Effect of ATP,EDTA and EGTA on the simultaneous binding of Na,K, Mg and Ca by rat liver microsomes

In the presence of Na, K, Mg and Ca at physiological pH, complexing agents can affect cation binding by rat liver microsomes in a manner not always readily predictable simply from a knowledge of individual formation constants. Increasing concentrations (0 to 20 mM) of the strong nonbiological complexing agent, ethylenediaminetetraacetate (EDTA), produced a sharp decrease almost to zero in bound Ca, an increase to a high plateau in bound Na and K and an initial increase followed by a sharp decrease in bound Mg. Increasing concentrations of the Ca-preferring analogue of EDTA, ethylene bisglycol (β-aminoethylether) tetraacetate (EGTA), produced similar changes except that bound Mg increased and remained elevated, indicating that this agent complexes Mg very weakly at physiological pH. The biological complexing agent, adenosine triphosphate (ATP), caused a gradual rectilinear and parallel decrease in bound Mg and Ca and a concomitant and parellel increase in bound Na and K at about 4°C and pH 6.4. Results with EDTA and EGTA suggest, however, that under different conditions, enhancement by ATP of divalent cation binding may be possible. Reactions of this nature may be of significance in ATP stimulated divalent cation uptake by subcellular particles.     

The Mg,Ca, EDTA and ATP dependence of Na binding by rat liver microsomes

The role of ionic interactions in the adenosinetriphosphate (ATP) dependent Na binding by rat liver microsomes was investigated. In the concentration range of 0 to 20 mM, Mg and Ca are demonstrated to compete strongly against Na for microsome binding sites. In the presence of Ca, the nonbiological complexing agent ethylenediaminetetraacetate (EDTA) produced a marked increase in Na binding accompanied by a concomitant decrease in Ca binding. Under similar conditions ATP, which is a weaker complexing agent than EDTA, produced quantitatively smaller but qualitatively similar changes in binding. The data show that the effect of ATP on Na binding is not dependent upon the formation of a hypothetical Na binding intermediate in the hydrolysis of ATP as other investigators have postulated. Rather, the effect of ATP is demonstrated to depend upon the presence of unhydrolyzed ATP and its ability to complex divalent cations, and thereby to reduce divalent cation competition against monovalent cations for membrane binding sites.     

Cation exchange binding of rubidium and cesium by rat liver cell microsomes

The rubidium and cesium binding characteristics of rat liver cell microsomes were studied by an equilibration, centrifugation and washing procedure. Concentration dependence experiments, in which microsomes were equilibrated in media containing 0 to 400 mM rubidium or cesium chloride at pH 6.9, yielded saturation type adsorption isotherms similar to those previously reported for sodium and potassium. Mass law analysis of the data yielded apparent dissociation constants of 21 × 10^?3 eq/liter and 19 × 10^?3 eq/liter for rubidium and cesium binding, respectively. The results indicate that cesium is bound slightly more strongly than rubidium, and that both these cations may be bound more strongly than sodium or potassium. The maximum binding capacity at pH 6.9 was approximately 1.3 meq rubidium or cesium/g nitrogen. Sodium, potassium, magnesium and calcium generally associated with the isolated microsomes decreased concomitantly with increasing bound rubidium or cesium, demonstrating the ion exchange nature of the binding. Results of pH-dependence experiments showed that following equilibration of the microsomes in media containing approximately 96 mM rubidium or cesium at various pH values, bound rubidium or cesium was essentially zero at pH less than five, increased sharply between pH 5 and 7, and tended to level off at higher pH. The present results further characterize the cation binding properties of the microsomal material.     

