Effect of Ca2+ on the dimeric structure of scallop sarcoplasmic reticulum

Scallop sarcoplasmic reticulum (SR), visualized in situ by freeze-fracture and deep-etching, is characterized by long tubes displaying crystalline arrays of Ca2+-ATPase dimer ribbons, resembling those observed in isolated SR vesicles. The orderly arrangement of the Ca2+-ATPase molecules is well preserved in muscle bundles permeabilized with saponin. Treatment with saponin, however, is not needed to isolate SR vesicles displaying a crystalline surface structure. Omission of ATP from the isolation procedure of SR vesicles does not alter the dimeric organization of the Ca2+-ATPase, although the overall appearance of the tubes seems to be affected: the edges of the vesicles are scalloped and the individual Ca2+-ATPase molecules are not clearly defined. The effect of Ca2+ on isolated scallop SR vesicles was investigated by correlating the enzymatic activity and calcium-binding properties of the Ca2+-ATPase with the surface structure of the vesicles, as revealed by electron microscopy. The dimeric organization of the membrane is preserved at Ca2+ concentrations where the Ca2+ binds to the high affinity sites (half-maximum saturation at pCa approximately 7.0 with a Hill coefficient of 2.1) and the Ca2+-ATPase is activated (half-maximum activation at pCa approximately 6.8 with a Hill coefficient of 1.84). Higher Ca2+ concentrations disrupt the crystalline surface array of the SR tubes, both in the presence and absence of ATP. We discuss here whether the Ca2+-ATPase dimer identified as a structural unit of the SR membrane represents the Ca2+ pump in the membrane.     

Ca2+-ATPase membrane crystals in sarcoplasmic reticulum. The effect of trypsin digestion

Vanadate induces the formation of two-dimensional crystalline arrays of Ca2+-ATPase molecules in sarcoplasmic reticulum. The Ca2+-ATPase membrane crystals are evenly distributed among the terminal cisternae and longitudinal tubules of sarcoplasmic reticulum, but very few crystals were observed in the T tubules. Tryptic cleavage of the Ca2+ transport ATPase into two major fragments (A and B) did not interfere with the vanadate-induced formation of membrane crystals. The ability of Ca2+-ATPase to crystallize was lost after further cleavage of the A fragment into the A1 and A2 subfragments that is known to be accompanied by loss of Ca2+ uptake. Vanadate (0.1-5 mM) inhibited the secondary cleavage of Ca2+-ATPase by trypsin suggesting that the susceptibility of the tryptic cleavage sites is influenced either by the conformation of the enzyme or by the formation of ATPase crystals.     

The regulation of ATPase-ATPase interactions in sarcoplasmic reticulum membrane. I. The effects of Ca2+, ATP, and inorganic phosphate

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in calcium-free medium (Dux, L., and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). The formation of Ca2+-ATPase crystals is inhibited by Ca2+ (2 microM), or ATP (5 mM), but not by ADP, 5'-adenylylimidodiphosphate, or adenylylmethylenediphosphonate. ATPase crystals did not form at 37 degrees C and exposure of preformed crystals to 37 degrees C for 1 h caused the disappearance of crystal lattice. Inorganic orthophosphate (1 mM at pH 6.0) promoted the formation of a distinct crystal form of Ca2+-ATPase, which was different from that produced by Na3VO4. These observations indicate that Ca2+, ATP, inorganic phosphate, pH, and temperature influence the interactions between ATPase molecules in the sarcoplasmic reticulum membrane.     

Crystallization of the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum. Inhibition by myotoxin a

Decavanadate produces extensive ordered arrays of Ca2+-ATPase molecules on sarcoplasmic reticulum (SR) vesicle surfaces [(1984) J. Bioenerg. Biomembranes 16, 491-505] and the basic unit of these crystalline structures seems to be a dimer of Ca2+-ATPase [(1983) J. Ultrastruct. Res. 24, 454-464; (1984) J. Mol. Biol. 174, 193-204]. Myotoxin a, isolated from the venom of the prairie rattlesnake Crotalus viridis viridis, is a muscle-degenerating polypeptide and its primary site of interaction is the SR membrane, where it uncouples CA2+-translocation from CA2+-dependent ATP hydrolysis [(1986) Arch. Biochem. Biophys. 246, 90-97]. The effect of myotoxin a on decavanadate-induced two-dimensional Ca2+-ATPase crystals of SR membranes has been investigated. The toxin inhibits the formation of two-dimensional SR-membrane crystals and disrupts previously formed crystals in a time- and concentration-dependent manner, which parallels the uncoupling of ATP hydrolysis from Ca2+ translocation. Two-dimensional crystalline arrays of the SR membrane have a typical diffraction pattern which, after myotoxin a treatment, displays a progressive loss of order. Decavanadate is an uncompetitive inhibitor of the Ca2+-ATPase enzyme-myotoxin a complex. The present results suggest that a Ca2+-ATPase dimer is required for coupling Ca2+ translocation to Ca2+-dependent ATP hydrolysis.     

