Reconstruction of purified Ca2+,Mg2+-ATPase from the myometrium sarcolemma into liposomes and its catalytic properties

The Ca2+, Mg2(+)-ATPase of the myometrium sarcolemma purified by the method of affinity chromatography on calmodulin sepharose is reconstituted into azolectin liposomes in the functionally active form by means of cholate dialysis. The ATPase-dependent accumulation of 45Ca is shown on the obtained model system. It makes up 95% of the total accumulation and may decrease to 43% under the effect of 0.8 microM A23187. Ca2+, Mg2(+)-ATPase reconstituted into azolectin liposomes is in the high affinity to Ca2+; Km for Ca2+ is equal to 0.88 +/- 0.22 microM, calmodulin practically does not change it. The highest activity of the reconstituted enzyme is observed at pH 7.0, temperature 50 degrees C, the Mg-ATP concentration 1-2 mM. The Km for substrate is 0.45 +/- 0.02 mM.     

A freeze-fracture study of the aggregation state of Ca2+,Mg2+-ATPase of sarcoplasmic reticulum in reconstituted vesicles at low and high temperature

Since it was possible for Ca2+,Mg2+-ATPase of sarcoplasmic reticulum (SR) to change its aggregation state in the membrane depending on temperature, and since the change could be the cause of the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity, the aggregation state of Ca2+,Mg2+-ATPase at 0 degrees C in the membrane was compared with that at 35 degrees C by freeze-fracture electron microscopy. These temperatures are below and above the break in the Arrhenius plot (about 18 degrees C), respectively. Two kinds of samples were used; fragmented SR vesicles and egg PC-ATPase vesicles, a reconstituted preparation from purified Ca2+,Mg2+-ATPase and egg yolk phosphatidylcholine (egg PC). For both the appearance of particles in the fracture faces of the samples fixed at 0 degrees C was similar to that at 35 degrees C, and phase separation between protein and lipid was not observed even at 0 degrees C. The size of the particles was measured and histograms of the sizes at 0 degrees C and 35 degrees C were made. The histogram at 0 degrees C was similar to that at 35 degrees C with a peak at 7.1 nm, which is 1-2 nm smaller than the value reported so far. The number of the particles per unit area of the membrane was also counted. The value at 0 degrees C was similar to that at 35 degrees C. These results indicate that Ca2+,Mg2+-ATPase of SR exists in the same aggregation state (estimated as oligomer based on the values obtained in this experiment) between 0 degrees C and 35 degrees C. Based on the results of this study we think that the break in the Arrhenius plot of Ca2+,Mg2+-ATPase activity in SR is not caused by the change in the aggregation state of Ca2+,Mg2+-ATPase.     

Thermal instability of rat muscle sarcoplasmic reticulum Ca(2+)-ATPase function

To examine the thermal instability and the role of sulfhydryl (SH) oxidation on sarcoplasmic reticulum (SR) Ca(2+)-ATPase function, crude homogenates were prepared from the white portion of the gastrocnemius (WG) adult rat muscles (n = 9) and incubated in vitro for < or =60 min either at a normal resting body temperature (37 degrees C) or at a temperature indicative of exercise-induced hyperthermia (41 degrees C) with DTT and without DTT (CON). In general, treatment with DTT resulted in higher Ca(2+)-ATPase and Ca(2+) uptake values (nmol. mg protein(-1). min(-1), P < 0.05), an effect that was not specific to time of incubation. Incubations at 41 degrees C resulted in lower (P < 0.05) Ca(2+) uptake rates (156 +/- 18 and 35.9 +/- 3.3) compared with 37 degrees C (570 +/- 54 and 364 +/- 26) at 30 and 60 min, respectively. At 37 degrees C, ryanodine (300 microM), which was used to block Ca(2+) release from the calcium release channel, prevented the time-dependent decrease in Ca(2+) uptake. A general inactivation (P < 0.05) of maximal Ca(2+)-ATPase activity (V(max)) in CON was observed with incubation time (0 > 30 > 60 min), with the effect being more pronounced (P < 0.05) at 41 degrees C compared with 37 degrees C. The Hill slope, a measure of co-operativity, and the pCa(50), the cytosolic Ca(2+) concentration required for half-maximal activation of Ca(2+)-ATPase activity, decreased (P < 0.05) at 41 degrees C only. Treatment with DTT attenuated the alterations in enzyme kinetics. The increase in V(max) with the Ca(2+) ionophore A-23187 was less pronounced at 41 degrees C compared with 37 degrees C. It is concluded that exposure of homogenates to a temperature typically experienced in exercise results in a reduction in the coupling ratio, which is mediated primarily by lower Ca(2+) uptake and occurs as a result of increases in membrane permeability to Ca(2+). Moreover, the decreases in Ca(2+)-ATPase kinetics in WG with sustained heat stress result from SH oxidation.     

