Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5-5 microM) follows an exponential curve described by the general formula y = a.ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 microM cholesterol glucoside, with a Hill coefficient of h = 2.98 +/- 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 microM) or its glucoside (up to 50 microM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 microM) incorporation. Cholesterol (5 microM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 +/- 1.1 degrees C in control SPM, which was elevated to 29.5 +/- 1.4 degrees C in SPM treated with cholesterol glucoside (50 microM) and abolished in SPM treated with cholesterol (5 microM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F- and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 microM) produced an increased packing of the bulk lipids, while cholesterol (5 microM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.     

Ouabain inhibition of kidney ATPase is altered by 9.14 GHz radiation.

At each of several stabilized temperatures between 7.0 and 43.8 degrees C, increases in dog-kidney, Na(+)-, K(+)-ATPase catalytic activity were usually observed in association with exposure for 5 min to 9.14 GHz CW microwave radiation in a thin tubular reactor. However, at 24.9 degrees C, a 23% decrease occurred. Comparisons of activity of ouabain-inhibited reactions revealed that the efficacy of the cardiac glycoside as an inhibitor of ATPase activity was severely diminished by the microwave field. The ouabain-site control mechanism may be a specific microwave target at this exposure frequency. Experimental results can be interpreted in terms of molecular structural changes or direct energy input. The estimated SAR of energy that was incident on preparations is 20 W/kg.     

Effect of amiodarone on membrane fluidity and Na+/K+ ATPase activity in rat-brain synaptic membranes

In rat-brain synaptic membranes at a fixed temperature (37 degrees C), amiodarone dose-dependently inhibits the Na+/K+ ATPase activity (IC50 approximately equal to 2.10(-5)M) and produces a linear increase in the degree of fluorescence depolarization (P) of 1,6-diphenylhexatriene embedded in the lipid matrix. Amiodarone has no effect on Mg++ ATPase and K+PNPase activity up to 3.10(-4)M. Studies carried out at different temperatures indicate that 10(-5)M amiodarone inhibits the Na+/K+ ATPase and decreases the lipid fluidity at all the temperatures studied (9 - 40 degrees C). The compound significantly displaces the temperature of transition observed around 20 degrees C in both Na+/K+ ATPase activity and lipid fluidity to 24 degrees C with no changes in slopes. The results suggest that part of the selective inhibition of Na+/K+ ATPase activity by amiodarone could be due to the effects of the drug on lipid dynamics.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Temperature-dependencies of various catalytic activities of membrane-bound Na+/K+-ATPase from ox brain, ox kidney and shark rectal gland and of C12E8-solubilized shark Na+/K+-ATPase

The temperature dependence of ouabain-sensitive ATPase and phosphatase activities of membrane fragments containing the Na+/K+-ATPase were investigated in tissue from ox kidney, ox brain and from shark rectal glands. The shark enzyme was also tested in solubilized form. Arrhenius plots of the Na+/K+-ATPase activity seem to be linear up to about 20 degrees C, and non-linear above this temperature. The Arrhenius plots of mammalian enzyme (ox brain and kidney) were steeper, especially at temperatures below 20-30 degrees C, than that of shark enzyme. The Na+-ATPase activity showed a weaker temperature-dependence than the Na+/K+-ATPase activity. The phosphatase reactions measured, K+-stimulated, Na+/K+-stimulated and Na+/K+/ATP-stimulated, also showed a weaker temperature-dependence than the overall Na+/K+-ATPase activity. Among the phosphatase reactions, the largest change in slope of the Arrhenius plot was observed with the Na+/K+/ATP)-stimulated phosphatase reaction. The Arrhenius plots of the partial reactions were all non-linear. Solubilization of shark enzyme in C12E8 did not change the curvature of Arrhenius plots of the Na+/K+-ATPase activity or the K+-phosphatase activity. Since solubilization involves a disruption of the membrane and an 80% delipidation, the observed curvature of the Arrhenius plot can not be attributed to a property of the membrane as such.     