The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

1. The intrinsic Na(+), K(+), Mg(2+) and Ca(2+) contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na(+) from 90+/-20 to 24+/-12, the bound K(+) from 27+/-3 to 7+/-2, the bound Mg(2+) from 20+/-2 to 3+/-1 and the bound calcium from 8+/-1 to <1nmol/mg of protein. 3. The activities of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase and the Na(+)-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5mum (ATP/protein ratio 12.5pmol/mug). 4. The Na(+)-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5mum-magnesium chloride and 2mum-potassium chloride. Addition of 2.5mum-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na(+)-dependent ATP hydrolysis was partly restored with 2.5mum-magnesium chloride; addition of K(+) in the range 2-10mum-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0 degrees C with 0.5nmol of K(+)/mg of protein so that the final added K(+) in the reaction mixture was 0.1mum restored the Na(+)-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [(42)K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K(+)/mg of protein was linear over a period of 20min and was inhibited by Na(+). Half-maximal inhibition of (42)K(+)-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na(+)-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K(+) and Mg(2+) of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K(+) from a solution of 0.5mum-potassium chloride.     

Specific effects of spermine on Na+,K+-adenosine triphosphatase

Specific effects of spermine on Na+,K+-ATPase were observed using an enzyme partially purified from rabbit kidney microsomes by extraction with deoxycholate. 1. Spermine competed with K+ for K+-dependent, ouabain-sensitive nitrophenylphosphatase. The K1 for spermine was 0.075 mm in the presence of 1 mM Mg2+ and 5 mM p-nitrophenylphosphate at pH 7.5. 2. spermine activated Na+,K+-ATPase over limited concentration ranges of K+ and Na+ in the presence of 0.05 mM ATP. The spermine concentration required for half maximal activation was 0.055 mM in the presence of 1 mM K+, 10 mM Na+, 1 mM Mg2+, and 0.05 mM ATP. 3. The activation of Na+,K4-ATPase was not due to substitution of spermine for K+, Na+, or Mg2+. 4. When the concentration of K+ or Na+ was extremely low, or in excess, spermine did not activate Na+,K+-ATPase, but inhibited it slightly. 5. Plots of 1/v vs. 1/[ATP] at various concentrations of spermine showed that spermine decreased the Km for ATP without changing the Vmax. 6. Plots of 1/v vs. 1/[ATP] at concentrations of K+ from 0.05 mM to 0.5 mM showed that K+ increased the Km for ATP with increase in the Vmax in the presence of 0.2 mM spermine similarly to that in the absence of spermine. The contradictory effects of spermine on this enzyme system suggest that the K+-dependent monophosphatase activity does not reflect the second half (the dephosphorylation step) of the Na+,K+-ATPase catalytic cycle.     

Na+ and K+ binding by glycerinated muscle fibers with reciprocal cation concentrations in the medium

The binding of Na+ and K+ by glycerinated muscle fibres was observed at reserve concentrations of NaCl in the medium. Under external concentrations of Na+ of K+ up to 0.4-0.5 mM, a constant fraction (0.15-0.25 mmoles/kg dry weight of the fibres) bound by glycerinated fibres was revealed. With the increase of NaCl or KCl concentration in the medium up to 10 mM the concentration of bound cations increased too. The parameters of Na+ and K+ sorption by glycerinated models were calculated. The values of Na+ and K+ binding limits were 4.4 and 1.8 mmole/kg dry weight of the fibres and those of affinity, 3.2 and 4.1 kcal/mol, respectively. The binding of one cation took place in conditions when its concentration was 10,000-20,000 fold less than that of the other cation. This points to the fact that Na+ and K+ binding is highly specific and is carried out by different centres. It is suggested that myosin ATPase is a substratum binding Na+ and K+ in glycerinated muscle fibres at reverse ratio concentrations of these cations in the medium.     

Pulmonary phosphatidic acid phosphatase. Properties of membrane-bound phosphatidate-dependent phosphatidic acid phosphatase in rat lung.