Reversible inhibition of sarcoplasmic reticulum Ca-ATPase by altered neuromuscular activity in rabbit fast-twitch muscle

A 50% decrease in both the initial rate and the total capacity of Ca2+ uptake by the sarcoplasmic reticulum (SR) occurred 2 days after the onset of chronic (10 Hz) nerve stimulation in rabbit fast-twitch muscle. Prolonged stimulation (up to 28 days) did not lead to further decreases. This reduction, which was detected in muscle homogenates using a Ca2+-sensitive electrode, was reversible after 6 days cessation of stimulation and was not accompanied by changes in the immunochemically (ELISA) determined tissue level or isozyme characteristics of the SR Ca2+-ATPase protein. However, as measured in isolated SR, it correlated with a reduced specific activity of the Ca2+-ATPase. Kinetic analyses demonstrated that affinities of the SR Ca2+-ATPase towards Ca2+ and ATP were unaltered. Positive cooperativity for Ca2+ binding (h = 1.5) was maintained. However, a 50% decrease in Ca2+-dependent phosphoprotein formation indicated the presence of inactive forms of Ca2+-ATPase in stimulated muscle. The reduced phosphorylation of the enzyme was accompanied by an approximately 50% lowered binding of fluorescein isothiocyanate, a competitor at the ATP-binding site. In view of the unaltered affinity for ATP, this finding suggests that active Ca2+-ATPase molecules coexist in stimulated muscle with inactive enzyme molecules, the latter displaying altered properties at the nucleotide-binding site.     

Three-dimensional reconstruction of negatively stained crystals of the Ca2+-ATPase from muscle sarcoplasmic reticulum

The structure of the Ca2+ transport ATPase from rabbit skeletal muscle sarcoplasmic reticulum has been determined to 25 A resolution by three-dimensional image reconstruction of crystalline membrane tubules induced through exposure to Na3VO4 and preserved for electron microscopy in negative stain. The crystalline arrays have projection symmetry p2 and consist of chains of Ca2+-ATPase dimers arranged in a right-handed helix. The density map shows protein features that project from the membrane surface into the cytoplasm. The luminal side of the membrane tubules is featureless, presumably because very little of the Ca2+-ATPase molecule projects into the luminal space. The cytoplasmic region of the Ca2+-ATPase molecule is pear-shaped, with a lobe oriented nearly parallel to the axis of the dimer ribbons, about 16 A above the surface of the membrane bilayer. The structure seen in the maps has a volume of 71,000 A3, corresponding to a molecular weight of 57,000. The two Ca2+-ATPase profiles that constitute a dimer are connected by a stain-excluding bridge that is oriented parallel with the axis of the tubule at a height of about 42 A above the surface of the bilayer.     

A spin-label and hydrogen-deuterium exchange reaction kinetics study of protein-lipid interactions in lipid-replaced Ca2+-ATPase of rabbit skeletal muscle sarcoplasmic reticulum