Temperature dependence of Na+/Ca2+ exchange activity in beef-heart sarcolemmal vesicles and proteoliposomes

Temperature dependence of Na+/Ca2+ exchange activity was studied in beef cardiac sarcolemmal vesicles in the absence and presence of the inhibitor amiloride and in proteoliposomes reconstituted with different lipid mixtures. Arrhenius plots for Na+/Ca2+ exchange activity in both control and amiloride-treated vesicles revealed an apparent energy of activation of 9665 +/- 585 (SE, n = 4) cal/mol, corresponding to a temperature coefficient (Q10) value of 1.70 +/- 0.05 (SE, n = 4) over the range 25-37 degrees C. When Na+/Ca2+ exchange was reconstituted into phosphatidylcholine (PC):phosphatidylserine (PS) (52:48, mol/mol), PC:PS:cholesterol (25:39:36, mol/mol), and PC:PS:distearoylphosphatidylcholine (DSPC) (31:48:21, mol/mol) proteoliposomes, the highest activity was found in PC:PS:cholesterol proteoliposomes. Arrhenius plots of Na+/Ca2+ exchange activity exhibited breakpoints at 23 degrees C (PC:PS), 33 degrees C (PC:PS:cholesterol), and 23 degrees C (PC:PS:DSPC). The increase in the thermotropic transition temperature with cholesterol could result from the condensing effect of this sterol, whereas the breaks observed with PC:PS and PC:PS:DSPC could be caused by a non-lipid-mediated membrane protein conformational change. These results indicate that the lipid microenvironment around the Na+/Ca2+ exchanger and the nature of the specific lipid-protein interactions influence the activity of this antiporter. Further evidence supporting the hypothesis that cholesterol behaves as a specific positive effector for the exchanger is also given.     

Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system.

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.     

Membrane-bound Na,K-ATPase: target size and radiation inactivation size of some of its enzymatic reactions

Frozen samples of membrane-bound pig kidney Na,K-ATPase were subjected to target size analysis by radiation inactivation with 10-MeV electrons at -15 degrees C. The various properties investigated decreased monoexponentially with radiation dose, and the decay constants, gamma, were independent of the presence of other proteins and of sucrose concentrations above 0.25 M. The temperature factor was the same as described by others. Irradiation of four proteins of known molecular mass, m, showed that gamma for protein integrity was proportional to m with a proportionality factor about 20% higher than that conventionally used. By this standard curve, glucose-6-phosphate dehydrogenase activity used as internal standard gave a radiation inactivation size of 110 +/- 5 kDa, very close to m = 104-108 kDa for the dimer, as expected. For Na+/K+-transporting ATPase the following target sizes and radiation inactivation size values were very close to m = 112 kDa for the alpha-peptide: peptide integrity of alpha, 115 kDa; unmodified binding sites for ATP and vanadate, 108 kDa; K+-activated p-nitrophenylphosphatase activity, 106 kDa. There was thus no sign of dimerization of the alpha-peptide or involvement of the beta-peptide. In contrast, optimal Na+/K+-transporting ATPase activity had a radiation inactivation size = 189 +/- 7 kDa, and total nucleotide binding capacity corresponded to 72 +/- 3 kDa. These latter results will be extended and discussed in a forthcoming paper.     

The Resting Potential of Mouse Leydig Cells: Role of an Electrogenic Na+/K+ Pump

Resting potentials (Vm) were measured in mouse Leydig cells, using the whole-cell patch-clamp technique. In contrast to conventional microelectrode measurements, where a biphasic potential was observed, we recorded a stable Vm around -32.2 +/- 1.2 mV (mean +/- SEM, n = 159), at 25 degrees C, and an input resistance larger than 2.7 x 109 W. Although Vm is sensitive to changes in the extracellular concentrations of potassium and chloride, the relationship between Vm and these ions' concentrations cannot be described by either the Goldman-Hodgkin-Katz or the Nernst equation. Perifusing cells with potassium-free solution or 10?3 M ouabain induced a marked depolarization averaging 20.1 +/- 3.2 mV (n = 9) and 23.1 +/- 2.8 mV, (n = 7), respectively. Removal of potassium or addition of ouabain with the cell voltage-clamped at its Vm, resulted in an inwardly directed current, due to inhibition of the Na+K+ATPase. The pump current increased with temperature with a Q10 coefficient of 2.3 and had an average value of -6.5 +/- 0.4 pA (n = 21) at 25 degrees C. Vm also varied strongly with temperature, reaching values as low as -9.2 +/- 1.2 mV (n = 22) at 15 degrees C. Taking the pump current at 25 degrees C and a minimum estimate for the membrane input resistance, we can see that the Na+K+ATPase could directly contribute with 17.7 mV to the Vm of Leydig cells, which is a major fraction of the ?32.2 +/- 1.2 mV (n = 159) observed.     