1. The membrane-bound phosphatidate-dependent phosphatidic acid phosphatase activity of rat lung has been investigated in cytosol and microsomal fractions using as a substrate [32P]phosphatidate bound to heat inactivated rat liver microsomes. Both activities demonstrated broad pH optima with a maximum of 7.4--8 for the cytosol and a maximum of 6.5--7.5 with microsomal preparations. 2. At low concentrations (0--5 mM) Mg2+ produced a slight stimulation of the cytosol activity but at higher concentrations an inhibition was observed. Low concentrations (1.0--2.0 mM) of EDTA abolished the cytosol activity and reduced the microsomal activity to half. In both cases, the addition of Mg2+ in the presence of EDTA resulted in an activity which was more than 2-fold greater than that observed in the absence of chelator or divalent cation. 3. The cytosol activity was relatively resistant to the addition of ionic and nonionic detergents. In general, the addition of a number of phosphate esters increased rather than decreased the release of 32Pi, indicating a relative specificity for phosphate groups associated with a hydrophobic environment. The addition of aqueous dispersions of phosphatidate, lysophosphatidic acid or phosphatidylglycerophosphate markedly reduced the hydrolysis of membrane-bound [32P]phosphatidate. The cytosol activity was slightly inhibited by the addition of phosphatidylcholine. 4. In an attempt to estimate the relative contributions of the cytosol and microsomal activities in vivo, these activities were assayed using [32P]phosphatidate endogenously generated on rat lung microsomes. With the 32P-labelled microsomes, the hydrolysis remained linear over the 45 min of the experiment. Addition of high speed supernatant produced a rapid release of 32Pi during the first 10 min followed by a more gradual release similar to that oberved with the microsomes alone. The cytosol activity remained greater than the microsomal activity at all times studied. 5. When [14C]phosphatidate-labelled microsomes were incubated in the presence of nonradioactive CDPcholine, the addition of cytosol markedly stimulated the incorporation of radioactivity into phosphatidylcholine. This observation suggests that the phosphatidic acid phosphatase activity associated with the cytosol has a role in phosphatidylcholine (and presumably surfactant) biosynthesis in rat lung.     

Phosphate binding by cerebral microsomes in relation to adenosine-triphosphatase activity

1. Microsomes prepared from guinea-pig and ox brain were incubated for periods of a few seconds with low concentrations of Mg-[(32)P]ATP, the reaction was stopped with trichloroacetic acid and determinations were made of the phosphate bound to the acid-washed, and in some cases solvent-extracted, residue. 2. At 20 mum-ATP, at 37 degrees and in the presence of Na(+) ions, 30-50 mumumoles of phosphate/mg. of microsomal protein were bound by the preparation within 1 sec. of starting the reaction; little further change in level occurred until hydrolysis of ATP exceeded 50%, when the bound phosphate began to decline fairly rapidly to the zero-time value. 3. At 20mum-ATP without Na(+) ions present or in the presence of K(+) ions, the level of bound phosphate increased gradually and did not decline as ATP hydrolysis approached completion. 4. Potassium ions either inhibited the formation of Na(+)-dependent bound phosphate or, when added during the course of the reaction, rapidly reduced its level. 5. At 200 mum-ATP the bound phosphate formed in the presence of Na(+) ions appeared to consist of a mixture of the unstable Na(+)-dependent type and the stable type requiring only Mg(2+) ions for its formation. 6. Non-radioactive ATP added during the course of the reaction at 20 mum-ATP with Na(+)ions present rapidly discharged virtually all the bound (32)P counts; at 200 mum-ATP only a proportion of the label was similarly discharged. The Na(+)-dependent bound phosphate is therefore turning over, in contrast with that formed in the absence of Na(+)ions, which proved more stable. 7. The Na(+)-dependent bound phosphate was not in the form of ATP; experiments with [(14)C]ATP instead of [(32)P]ATP showed a small and invariable binding of ATP by the preparation unaffected by Na(+) ions or time of incubation. 8. Under the usual conditions employed in this work ouabain stimulated formation of Na(+)-dependent bound phosphate when Na(+) ions were suboptimum and inhibited it when optimum Na(+) ions were present. 9. The Na(+)-dependent binding reaction under present conditions did not involve incorporation into phosphorylserine groups. 10. The relation of the findings to the (Na(+),K(+))-ATPase of the preparation, and to observations in brain slices appearing to implicate phosphorylserine groups in cation transport, is discussed.     