The Ca2+-ATPase of sarcoplasmic reticulum from rabbit skeletal muscle was incorporated into vesicles made from dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine. The Ca2+-ATPase activity of these reconstituted membranes became appreciable above 20 degrees C and 30 degrees C, respectively, in accord with the results of previous investigators. Measurement by the spin-labeling technique of the fluidity of the bulk lipid revealed the gel-to-liquid crystalline phase transition at 29 degrees C and 39 degrees C, respectively, while the fluidity of the boundary lipid in both samples was found to be low throughout the temperature range studied. The rotational mobility of the Ca2+-ATPase protein in both samples, measured by saturation transfer electron spin resonance, was also very low throughout the temperature range studied and its temperature-dependence did not show any break or jump corresponding to the phase transition of the bulk lipid. On the other hand, the structural fluctuation of the Ca2+-ATPase protein in dimyristoylphosphatidylcholine-recombinant, measured in terms of hydrogen-deuterium exchange reaction kinetics, showed a jump at about 27 degrees C, apparently in accordance with the phase transition of the bulk lipid. Results obtained in this study suggested that the Ca2+-ATPase protein molecules are in an aggregated state in these reconstituted membranes and that the Ca2+-ATPase activity is neither directly correlated to the fluidity of the boundary lipid nor to the rotational mobility of the Ca2+-ATPase, contrary to the suggestions of previous investigators (Hesketh et al. (1976) Biochemistry 15, 4145-4151; Hidalgo et al. (1978) J. Biol. Chem. 253, 6879-6887).     

Interaction of myotoxin a with the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum

Myotoxin a is a muscle-damaging toxin isolated from the venom of Crotalus viridis viridis. Its interaction with the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles purified from rabbit skeletal muscle was investigated. Myotoxin a inhibited Ca2+ loading and stimulated Ca2+-dependent ATPase without affecting unidirectional Ca2+ efflux. Its action was dose, time, and temperature dependent. Myotoxin a partially blocked the binding of specific anti-(rabbit SR Ca2+-ATPase) antibodies. It is concluded that myotoxin a attaches to the SR Ca2+-ATPase and uncouples Ca2+ uptake from Ca2+-dependent ATP hydrolysis. Myotoxin a also prevented the formation of decavanadate-induced two-dimensional crystalline arrays of the SR Ca2+-ATPase.     

Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin

The octaethyleneglycol mono-n-dodecyl ether solubilized Ca2+-ATPase purified from human erythrocytes has been studied to determine the physical mechanism of its activation by calmodulin. The dependence of Ca2+-ATPase activity on the enzyme concentration shows a transformation from a calmodulin-dependent to a fully active calmodulin-independent form. The transformation is cooperative with a half-maximal activation at 10-20 nM enzyme. This suggests that at higher enzyme concentrations interactions between Ca2+-ATPase polypeptide chains substitute for calmodulin-enzyme interactions, resulting in activation. In support of this interpretation, the inclusion of higher octaethyleneglycol mono-n-dodecyl ether concentrations shifts the half-maximal transformation to higher enzyme concentrations. Regardless of the detergent concentration, calmodulin decreases by about 2-fold the enzyme concentration required to observe half-maximal Ca2+-ATPase activation, without affecting the maximal velocity or cooperativity. This indicates that calmodulin facilitates interactions between enzyme molecules. The fluorescein-5'-isothiocyanate-modified Ca2+-ATPase shows an increase in fluorescence polarization which occurs over the same narrow concentration range that is seen with the Ca2+-ATPase activity, confirming association of enzyme molecules. Stimulation of the Ca2+-ATPase activity by calmodulin has revealed a stoichiometry of 0.73, with a dissociation constant of 1.6 nM calmodulin. We have demonstrated by use of calmodulin-Sepharose chromatography that both the calmodulin-dependent and independent Ca2+-ATPase forms bind calmodulin, even though stimulation of activity is seen only with the former one. Our data suggest the following two mechanisms for the Ca2+-ATPase activation: self-association of enzyme molecules or interaction with calmodulin.     

Electron microscope observations on Ca2+-ATPase microcrystals in detergent-solubilized sarcoplasmic reticulum

Crystalline arrays of Ca2+-ATPase molecules develop in detergent-solubilized sarcoplasmic reticulum during incubation for several weeks at 2 degrees C under nitrogen in a medium of 0.1 M KCl, 10 mM K-3-(N-morpholino)propanesulfonate, pH 6.0, 3 mM MgCl2, 20 mM CaCl2, 20% glycerol, 3 mM NaN3, 5 mM dithiothreitol, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 2 mg/ml protein, and 2-4 mg of detergent/mg of protein. Electron microscopy of sectioned, negatively stained, freeze-fractured, and frozen-hydrated Ca2+-ATPase crystals indicates that they consist of stacked lamellar arrays of Ca2+-ATPase molecules. Prominent periodicities of ATPase molecules within the lamellae arise from a centered rectangular lattice of dimensions 164 x 55.5 A. The association of lamellae into three-dimensional stacks is assumed to involve interactions between the exposed hydrophilic headgroups of ATPase molecules, that is promoted by glycerol and 20 mM Ca2+. Similar Ca2+-induced crystals were observed with purified or purified and delipidated Ca2+-ATPase preparations at lower detergent/protein ratios. Cross-linking of Ca2+-ATPase crystals with glutaraldehyde protects the structure against conditions such as low Ca2+, high pH, elevated temperature, SH group reagents, high concentration of detergents, and removal of phospholipids by extraction with organic solvents that disrupt unfixed preparations.     

Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum

The interaction between Ca2+-ATPase molecules in the native sarcoplasmic reticulum membrane and in detergent solutions was analyzed by chemical crosslinking, high performance liquid chromatography (HPLC), and by the polarization of fluorescence of fluorescein 5'-isothiocyanate (FITC) covalently attached to the Ca2+-ATPase. Reaction of sarcoplasmic reticulum vesicles with glutaraldehyde causes the crosslinking of Ca2+-ATPase molecules with the formation of dimers, tetramers and higher oligomers. At moderate concentrations of glutaraldehyde solubilization of sarcoplasmic reticulum by C12 E8 or Brij 36T (approximately equal to 4 mg/mg protein) decreased the formation of higher oligomers without significant interference with the appearance of crosslinked ATPase dimers. These observations are consistent with the existence of Ca2+-ATPase dimers in detergent-solubilized sarcoplasmic reticulum. Ca2+ (2-20 mM) and glycerol (10-20%) increased the degree of crosslinking at pH 6.0 both in vesicular and in solubilized sarcoplasmic reticulum, presumably by promoting interactions between ATPase molecules; at pH 7.5 the effect of Ca2+ was less pronounced. In agreement with these observations, high performance liquid chromatography of sarcoplasmic reticulum proteins solubilized by Brij 36T or C12 E10 revealed the presence of components with the expected elution characteristics of Ca2+-ATPase oligomers. The polarization of fluorescence of FITC covalently attached to the Ca2+-ATPase is low in the native sarcoplasmic reticulum due to energy transfer, consistent with the existence of ATPase oligomers (Highsmith, S. and Cohen, J.A. (1987) Biochemistry 26, 154-161); upon solubilization of the sarcoplasmic reticulum by detergents, the polarization of fluorescence increased due to dissociation of ATPase oligomers. Based on its effects on the fluorescence of FITC-ATPase, Ca2+ promoted the interaction between ATPase molecules, both in the native membrane and in detergent solutions.     

The binding of monoclonal and polyclonal antibodies to the Ca2(+)-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules.

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATPase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2(+)-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2(+)-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2(+)-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2(+)-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2(+)-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2(+)-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2(+)-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2(+)-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2(+)-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2(+)-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.     

Regulation of the (Na+ + K+)-ATPase in cultured chick skeletal muscle. Modulation of expression by the demand for ion transport

The levels of (Na+ + K+)-ATPase expression during muscle development and in response to modulation of demand for ion transport were studied in chick skeletal muscle cells in culture. The number of (Na+ + K+)-ATPase molecules on the myogenic cell surface, quantified with 125I-labeled monoclonal antibodies, increased 20-fold during muscle differentiation, with a substantial increase in (Na+ + K+)-ATPase molecules/unit area of membrane. The demand for sodium ion transport by the (Na+ + K+)-ATPase was modulated by activating voltage-sensitive sodium channels with veratridine or exposing cultures to low [K+]o (0.5 mM). Exposure to veratridine (10 microM) resulted in a 60-100% increase in cell surface and a smaller increase in intracellular (Na+ + K+)-ATPase over a 24-36-h period. Neither high [K+]o (50 mM) nor Ca2+ ionophore A23187 (1 microM) produced any such change, suggesting that neither membrane depolarization nor elevated cytosolic calcium was mediating the effect of veratridine. Veratridine stimulated up-regulation was specific for the (Na+ + K+)-ATPase, blocked by tetrodotoxin, and completely reversible. The kinetics of the reversal (down-regulation) process were much faster (t1/2 = 3 h) than those of up-regulation (t1/2 = 18 h). Up-regulation of the (Na+ + K+)-ATPase by veratridine occurred by a combination of two mechanisms: the first an early phase involving a stimulated biosynthesis of the (Na+ + K+)-ATPase and a later phase in which the biosynthetic rate returned to approximately control levels while the degradation rate slowed (t1/2 control = 31 h, t1/2 veratridine = 64 h).     