Protein kinase C-dependent phosphorylation of sarcolemmal Ca2(+)-ATPase isolated from bovine aortic smooth muscle

We examined the effect of protein kinase C (PKC)-dependent phosphorylation on Ca2+ uptake and ATP hydrolysis by microsomal as well as purified sarcolemmal Ca2(+)-ATPase preparations isolated from bovine aortic smooth muscle. The phosphorylation was performed by treating these preparations with PKC and saturating concentrations of ATP (or ATP-gamma S), Ca2+, and 12-O-tetradecanoyl phorbol-13-acetate (TPA) at 37 degrees C for 10 min. In microsomes, treatment with PKC enhanced a portion of the Ca2+ uptake activity inhibitable by 10 microM vanadate, by up to about 30%. On the other hand, Ca2(+)-dependent ATPase activity in the purified Ca2(+)-ATPase preparation was stimulated by up to twofold. Up to twofold stimulation by PKC was also observed for the Ca2+ uptake by proteoliposomes reconstituted from purified sarcolemmal Ca2(+)-ATPase and phospholipids. Since these effects were evident only at Ca2+ concentrations between 0.1 to 1.0 microM, we concluded that it was the affinity of the Ca2(+)-ATPase for Ca2+ that was increased by the PKC treatment. Under conditions in which PKC increased Ca2+ pump activity, the sarcolemmal Ca2(+)-ATPase was phosphorylated to a level of about 1 mol per mol of the enzyme. There was good parallelism between the ATPase phosphorylation and the extent of enzyme activation. These results strongly suggest that the activity of the sarcolemmal Ca2+ pump in vascular smooth muscle is regulated through its direct phosphorylation by PKC.     

Selective inhibition by lasalocid of hydrolysis of the ADP-insensitive phosphoenzyme in the catalytic cycle of sarcoplasmic reticulum Ca2(+)-ATPase

The effect of a carboxylic ionophore (lasalocid) on the sarcoplasmic reticulum Ca2(+)-ATPase was investigated. The purified enzyme was preincubated with lasalocid in the presence of Ca2+ and the absence of K+ at pH 7.0 and 0 degrees C for 2 h. The Ca2(+)-dependent ATPase activity was strongly inhibited by this preincubation, whereas the activity of the contaminant Mg2(+)-ATPase was unaffected. The steady-state level of the phosphoenzyme (EP) intermediate remained constant over the wide range of lasalocid concentrations. The Ca2(+)-induced enzyme activation was unaffected. The kinetics of phosphorylation of the Ca2(+)-activated enzyme by ATP as well as the rate of conversion of ADP-sensitive EP to ADP-insensitive EP were also unaffected. Accumulation of ADP-insensitive EP was greatly enhanced, and almost all of the EP accumulating at steady state was ADP-insensitive. Hydrolysis of ADP-insensitive EP was strongly inhibited. A similar strong inhibition of the Ca2(+)-dependent ATPase activity by lasalocid was found with sarcoplasmic reticulum vesicles. To examine the effect of lasalocid on the conformational change in each reaction step, the Ca2(+)-ATPase of sarcoplasmic reticulum vesicles was labeled with a fluorescent probe (N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine) without a loss of catalytic activity and then preincubated with lasalocid as described above. The conformational changes involved in hydrolysis of ADP-insensitive EP and in the reversal of this hydrolysis were appreciably retarded by lasalocid. The conformational changes involved in other reaction steps were unaffected. These results demonstrate that hydrolysis of ADP-insensitive EP in the catalytic cycle of this enzyme is selectively inhibited by lasalocid.     