The kinetics and divalent cation inhibition of plasma membrane ATPase in the yeast Candida albicans

The kinetics of ATP hydrolysis and cation effects on ATPase activity in plasma membrane from Candida albicans ATCC 10261 yeast cells were investigated. The ATPase showed classical Michaelis-Menten kinetics for the hydrolysis of Mg X ATP, with Km = 4.8 mM Mg X ATP. Na+ and K+ stimulated the ATPase slightly (9% at 20 mM). Divalent cations in combination with ATP gave lower ATPase activity than Mg X ATP (Mg greater than Mn greater than Co greater than Zn greater than Ni greater than Ca). Divalent cations inhibited the Mg X ATPase (Zn greater than Ni greater than Co greater than Ca greater than Mn). Free Mg2+ inhibited Mg X ATPase weakly (20% inhibition at 10 mM). Computed analyses of substrate concentrations showed that free Zn2+ inhibited Zn X ATPase, mixed (Zn2+ + Mg2+) X ATPase, and Mg X ATPase activities. Zn X ATP showed high affinity for ATPase (Km = 1.0 mM Zn X ATP) but lower turnover (52%) relative to Mg X ATP. Inhibition of Mg X ATPase by (free) Zn2+ was noncompetitive, Ki = 90 microM Zn2+. The existence of a divalent cation inhibitory site on the plasma membrane Mg X ATPase is proposed.     

Effects of calcium binding and of EDTA and CaEDTA on the clotting of bovine fibrinogen by thrombin

Studies were carried out at pH 7.0 and gamma/2 0.15 before addition of CaCl2 or EDTA. Clotting time, tau, at 3.03 microM fibrinogen and 0.91 u/ml thrombin was determined for equilibrium systems. With added Ca2+, tau decreases, from tau 0 at 0 added Ca2+ (mean, 29.7 +/- 3 s), by approximately 3 s at 5 mM added Ca2+. With added EDTA, tau increases sigmoidally from tau 0 at 0 EDTA to a maximum (mean tau m = 142 +/- 23 s) at approximately 200 microM EDTA. tau then decreases slightly to a minimum at approximately 1.3 mM and finally increases to infinity at approximately 10 mM EDTA. Between 0 and 1.3 mM EDTA, effects on clotting time are completely reversed by adding Ca2+ and, after equilibration at 400 microM EDTA, tau is independent of EDTA concentration. Thus, up to 400 microM EDTA, effects on clotting time are attributed to decreasing fibrinogen bound Ca2+. Between 5 mM Ca2+ and 200 microM EDTA it is assumed that an equilibrium distribution of fibrinogen species having 3, 2, 1, or 0 bound calcium ions is established and that a clotting time is determined by the sum of products of species fractional abundance and pure species clotting time. Analysis indicates that pure species clotting times increase proportionately with decreasing Ca2+ binding, binding sites are nearly independent, and the microscopic association constant for the first bound Ca2+ is approximately 4.9 X 10(6) M-1. Effects of adding Ca2+ at times t1 after thrombin addition to systems initially equilibrated at 200 microM EDTA were determined. Analysis of the relation between tau and t1 indicates that as Ca2+ binding decreases, rate constants for release of B peptides decrease less than those for release of A peptides. As EDTA concentration is increased above 1.3 mM, inhibitory effects of EDTA and CaEDTA progressively increase.     

trans-1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid is superior to ethylenediamine-N,N,N',N'-tetraacetic acid for sequestering Mg2+ in 31P NMR experiments involving ATP spectra at neutral and acidic pH

The use of ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) to sequester Mg2+ from samples containing ATP at acidic or neutral pH prior to 31P NMR spectroscopic analysis leads to significant broadening of the gamma- and beta-phosphorus resonances of ATP as compared to ATP alone. It was found that the use of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) reduces the broadening of the ATP resonances. At pH 7.0, 30 mM EDTA in the presence of 5 mM ATP and 7 mM Mg2+ leads to a threefold increase in the peak width of the gamma phosphorus of ATP as compared to 5 mM ATP alone. When 30 mM CDTA is used in the place of EDTA, the peak width decreased to about 80% of the peak width of ATP alone. When the experiment is repeated at pH 8.5, both EDTA and CDTA lead to narrow peak widths with no significant difference between the two spectra. At pH 6.0, the use of EDTA leads to a spectrum that is very noisy, with a 10-fold increase in the peak width as compared to ATP in the absence of Mg2+ at this pH, whereas the increase with CDTA is only 50%. These results do not reflect the difference in chelating strength between EDTA and CDTA: The free Mg2+ concentration in the presence of each chelator, as calculated by the computer program given in the Appendix, was nearly equal at each pH. The results, however, reflect a difference in the lability of the metal-ligand bond between EDTA and CDTA.     