Comparison of the catalytic and structural properties of Ca2+-ATPase in longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum

The catalytic behavior and structural features of Ca2+-ATPase in the vesicles of longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum isolated from rabbit skeletal muscles was analysed. pH measurements have shown under optimal conditions Ca2+-ATPase has similar catalytic behavior both in the fractions of longitudinal tubules and terminal cisternae. Under non-optimal conditions, the behavior similarity was not observed. The specific activity of the ATPase enzyme under optimal conditions was shown to be much higher in the fraction of longitudinal tubules than in the fraction of terminal cisternae. Caffeine added to both fractions had no effect on the catalytic behavior of Ca2+-ATPase. As judged from fluorescence analysis, the structure of Ca2+-ATPase of longitudinal tubules differs from that structure of terminal cisternae. In sarcoplasmic reticulum membrane, at least half of the tryptophan residues of Ca2+-ATPase was shown to be buried in the lipid bilayer. Our findings suggest that in terminal cisternae some of the Ca2+-ATPase molecules exist as an oligomeric protein and do not participate in ATP hydrolysis (named "silent" Ca2+-ATPase).     

Characterisation of a high affinity Ca2+-stimulated, Mg2+-dependent ATPase in the rat parotid plasma membrane

Two Ca2+-stimulated ATPase activities have been identified in the plasma membrane of rat parotid: (a) a (Ca2+ + Mg2+)-ATPase with high affinity for free Ca2+ (apparent Km = 208 nM, Vmax = 188 nmol/min per mg) and requiring micromolar concentration of Mg2+ and (b) a (Ca2+ or Mg2+)-ATPase with relatively low affinity for free Ca2+ (K0.5 = 23 microM) or free Mg2+ (K0.5 = 26 microM). The low-affinity (Ca2+ or Mg2+)-ATPase can be maximally stimulated by Ca2+ alone or Mg2+ alone. The high-affinity (Ca2+ + Mg2+)-ATPase exhibits sigmoidal kinetics with respect to ATP concentration with K0.5 = 0.4 mM and a Hill coefficient of 1.91. It displays low substrate specificity with respect to nucleotide triphosphates. Although trifluoperazine inhibits the activity of the high affinity (Ca2+ + Mg2+)-ATPase only slightly, it inhibits the activity of the low-affinity (Ca2+ or Mg2+)-ATPase quite potently with 22 microM trifluoperazine inhibiting the enzymic activity by 50%. Vanadate, inositol 1,4,5-trisphosphate, phosphatidylinositol 4,5-bisphosphate, Na+,K+ and ouabain had no effect on the activities of both ATPases. Calmodulin added to the plasma membranes does not stimulate the activities of both ATPases. The properties of the high-affinity (Ca2+ + Mg2+)-ATPase are distinctly different from those of the previously reported Ca2+-pump activity of the rat parotid plasma membrane.     

Correlation between Ca2+-stimulated adenosine triphosphatase activity and Ca2+ uptake in microsomal fraction of rat submandibular gland

Both the Ca2+-ATPase activity and the Ca2+ uptake in a microsomal fraction of rat submandibular gland were inhibited by the addition of indomethacin in vitro. The decrease of both the Ca2+-ATPase activity and the Ca2+ uptake caused by the drug closely paralleled each other (r = 0.97). The inhibitory manner of indomethacin on Ca2+-ATPase and Ca2+ uptake was noncompetitive for Ca2+. These results suggest that the Ca2+-ATPase in the microsomal fraction of rat submandibular gland is a Ca2+ pump in this tissue.     

A Mg2+-independent high-affinity Ca2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg2+

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca2+-stimulated ATPase activity in the absence of Mg2+. The Ca2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity (Km = 0.4 microM) and a low affinity (Km = 0.6 mM) for Ca2+. Distribution of these high-affinity and low-affinity Ca2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg2+ also stimulates the ATPase in the absence of Ca2+. Unlike the Mg2+-ATPase and low-affinity Ca2+-ATPase, the plasmalemmal high-affinity Ca2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca2+-ATPase is noncompetitively inhibited by Mg2+ with respect to Ca2+ stimulation. Such an inhibitory effect of Mg2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg2+-independent, high-affinity Ca2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg2+-dependent, high-affinity Ca2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca2+-pump.     