Low temperature limits photoperiod control of smolting in atlantic salmon through endocrine mechanisms

We have examined the interaction of photoperiod and temperature in regulating the parr-smolt transformation and its endocrine control. Atlantic salmon juveniles were reared at a constant temperature of 10 degrees C or ambient temperature (2 degrees C from January to April followed by seasonal increase) under simulated natural day length. At 10 degrees C, an increase in day length [16 h of light and 8 h of darkness (LD 16:8)] in February accelerated increases in gill Na(+)-K(+)-ATPase activity, whereas fish at ambient temperature did not respond to increased day length. Increases in gill Na(+)-K(+)-ATPase activity under both photoperiods occurred later at ambient temperature than at 10 degrees C. Plasma growth hormone (GH), insulin-like growth factor, and thyroxine increased within 7 days of increased day length at 10 degrees C and remained elevated for 5-9 wk; the same photoperiod treatment at 2 degrees C resulted in much smaller increases of shorter duration. Plasma cortisol increased transiently 3 and 5 wk after LD 16:8 at 10 degrees C and ambient temperature, respectively. Plasma thyroxine was consistently higher at ambient temperature than at 10 degrees C. Plasma triiodothyronine was initially higher at 10 degrees C than at ambient temperature, and there was no response to LD 16:8 under either temperature regimen. There was a strong correlation between gill Na(+)-K(+)-ATPase activity and plasma GH; correlations were weaker with other hormones. The results provide evidence that low temperature limits the physiological response to increased day length and that GH, insulin-like growth factor I, cortisol, and thyroid hormones mediate the environmental control of the parr-smolt transformation.     

Temperature and Ca(2+)-dependence of the sarcoplasmic reticulum Ca(2+)-ATPase in haddock,salmon, rainbow trout and zebra cichlid

Temperature dependence of Ca(2+)-ATPase from the sarcoplasmic reticulum (SR) in rabbit muscle has been widely studied, and it is generally accepted that a break point in Arrhenius plot exist at approximately 20 degrees C. Whether the break point arises as a result of temperature dependent changes in the enzyme or its membrane lipid environment is still a matter of discussion. In this study we compared the temperature dependence and Ca(2+)-dependence of SR Ca(2+)-ATPase in haddock (Melanogrammus aeglefinus), salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and zebra cichlid (Cichlasoma nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degrees C, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the temperature area, 6-14 degrees C. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca(2+)-ATPase activity. The temperature range of the plateau was 14-21 and 18-25 degrees C in salmon and rainbow trout, respectively. Ca(2+)-dependence in the four different fish species investigated was very similar with half maximal activation (K(0.5)) between 0.2 and 0.6 micro M and half maximal inhibition (I(0.5)) between 60 and 250 micro M. Results indicated that interaction between SR Ca(2+)-ATPase and its lipid environment may play an important role for the different Arrhenius plot of the different types of fish species investigated.     

The effect of temperature on juvenile Mozambique tilapia hybrids (Oreochromis mossambicus x O. urolepis hornorum) exposed to full-strength and hypersaline seawater

The effects of temperature on the salinity tolerance of Mozambique-Wami tilapia hybrids (Oreochromis mossambicus x O. urolepis hornorum) were investigated by transferring 35 g/l, 25 degrees C-acclimated fish to 35, 43, 51 or 60 g/l salinity at 15, 25 or 35 degrees C for 24 h, and by assaying gill tissue for branchial Na(+), K(+)-ATPase activity at the three temperatures after acclimating the fish to 15, 25 or 35 degrees C for 2 weeks. Tilapia survived all salinities at 25 and 35 degrees C; however, at 15 degrees C, mortality was 85.7% and 100% in the 51 g/l and 60 g/l groups, respectively. There was a significant interaction between temperature and salinity, as plasma osmolality, [Na(+)] and [Cl(-)] were significantly increased at 51 and 60 g/l salinity in 35 degrees C water (P<0.001). Additionally, muscle water content was significantly reduced at 43 g/l, 15 degrees C relative to pre-transfer values (P<0.001). Branchial Na(+), K(+)-ATPase activity was reduced at 15 degrees C regardless of acclimation temperature, and 25 degrees C-acclimated gill tissue did not show an increase in activity when assayed at 35 degrees C. Results indicate that the effects of a combined temperature-salinity transfer on plasma osmolality and ion concentrations, as well as muscle water content, are greater than when either challenge is given alone. Additionally, branchial Na(+), K(+)-ATPase activity is altered when assayed at varying temperatures; in the case of 15 degrees C, regardless of acclimation temperature. Our enzyme activity data may indicate the presence of a high temperature isoform of branchial Na(+), K(+)-ATPase enzyme.     