Effects of ATP and monovalent cations on Mg2+ inhibition of (Na,K)-ATPase

The hydrolysis of ATP catalyzed by purified (Na,K)-ATPase from pig kidney was more sensitive to Mg2+ inhibition when measured in the presence of saturating Na+ and K+ concentrations [(Na,K)-ATPase] than in the presence of Na+ alone, either at saturating [(Na,Na)-ATPase] or limiting [(Na,0)-ATPase] Na+ concentrations. This was observed at two extreme concentrations of ATP (3 mM where the low-affinity site is involved and 3 microM where only the catalytic site is relevant), although Mg2+ inhibition was higher at low ATP concentration. In the case of (Na,Na)-ATPase activity, inhibition was barely observed even at 10 mM free Mg2+ when ATP was 3 mM. When (Na,K)-ATPase activity was measured at different fixed K+ concentrations the apparent Ki for Mg2+ inhibition was lower at higher monovalent cation concentration. When K+ was replaced by its congeners (Rb+, NH+4, Li+), Mg2+ inhibition was more pronounced in those cases in which the dephosphorylating cation forms a tighter enzyme-cation complex after dephosphorylation. This effect was independent of the ATP concentration, although inhibition was more marked at lower ATP for all the dephosphorylating cations. The K0.5 for ATP activation at its low-affinity site, when measured in the presence of different dephosphorylating cations, increased following the sequence Rb+ greater than K+ greater than NH+4 greater than Li+ greater than none. The K0.5 values were lower with 0.05 mM than with 10 mM free Mg2+ but the order was not modified. The trypsin inactivation pattern of (Na,K)-ATPase indicated that Mg2+ kept the enzyme in an E1 state. Addition of K+ changed the inactivation into that observed with the E2 enzyme form. On the other hand, K+ kept the enzyme in an E2 state and addition of Mg2+ changed it to an E1 form. The K0.5 for KCl-induced E1-to-E2 transformation (observed by trypsin inactivation profile) in the presence of 3 mM MgCl2 was about 0.9 mM. These results concur with two mechanisms for free Mg2+ inhibition of (Na,K)-ATPase: "product" and dead-end. The first would result from Mg2+ interaction with the enzyme in the E2(K) occluded state whereas the second would be brought about by a Mg2+-enzyme complex with the enzyme in an E1 state.     

Functional Approach to the Catalytic Site of the Sarcoplasmic Reticulum Ca2+-ATPase: Binding and Hydrolysis of ATP in the Absence of Ca2+

Isolated sarcoplasmic reticulum vesicles in the presence of Mg(2+) and absence of Ca(2+) retain significant ATP hydrolytic activity that can be attributed to the Ca(2+)-ATPase protein. At neutral pH and the presence of 5 mM Mg(2+), the dependence of the hydrolysis rate on a linear ATP concentration scale can be fitted by a single hyperbolic function. MgATP hydrolysis is inhibited by either free Mg(2+) or free ATP. The rate of ATP hydrolysis is not perturbed by vanadate, whereas the rate of p-nitrophenyl phosphate hydrolysis is not altered by a nonhydrolyzable ATP analog. ATP binding affinity at neutral pH and in a Ca(2+)-free medium is increased by Mg(2+) but decreased by vanadate when Mg(2+) is present. It is suggested that MgATP hydrolysis in the absence of Ca(2+) requires some optimal adjustment of the enzyme cytoplasmic domains. The Ca(2+)-independent activity is operative at basal levels of cytoplasmic Ca(2+) or when the Ca(2+) binding transition is impeded.     

(Na+, K+)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Steady-state analysis.