Freeze-fracture study of water-soluble, standard proteins and of detergent-solubilized forms of sarcoplasmic reticulum Ca2+-ATPase

Conventional freeze-fracturing electron microscopy was used to study water-soluble proteins and different forms of Ca2+-ATPase-detergent complexes. Freeze-fracture images of solutions containing proteins larger than myoglobin showed the presence of distinct, randomly dispersed particles on smooth fracture surfaces. The distribution of sizes of these particles was closely to Gaussian, with a mean size which was correlated to the Stokes diameter. Monomeric Ca2+-ATPase from sarcoplasmic reticulum, solubilized by deoxycholate or a non-ionic detergent, showed a bimodal distribution of particle sizes. Even more complex distributions were found for dimeric and trimeric preparations of Ca2+-ATPase. The results can be interpreted on the assumption that the Ca2+-ATPase molecule is elongated, with an overall length of about 110 A and a width in its largest part of about 75 A. It is concluded on the basis of the presented results that freeze-fracture electron microscopy can be successfully used for morphological studies of protein molecules in solution.     

The plasma membrane Ca2+-ATPase protein from red blood cells is not modified in preeclampsia

Plasma membrane Ca2+-ATPase activity diminishes by about 50% in red blood cells during preeclampsia. We investigated whether the number of Ca2+-ATPase molecules is modified in red cell membranes from preeclamptic pregnant women by measuring the specific phosphorylated intermediate of this enzyme. Also, we isolated the Ca2+-ATPase protein from both normotensive and preeclamptic pregnant women and estimated its molecular weight, and its cross-reactions with specific polyclonal and monoclonal (5F10) antibodies against it. We measured the Ca2+-ATPase activity in a purified state and the effect of known modulators of this ATPase. It was found that the phosphorylated intermediate associated with PMCA is similar for red cell ghosts from normotensive and preeclamptic women, suggesting a similar number of ATPase molecules in these membranes. The molecular weight of the Ca2+-ATPase is around 140 kDa for both normotensive and preeclamptic membranes, and its cross-reactions with specific antibodies is similar, suggesting that the protein structure remains intact in preeclampsia. Calmodulin, ethanol, or both calmodulin plus ethanol, stimulated the Ca2+-ATPase activity to the same extent for both normotensive and preeclamptic preparations. Our results showed that the reduced Ca2+-ATPase activity of the red cell membranes from preeclamptic women is not associated with a defective enzyme, but rather with a high level of lipid peroxidation.     

Gel to liquid crystalline phase transition promotes a conformational reorganization of Ca2+, Mg2+-ATPase from sarcoplasmic reticulum in dimyristoylphosphatidylcholine reconstituted systems

Sarcoplasmic reticulum Ca2+, Mg2+-ATPase has been reconstituted in membranes highly enriched in dimyristoylphosphatidylcholine. According to electron microscopy data these membranes form vesicles of an average diameter of 1000 +/- 200 A. These reconstituted membranes show hysteretic behavior in some physical-chemical properties, such as light scattering and fluorescence when labeled with iodoacetamidofluorescein and with N-iodoacetyl-N'-(5-sulfo-1-naphthyl) ethylenediamine. Hysteretic behavior in catalytic activity can also be inferred from the kinetic data presented in this paper, because the temperature dependence of the Ca2+, Mg2+-ATPase activity is altered by a mild thermal pretreatment of the samples. Furthermore, it was noticed that the Ca2+-dependent ATPase activity of these complexes, when assayed above the phase transition temperature (Tc) of the lipid matrix, showed a lag phase in the minute time scale range. On the basis of these findings, it is suggested that the gel-to-liquid crystalline phase transition of the lipid is able to shift the conformational equilibrium E----E* of Ca2+, Mg2+-ATPase. The fact that the -SH reactivity against 5,5'-dithio-bis-nitrobenzoic acid of these complexes is also altered by preincubation above Tc for several minutes also supports that lipid melting induces a conformational change in Ca2+, Mg2+-ATPase.