Insulin activation of (Na+,K+)-adenosinetriphosphatase exhibits a temperature-dependent lag time. Comparison to activation of the glucose transporter

The time course of insulin activation of sodium and potassium ion activated adenosinetriphosphatase [(Na+,K+)ATPase] was studied in the rat adipocyte and was compared to activation of the glucose transporter. Under conditions in which the binding of insulin to its cell surface receptor was not rate limiting, a distinct time lag was apparent between insulin addition and stimulation of transport activity. At 37 degrees C, 40-50 s elapsed before an increase in Rb+ uptake [a measure of (Na+,K+)ATPase transport activity] or 2-deoxyglucose uptake could be observed. This lag time increased in an identical manner for both transport processes as the temperature was lowered to 23 degrees C. Addition of the insulinomimetic agent hydrogen peroxide also produced a lag time similar to that for insulin before activation of Rb+ and 2-deoxyglucose uptakes was detected. These data provide the first evidence of a discrete time lag involved during stimulation of the adipocyte (Na+,K+)ATPase. A model for the molecular mechanism of insulin activation of (Na+,K+)ATPase is presented that incorporates these results into the hypothesis of insulin mediated "translocation" of glucose transporters to the plasma membrane.     

Insulin regulates ionic metabolism in a fresh water teleost, anabas testudineus (bloch)

Short term effects of insulin on total brain and branchial Na+K+ ATPase, Ca2+ ATPase and Na+, K+ and Ca2+ ions were investigated in A. testudineus. The increase in brain Ca2+ ATPase after alloxan treatment may account for an increased amount of intracellular calcium required for biochemical events taking place inside the cells. Branchial Na+K+ATPase was significantly stimulated while Ca2+ ATPase significantly inhibited after alloxan treatment. This suggests that alloxan exerts its inhibitory effect on the ATP-driven Ca2+ transport via; its action on the Ca2+ pump protein rather than the membrane permeability to Ca2+. The increased activity of brain Na+K+ ATPase at 3 and 24 hr by insulin to alloxan pretreated fish may account for the stimulated co-transport of glucose and its utilization for energy requirements and the excitatory action on neurons in the brain. The elevated brain Ca2+ ATPase may be due to the role of calcium as a second messenger in hormone action. At 24 hr, the activity of branchial Na+K+ ATPase and Ca2+ ATPase in alloxan pretreated specimens was significantly stimulated by insulin. This may be due to increased synthesis of these enzyme units. Administration of insulin (lU/fish) in normal fish significantly inhibited the activity of brain and branchial Na+K+ ATPase while brain Ca2+ ATPase showed a stimulatory effect at 3 and 24 hr compared to control. Inhibition of total branchial Ca2+ ATPase activity by insulin may be due to increased Ca2+ concentration. Higher plasma glucose level in alloxan treated groups confirms the diabetic effect of alloxan. Insulin reverses this effect. The possible mechanism by which insulin controls Na+K+ ATPase activity appears to be tissue specific. The results seem to be the first report on the effect of insulin on ATPase activity in a teleost. These data are consistent with the hypothesis that insulin performs a role in hydro mineral regulation in freshwater teleosts.     

Na+/K+ ATPase and its functional coupling with Na+/Ca2+ exchanger in mouse embryonic stem cells during differentiation into cardiomyocytes