1. The ATP sites. Homotropic interactions between ATP sites have been studied in a very large range of Na+ and K+ concentrations. The ( Na+, K+)-activated ATPase displays Michaelis-Menten kinetics for ATP under standard concentration conditions of Na+ (100 mM) and K+ (10 mM). The steady-state kinetics behavior changes at very low concentrations of K+ where negative cooperativity is observed. The existence of a high affinity and a low affinity site for ATP was clearly demonstrated from the study of the ATP stimulated hydrolysis of p-nitrophenylphosphate in the presence of Na+ and K+. The ratio of apparent affinities of high and low affinity sites for ATP is 86 at pH 7.5. 2. The Na+ sites. The binding of Na+ to its specific stimulatory sites (internal sites) is characterized by positive cooperativity with a Hill coefficient n(H(Na+))=2.0. Homotropic interactions between Na+ sites are unaffected by variations of the K+ concentration. 3. The K+ sites. (a) Binding of K+ to the (external) stimulatory site of the ATPase has been analyzed by following the (Na+, K+)-ATPase activity as well as the p-nitrophenylphosphatase activity in the presence of Na+ and K+ (with or without ATP). Binding is characterized by a Hill coefficient of 1.0 and a K(0.5(K+))=0.1 to 0.8 mM. The absence of positive or negative cooperativity persists between 5 mM and 100 mM Na+. (b) The analysis of the p-nitrophenylphosphatase or of the 2, 4 dinitrophenylphosphatase activity in the presence of K+ alone indicates the existence of low affinity sites for K+ with positive homotropic interactions. The characteristics of stimulation in that case are, K(0.5)=5 mM, n(H)=1.9. The properties of this family of site(s) are the following: firstly, saturation of the low affinity site(s) by K+ prevents ATP binding to its high affinity internal site. Secondly, saturation of the low affinity sites for K+ prevents binding of Na+ to its internal sites. Thirdly, this family of sites disappears in the presence of ATP, p-nitrophenylphosphate or of both substrates, when Na+ binds to its internal sites. Na+ binding to its specific stimulatory sites provokes the formation of the high affinity type of site for K+. 4. Mg2+ stimulation of the (Na+, K+)-ATPase is characterized by a Hill coefficient n(H(Mg2+))=1.0 and a K(0.5(Mg2+))=1 mM stimulation is essentially a V effect. Heterotropic effects between binding of Mg2+ and substrate to their respective sites are small. Heterotropic interactions between the Ms2+, Na+ and K+ sites are also small. 5. The fluidity of membrane lipids also controls the (Na+, K+)-ATPase activity. Phase transitions or separations in the membrane hardly affect recognition properties of substrates, Na+, K+ and Mg2+ for their respective sites on both sides of the membrane. Only the rate of the catalytic transformation is affected.     

Estimation of polyamine binding to macromolecules and ATP in bovine lymphocytes and rat liver.

To estimate the polyamine distribution in bovine lymphocytes and rat liver, the binding constants (K) for DNA, RNA, phospholipid, and ATP were determined under the conditions of 10 mM Tris-HCl, pH 7.5, 2 mM Mg2+, and 150 mM K+. The binding constants of spermine for calf thymus DNA, Escherichia coli 16 S rRNA, phospholipid in rat liver microsomes and ATP were 1.15 x 10(2), 6.69 x 10(2), 2.22 x 10(2), and 5.95 x 10(2) M-1, respectively. From these binding constants and experimentally determined cellular concentrations of macromolecules, ATP, and polyamines, spermine distribution in the cells was estimated. In bovine lymphocytes, the mols of spermine bound to DNA, RNA, phospholipid, and ATP were 0.79, 3.7, 0.23, and 4.3 per 100 mol of phosphate of macromolecules or ATP, respectively. In rat liver, they were 0.19, 1.0, 0.05, and 0.97/100 mol of phosphate of macromolecules or ATP, respectively. The binding constants of spermidine for macromolecules and ATP were smaller than those of spermine, but a similar tendency was observed with spermidine distribution among macromolecules and ATP in the above two cells. The amount of polyamine bound to DNA and phospholipid was significantly lower than that to RNA. When either the Mg2+ or K+ concentration increased, the amount of free spermine and that bound to RNA and ATP increased, but the amount of spermine bound to DNA and phospholipid decreased. The results indicate that most polyamines exist as a polyamine-RNA complex in cells. Under the conditions that globin synthesis is stimulated by spermine in a rabbit reticulocyte cell-free system, the amount of spermine bound to RNA was very close to the value estimated in the cells.     

