluidity suggests that melittin interacts primarily with the membrane surface. For all of these melittin effects (on enzymatic activity, protein mobility, and fluidity), increasing the ionic strength lessened the effect of melittin but did not alleviate it entirely. This is consistent with a melittin-SR interaction characterized by both hydrophobic and electrostatic forces. Since the effect of melittin on lipid fluidity alone is too small to account for the large inhibition of Ca-ATPase rotational mobility and enzymatic activity, we propose that melittin inhibits the ATPase primarily through its capacity to aggregate the enzyme, consistent with previous observations of decreased Ca-ATPase activity under conditions that decrease protein rotational mobility.