The state of lipid peroxidation and enzymes of calcium transport system in sarcoplasmic reticulum of ischemic myocardium

In 30 experiments on mongrel dog hearts it was shown that 30 min of total ischemia (37 degrees C) followed by accumulation of MDA in the SR membranes and decrease of their Ca2+-uptake, but had no effect on activity Ca2+-ATPase. After 60-120 min ischemia marked a decrease of Ca2+-uptake and activity Ca2+-ATPase took place, MDA content remained at the increased level. The results show that lipid peroxidation take part in the increase of the permeability of SR membranes for Ca2+ and inhibiting of Ca2+-ATPase.     

Effects of bacterial lipopolysaccharide and calmodulin on Ca2(+)-ATPase and calcium in human natural killer cells, studied by a combined technique of immunoelectron microscopy and ultracytochemistry

Our previous studies indicate that bacterial lipopolysaccharide (LPS) enhances natural killer (NK) cell-mediated cytotoxicity and increases intracellular calcium (Ca2+) in hepatocytes. Calmodulin (CAM) regulates Ca2(+)-ATPase activity, intracellular Ca2+, and is also implicated in NK cell-mediated cytolysis. In the present work, the effects of LPS and CAM on Ca2(+)-ATPase and intracellular Ca2+ in human NK cells were studied by a combined technique of immunogold electron microscopy and ultracytochemistry. Peripheral blood mononuclear cells were treated with 100 micrograms/ml E. coli (0111:B4) LPS and/or 5 micrograms/ml CAM in RPMI 1640 medium at 37 degrees C for 1 or 4 hr. NK cells labeled with monoclonal anti-Leu-11a (CD16) antibody and colloidal gold-conjugated anti-mouse IgG were processed for cytochemical localization of Ca2(+)-ATPase and Ca2+. Ca2(+)-ATPase was localized in the plasma membrane of NK cells, and its activity was suppressed by LPS but was enhanced by CAM. However, no apparent changes in the enzyme reaction were observed when cells were exposed to CAM concomitantly with LPS or stimulated with LPS before CAM. Apparent reduction of the enzyme reaction was observed when LPS stimulation was preceded by CAM. Ca2(+)-ATPase reaction in mitochondria was observed only in NK cells exposed to CAM. Computer image analysis showed no changes in the intracellular Ca2+ in NK cells treated with LPS for 1 hr, whereas a significant increase in Ca2+ was found in cells exposed to LPS for 4 hr. The intracellular Ca2+ significantly decreased in NK cells treated with CAM or with a combination of LPS and CAM as compared to that of controls (p less than 0.05). The results indicate that CAM is capable of blocking or reversing the inhibitory effect of LPS on Ca2(+)-ATPase, and suggest that in human NK cells the plasma membrane-associated Ca2(+)-ATPase is responsible for extrusion of intracellular Ca2+.     

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).     

Erythrocyte-ghost Ca2+-stimulated Mg2+-dependent adenosine triphosphatase in Duchenne muscular dystrophy.

The Ca2+-stimulated Mg2-dependent ATPase activities (Ca2+-ATPase) of erythrocyte-ghost membranes from patients with Duchenne muscular dystrophy (DMD) and carriers of DMD were compared with activities of normal controls. The Ca2+-ATPase activity of DMD-patient ghost preparations was found to follow the same pattern of activation by Ca2+ as the control membranes. However, the Ca2+-ATPase activity in DMD and some DMD-carrier preparations was substantially elevated compared with controls. To characterize further the elevated Ca2+-ATPase activity found in DMD-patient ghost membrane preparations, we estimated kinetic parameters using both fine adjustment and weighting methods to analyse our experimental data. It was established that in both DMD and DMD-carrier preparations the increase in Ca2+-ATPase activity was reflected by a significant increase in Vmax. rather than by any change in Km. The response of the membrane Ca2+-ATPase activity to changes in temperature was also investigated. In all preparations a break in the Arrhenius plot occurred at 20 degrees C, and in DMD and DMD-carrier preparations an elevated Ca2+-ATPase activity was detected at all temperatures. Above 20 degrees C the activation energy for all types of preparation was the same, whereas below this temperature there appeared to be an elevated activation in DMD and DMD-carrier preparations compared with normal controls. The concept that a generalized alteration in the physicochemical nature of the membrane lipid domain may be responsible for the many abnormal membrane properties reported in DMD is discussed.     