Interaction of sodium and potassium ions with Na+,K(+)-ATPase. IV. Affinity change for K+ and Na+ of Na+,K(+)-ATPase in the cycle of the ATP hydrolysis reaction

The Kd for ouabain-sensitive K+ or Rb+ binding to Na+,K(+)-ATPase was determined by the centrifugation method with radioactive K+ and Rb+ in the presence of various combinations of Na+, ATP, adenylylimidodiphosphate (AMPPNP), adenylyl-(beta,gamma-methylene)diphosphonate (AMPPCP), Pi, and Mg2+. From the results of the K+ binding experiments, Kd for Na+ was estimated by using an equation describing the competitive inhibition between the K+ and Na+ binding. 1) The Kd for K+ binding was 1.9 microM when no ligand was present. Addition of 2 mM Mg2+ increased the Kd to 15-17 microM. In the presence of 2 mM Mg2+, addition of 3 mM AMPPCP with or without 3 mM Na+ increased the Kd to 1,000 or 26 microM, respectively. These Kds correspond to those for K+ of Na.E1.AMPPCPMg or E1.AMPPCPMg, respectively. 2) Addition of 4 mM ATP with or without 3 mM Na+ decreased the Kd from 15-17 microM to 5 or 0.8 microM, respectively. Because the phosphorylated intermediate was observed but ATPase activity was scarcely observed in the K+ binding medium containing 3 mM ATP and 2 mM Mg2+ in the absence of Na+ as well as in the presence of Na+ at 0 degrees C, it is suggested that K+ binds to E2-P.Mg under these ligand conditions. 3) The Kd for Na+ of the enzyme in the presence of 3 mM AMPPCP or 4 mM ATP with Mg2+ was estimated to be 80 or 570 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation from adenosine triphosphate of sodium- and potassium-activated adenosine triphosphatase. Comparison of enzyme-ligand complexes as precursors to the phosphoenzyme.

The relative effectiveness of the ligands Mg2+, Na+, and ATP in preparing sodium plus potassium ion transport adenosine triphosphatase for phosphorylation was studied by means of a rapid mixing apparatus. Addition of 2 mM MgC12, 120 mM NaC1, and 5 muM [gamma-32P]ATP simultaneously to the free enzyme gave an initial phosphorylation rate of about 0.3 mu mol-mg-1-min-1 at 25 degrees and pH7.4. Addition of Mg2+ to the enzyme beforehand, separately or in combination with Na+ or ATP, had little effect on the initial rate. Addition of Na+ only to the enzyme beforehand increased this rate 1.5- to 3-fold. Early addition of ATP 130 ms before Na+ plus Mg2+ increased the rate 6- to 7-fold. Early addition of Na+ plus ATP was most effective; it increased the rate about 10-fold. The data indicate that Na+ and ATP bind in a random order and that each ligand potentiates the effect of the other. The rate of dissociation of ATP from the enzyme was estimated by a chase of unlabeled ATP of variable duration. This rate was slowest in the presence of Mg2+ (k = 540 min-1), most rapid in the presence of Na+ (k = 2000 min-1), and intermediate (k = 1100 min-1) in the absence of metal ions. The effect of Na+ concentration on the rate of phosphorylation was estimated when Na+ with Mg2+ was added to the enzyme-ATP complex. The rate followed Michaelis-Menten kinetics with a maximum of 2.9 mu mol-mg-1 and a Km of 8 mM. The effect of Na+ concentration was also estimated on the increment in the rate of phosphorylation produced by the presence of Na+ with the enzyme-ATP complex beforehand. The increment followed the same kinetics with a maximum of 3.75 mu mol-mg-1-min-1 and a Km of 5.4 mM. In both cases estimation of the Hill coefficient failed to show cooperativity between binding sites for Na+. In contrast, the dependence of ouabain-sensitive ATPase activity on Na+ concentration in the absence of K+ indicated two sites for Na+ with apparent Km values of 0.16 and 8.1 mM, respectively.     

Differential dynamic behavior of actin filaments containing tightly-bound Ca2+ or Mg2+ in the presence of myosin heads actively hydrolyzing ATP.