Cardiomyocytes derived from mouse embryonic stem (mES) cells have been demonstrated to exhibit a time-dependent expression of ion channels and signal transduction pathways in electrophysiological studies. However, ion transporters, such as Na+/K+ ATPase (Na+ pump) or Na+/Ca2+ exchanger, which play crucial roles for cardiac function, have not been well studied in this system. In this study, we investigated the functional expression of Na+/K+ ATPase and Na+/Ca2+ exchanger in mES cells during in vitro differentiation into cardiomyocytes, as well as the functional coupling between the two transporters. By measuring [Na+]i and Na+ pump current (Ip), it was shown that an ouabain-high sensitive Na+/K+ ATPase was expressed functionally in undifferentiated mES cells and these activities increased during a time course of differentiation. Using RT-PCR, the expression of mRNA for alpha1-subunit and alpha3-subunit of the Na+/K+ ATPase could be detected in both undifferentiated mES cells and derived cardiomyocytes. In contrast alpha2-subunit mRNA could be detected only in derived cardiomyocytes but not in undifferentiated mES cells. mRNA for the Na+/Ca2+ exchanger 1 isoform (NCX1) could be detected in undifferentiated mES cells and its expression levels seemed to gradually increase throughout the differentiation accompanied by increasing its Ca2+ extrusion function. At the middle stages of differentiation (after 10-day induction), more than 75% derived cardiomyocytes exhibited [Ca2+]i oscillations by blocking of Na+/K+ ATPase, suggesting the functional coupling with Na+/Ca2+ exchanger. From these results and RT-PCR analysis, we conclude that alpha2-subunit Na+/K+ ATPase mainly contributes to establish the functional coupling with NCX1 at the middle stages of differentiation of cardiomyocytes.     

The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at [NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of [Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes.     

Is Na + K ATPase a Myelin-Associated Enzyme?

The Na + K ATPase activity associated with purified myelin has been investigated. On the basis of marker enzyme studies, the Na + K ATPase activity of myelin was higher than could be accounted for by microsomal contamination. Fractions prepared from white matter-enriched areas of rat brain showed a threefold enrichment in Na + K ATPase activity in myelin as compared with the white matter homogenate. The ATPase activity in myelin was stimulated fourfold by treatment with sodium deoxycholate, but the activity in the whole brain homogenate and the microsomal fraction was only doubled. This discontinuity temperature for Na + K ATPase activity was significantly higher for the myelin fraction (29 degrees C) than for the microsomal fraction (21 degrees C), but the energies of activation, both above and below the discontinuity temperature, were the same for both fractions, Myelin Na + K ATPase had a lower affinity for strophanthidin than the microsomal enzyme, but both fractions were inhibited to the same extent by 10-3 M-strophanthidin. The evidence thus indicated that much of the ATPase activity of myelin is not the result of microsomal contamination. Although the possibility of axolemmal contamination cannot be ruled out conclusively, indirect evidence suggest that this is not a significant factor and that Na + K ATPase may be a myelin-associated enzyme.     

Erythrocyte hemolysis by radiofrequency fields

A field-strength-dependent hemolytic effect of continuous-wave radiofrequency (RF) exposure in vitro has been demonstrated. Erythrocytes in whole heparinized rabbit blood were hemolyzed by a 2-h exposure to 50- or 100-MHz RF fields at field strengths of greater than 4 V/cm. An effect of comparable magnitude resulted from exposure to 10-MHz RF at a field strength of 9 V/cm. Sample temperatures were maintained at 22.5 degrees +/- 0.2 degrees C. There was no apparent involvement of heating or temperature gradients, nor were there any RF exposure effects on cellular K+ or Na+ concentration, nor on pH. The mechanism of the hemolytic effect is not known. Since the percentage of lysed erythrocytes was less than 1% and there was an absence of effects on cellular cation concentrations, RF radiation may have irreversibly altered the plasma membrane permeability of a sensitive subpopulation of red cells (possibly aged cells) leading to osmotic lysis. RF radiation at these frequencies appears to affect red cells in a manner that is qualitatively and quantitatively different from microwave radiation.     

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.     

Branchial and renal (Na+/K+) ATPase and carbonic anhydrase activities in a eurythermal freshwater teleost, Carassius auratus L

1. Plasma sodium and chloride levels were determined in goldfish, Carassius auratus L., following acclimation to 5, 15, 25 and 35 degrees C. Carbonic anhydrase and (Na+/K+)-stimulated ATPase activities of gill and kidney were also assayed at both acclimation temperature and 41 degrees C. 2. Consistent with earlier findings this eurythermal species exhibits more variation in plasma composition with temperature than do the more stenothermal salmonids. Seasonal changes were also observed. 3. Despite differences in detail the overall pattern of transport enzyme activity change with acclimation was comparable to that previously observed in trout. The goldfish is, however, notable for high levels of renal carbonic anhydrase activity, and presumably employs this system more than does the trout to drive urinary recovery of ions by H+:Na+ and HCO3-:Cl- exchanges.     