Variation of scallop sarcoplasmic reticulum Ca2+-ATPase activity with temperature

Methods for preparing native scallop sarcoplasmic reticulum vesicles, largely purified membranous scallop sarcoplasmic reticulum Ca2+-ATPase, and nonionic detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase are described. The effect of a range of polyoxyethylene-based detergents on the solubilized Ca2+-ATPase was tested. Decaethylene glycol dodecyl ether (C12E10) supported the highest levels of activity, although C12E8 and C12E9 were more routinely used. Arrhenius plots of Ca2+-ATPase activity, where the assays were carried out with the same pH at all temperatures (7.4), showed a region of nonlinearity at 10 degrees C. A very similar plot was obtained when no compensation was made for pH variation with temperature. Both the break in the Arrhenius plot and the activation energies for the scallop sarcoplasmic reticulum above and below the break were very similar to those found for lobster sarcoplasmic reticulum (Madeira, V. M. C., Antunes-Madeira, M. C., and Carvalho, A. R. (1974) Biochem. Biophys. Res. Commun. 65, 997-1003). The Arrhenius plot of the scallop Ca2+-ATPase in C12E8 no longer showed the nonlinearity at 10-12 degrees C seen with the native sarcoplasmic reticulum, but instead a break now appeared at 20-21 degrees C. This is close to the Arrhenius break temperature of rabbit Ca2+-ATPase in C12E8 and of a perturbation in C12E8 (Dean, W. L. (1982) Biophys. J. 37, 56-57).     

Emerging views on the structure and dynamics of the Ca2(+)-ATPase in sarcoplasmic reticulum

The ATP-dependent Ca2+ transport in sarcoplasmic reticulum involves transitions between several structural states of the Ca2(+)-ATPase, that occur without major changes in the secondary structure. The rates of these transitions are modulated by the lipid environment and by interactions between ATPase molecules. Although the Ca2(+)-ATPase restricts the rotational mobility of a population of lipids, there is no evidence for specific interaction of the Ca2(+)-ATPase with phospholipids. Fluorescence polarization and energy transfer (FET) studies, using site specific fluorescent indicators, combined with crystallographic, immunological and chemical modification data, yielded a structural model of Ca2(+)-ATPase in which the binding sites of Ca2+ and ATP are tentatively identified. The temperature dependence of FET between fluorophores attached to different regions of the ATPase indicates the existence of 'rigid' and 'flexible' regions within the molecule characterized, by different degrees of thermally induced structural fluctuations.     

Adenosine triphosphatases during maturation of cultured human skeletal muscle cells and in adult human muscle.

Na+/K(+)-ATPase, Mg(2+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase are examined in cultured human skeletal muscle cells of different maturation grade and in human skeletal muscle. Na+/K(+)-ATPase is investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase). SR Ca(2+)-ATPase is examined by ELISA, Ca(2+)-dependent phosphorylation and its activities on ATP and 3-O-methylfluorescein phosphate. Na+/K(+)-ATPase and SR Ca(2+)-ATPase are localized by immunocytochemistry. The activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase show a good correlation with the other assayed parameters of these ion pumps. All ATPase parameters investigated increase with the maturation grade of the cultured muscle cells. The number of ouabain-binding sites and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-MFPase are significantly higher in cultured muscle cells than in muscle. The Mg(2+)-ATPase activity, the content of SR Ca(2+)-ATPase and the activities of SR Ca(2+)-ATPase and Ca(2+)-dependent 3-O-MFPase remain significantly lower in cultured cells than in muscle. The ouabain-binding constant and the molecular activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase are equal in muscle and cultured cells. During ageing of human muscle the activity as well as the concentration of SR Ca(2+)-ATPase decrease. Thus the changes of the activities of the ATPases are caused by variations of the number of their molecules. Na+/K(+)-ATPase is localized in the periphery of fast- and slow-twitch muscle fibers and at the sarcomeric I-band. SR Ca(2+)-ATPase is predominantly confined to the I-band, whereas fast-twitch fibers are much more immunoreactive than slow-twitch fibers. The presence of cross-striation for Na+/K(+)-ATPase and SR Ca(2+)-ATPase in highly matured cultured muscle cells indicate the development and subcellular organization of a transverse tubular system and SR, respectively, which resembles the in vivo situation.     

Ca2+ stimulates the Mg2(+)-ATPase activity of brush border myosin I with three or four calmodulin light chains but inhibits with less than two bound.