Metabolic effects of microwave radiation and convection heating on human mononuclear leukocytes

The effects of microwave radiation (2450 MHz, continuous wave, mean specific absorption rate of 103.5 +/- 4.2 W/kg) and convection heating on the nonphosphorylating oxidative metabolism of human peripheral mononuclear leukocytes (96% lymphocytes, 4% monocytes) at 37 degrees C were investigated. Metabolic activity, determined by chemiluminescence (CL) of cells challenged with luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) linked to bovine serum albumin, was detected with a brightness photometer. A significant stimulation after microwave exposure (p less than 0.005) over total CL of matched 37 degrees C incubator controls was observed. A similar degree of stimulation compared to incubator controls was also detected after sham treatment. There was no significant difference between changes in total CL or stimulation indices of the microwave and sham exposed groups. It appears that exposure to microwave radiation, under normothermic (37 +/- 0.03 degrees C) conditions, has no effect on the oxidative metabolic activity of human peripheral mononuclear leukocytes. However, the significant differences between microwave or sham exposed cells and their respective incubator controls occurred because the temperature of the incubator controls did not exceed 35.9 degrees C and this temperature required 39 minutes to reach from 22 degrees C. Slow heating of incubator controls must be accounted for in thermal and radiofrequency radiation studies in vitro.     

Microsomal (Na- +K+)-activated ATPase from frog skin epithelium. Cation activations and some effects of inhibitors.

A method is described for the extraction of microsomal ouabain-sensitive (a- + K+)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na+ K+)-stimulated activity in the range of 30- 40 nmol - mg -1 - min -1 at 26 degrees C. This portion which is also ouabain sensitive, is about half of the total activity in media containing Mg2+, Na+ and K+. These preparations also contain Mg2+-dependent or Ca2+-dependent activities which are not additive and which are not significantly affected by ouabain, Na+, K+ or Li+. The activations of the ouabain-sensitive ATPase activity by Mg2+, Na+, and K+ are similar to those described in other tissues. It is found that Li+ does not substitute for Na+ as an activator but in high concentrations does produce partial activation in the presence of Na+ with no K+. These results are pertinent to the reported observations of ouabain-sensitive Li+ flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li+ on the sodium pump.     

Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments.

We measured the concentration of calmodulin required to reverse inhibition by caldesmon of actin-activated myosin MgATPase activity, in a model smooth-muscle thin-filament system, reconstituted in vitro from purified vascular smooth-muscle actin, tropomyosin and caldesmon. At 37 degrees C in buffer containing 120 mM-KCl, 4 microM-Ca2+-calmodulin produced a half-maximal reversal of caldesmon inhibition, but more than 300 microM-Ca2+-calmodulin was necessary at 25 degrees C in buffer containing 60 mM-KCl. The binding affinity (K) of caldesmon for Ca2+-calmodulin was measured by a fluorescence-polarization method: K = 2.7 x 10(6) M-1 at 25 degrees C (60 mM-KCl); K = 1.4 x 10(6) M-1 at 37 degrees C in 70 mM-KCl-containing buffer; K = 0.35 x 10(6) M-1 at 37 degrees C in 120 mM-KCl- containing buffer (pH 7.0). At 37 degrees C/120 mM-KCl, but not at 25 degrees C/60 mM-KCl, Ca2+-calmodulin bound to caldesmon bound to actin-tropomyosin (K = 2.9 x 10(6) M-1). Ca2+ regulation in this system does not depend on a simple competition between Ca2+-calmodulin and actin for binding to caldesmon. Under conditions (37 degrees C/120 mM-KCl) where physiologically realistic concentrations of calmodulin can Ca2+-regulate synthetic thin filaments, Ca2+-calmodulin reverses caldesmon inhibition of actomyosin ATPase by forming a non-inhibited complex of Ca2+-calmodulin-caldesmon-(actin-tropomyosin).