Brush border myosin I from chicken intestinal microvilli is a membrane-associated, single-headed myosin composed of a 119-kDa heavy chain and several calmodulin light chains. We first describe in detail a new procedure for the rapid purification of brush border myosin I in greater than 99% purity with a yield of 40%, significantly higher than for previous methods. The subunit stoichiometry was determined to be 4 calmodulin light chains/myosin I heavy chain by amino acid compositional analysis of the separated subunits. We have studied the effects of Ca2+ and temperature on dissociation of calmodulin from myosin I and on myosin I Mg2(+)-ATPase and contractile activities. At 30 degrees C the actin-activable ATPase activity is stimulated 2-fold at 10-700 microM Ca2+. Dissociation of 1 calmodulin occurs at 25-50 microM Ca2+, but this has no effect on actin activation. The contractile activity of myosin I, expressed as superprecipitation, is greatly enhanced by Ca2+ under conditions in which 1 calmodulin is dissociated. This calmodulin is thus not essential for actin activation or superprecipitation. Myosin I was found to be highly temperature-sensitive, with an increase to 37 degrees C resulting in dissociation of 1 calmodulin at below 10(-7) M Ca2+ and an additional 1.5 calmodulins at 1-10 microM Ca2+. A complete loss of actin activation accompanies the Ca2(+)-induced calmodulin dissociation at 37 degrees C. Our conclusion is that physiological levels of Ca2+ can either stimulate or inhibit the mechanoenzyme activities of brush border myosin I in vitro, with the mode of regulation determined by the number of associated calmodulin light chains.     

Interaction of ATP with sarcoplasmic reticulum Ca2+-ATPase; effect on the conformational state of the enzyme

It was studied how temperature influences the NBD-Cl inactivation of sarcoplasmic reticulum Ca2+-ATPase and the protective effect of ATP under conditions preventing ATP hydrolysis. Two types of ATP-binding sites with Kd equal to 30 and 220 microM at 37 degrees C were found. ADP interacts with these sites with the (K'd = 20 and 200 microM). The temperature decrease from 25 degrees to 5 degrees C induces the abrupt increase in the Kd for the low affinity site. The possible reasons for heterogeneity of ATP-binding sites are discussed. The conclusion is made that interaction of monomers in oligomeric complex of Ca2+-ATPase induces heterogeneity of ATP-binding sites.     

Interaction of the local anesthetics dibucaine and tetracaine with sarcoplasmic reticulum membranes. Differential scanning calorimetry and fluorescence studies

The local anesthetics dibucaine and tetracaine inhibit the (Ca2+ + Mg2+)-ATPase from skeletal muscle sarcoplasmic reticulum [DeBoland, A. R., Jilka, R. L., & Martonosi, A. N. (1975) J. Biol. Chem. 250, 7501-7510; Suko, J., Winkler, F., Scharinger, B., & Hellmann, G. (1976) Biochim. Biophys. Acta 443, 571-586]. We have carried out differential scanning calorimetry and fluorescence measurements to study the interaction of these drugs with sarcoplasmic reticulum membranes and with purified (Ca2+ + Mg2+)-ATPase. The temperature range of denaturation of the (Ca2+ + Mg2+)-ATPase in the sarcoplasmic reticulum membrane, determined from our scanning calorimetry experiments, is ca. 45-55 degrees C and for the purified enzyme ca. 40-50 degrees C. Millimolar concentrations of dibucaine and tetracaine, and ethanol at concentrations higher than 1% v/v, lower a few degrees (degrees C) the denaturation temperature of the (Ca2+ + Mg2+)-ATPase. Other local anesthetics reported to have no effect on the ATPase activity, such as lidocaine and procaine, did not significantly alter the differential scanning calorimetry pattern of these membranes up to a concentration of 10 mM. The order parameter of the sarcoplasmic reticulum membranes, calculated from measurements of the polarization of the fluorescence of diphenylhexatriene, is not significantly altered at the local anesthetic concentrations that shift the denaturation temperature of the (Ca2+ + Mg2+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic properties of purified Ca2+,Mg2+-ATPase from the myometrium sarcolemma

The catalytic properties of myometrium sarcolemmal Ca2+, Mg2(+)-ATPase purified from plasma membrane solubilizate by affinity chromatography on calmodulin-Sepharose were investigated. The enzyme isolated in the presence of azolectin revealed a calmodulin-independent affinity for Ca2+ (Km = 0.17 microM). Purified Ca2+, Mg2(+)-ATPase displayed a strict substrate specificity, was inhibited by low concentrations of o-vanadate and was insensitive to oxytocin and prostaglandins E2 and F2 alpha. The enzyme activity was maximal at 45 degrees C, pH 7.5-8.0, and at Mg-ATP and Ca2+ concentrations of 1.5-2.5 mM and 5-20 microM, respectively.