We have investigated how Ca2+ or Mg2+ bound at the high-affinity cation binding site in F-actin modulates the dynamic response of these filaments to ATP hydrolysis by attached myosin head fragments (S1). Rotational motions of the filaments were monitored using steady-state phosphorescence emission anisotropy of the triplet probe erythrosin-5-iodoacetamide covalently attached to cysteine 374 of actin. The anisotropy of filaments containing only Ca2+ increased from 0.080 to 0.137 upon binding S1 in a rigor complex and decreased to 0.065 in the presence of ATP, indicating that S1 induced additional rotational motions in the filament during ATP hydrolysis. The comparable anisotropy values for Mg(2+)-containing filaments were 0.067, 0.137, and 0.065, indicating that S1 hydrolysis did not induce measurable rotational motions in these filaments. Phalloidin, a fungal toxin which stabilizes F-actin and increases its rigidity, increased the anisotropy of F-actin containing either Ca2+ or Mg2+ but not the anisotropy of the 1:1 S1-actin complexes of these filaments. Mg(2+)-containing filaments with phalloidin bound also displayed increased rotational motions during S1 ATP hydrolysis. A strong positive correlation between the phosphorescence anisotropy of F-actin under specific conditions and the extent of the rotational motions induced by S1 during ATP hydrolysis suggested that the long axis torsional rigidity of F-actin plays a crucial role in modulating the dynamic response of the filaments to ATP hydrolysis by S1. Cooperative responses of F-actin to dynamic perturbations induced by S1 during ATP hydrolysis may thus be physically mediated by the torsional rigidity of the filament.     

The Na(+)-translocating F₁F₀-ATPase from the halophilic, alkalithermophile Natranaerobius thermophilus

Natranaerobius thermophilus is an unusual anaerobic extremophile, it is halophilic and alkalithermophilic; growing optimally at 3.3-3.9M Na(+), pH(50°C) 9.5 and 53°C. The ATPase of N. thermophilus was characterized at the biochemical level to ascertain its role in life under hypersaline, alkaline, thermal conditions. The partially purified enzyme (10-fold purification) displayed the typical subunit pattern for F-type ATPases, with a 5-subunit F(1) portion and 3-subunit-F(O) portion. ATP hydrolysis by the purified ATPase was stimulated almost 4-fold by low concentrations of Na(+) (5mM); hydrolysis activity was inhibited by higher Na(+) concentrations. Partially purified ATPase was alkaliphilic and thermophilic, showing maximal hydrolysis at 47°C and the alkaline pH(50°C) of 9.3. ATP hydrolysis was sensitive to the F-type ATPase inhibitor N,N'-dicylohexylcarbodiimide and exhibited inhibition by both free Mg(2+) and free ATP. ATP synthesis by inverted membrane vesicles proceeded slowly and was driven by a Na(+)-ion gradient that was sensitive to the Na(+)-ionophore monensin. Analysis of the atp operon showed the presence of the Na(+)-binding motif in the c subunit (Q(33), E(66), T(67), T(68), Y(71)), and a complete, untruncated ε subunit; suggesting that ATP hydrolysis by the enzyme is regulated. Based on these properties, the F(1)F(O)-ATPase of N. thermophilus is a Na(+)-translocating ATPase used primarily for expelling cytoplasmic Na(+) that accumulates inside cells of N. thermophilus during alkaline stress. In support of this theory are the presence of the c subunit Na(+)-binding motif and the low rates of ATP synthesis observed. The complete ε subunit is hypothesized to control excessive ATP hydrolysis and preserve intracellular Na(+) needed by electrogenic cation/proton antiporters crucial for cytoplasmic acidification in the obligately alkaliphilic N. thermophilus.     

Sarcoplasmic reticulum Ca-ATPase: distinction of phosphoenzymes formed from MgATP and CaATP as substrates and interconversion of the phosphoenzymes by Mg2+ and Ca2+

In order to characterize the phosphoenzymes (EPs) formed from MgATP and CaATP as substrates, the effects of Mg2+ and Ca2+ outside SR vesicles on the hydrolysis rates of EPs were examined by using purified and unpurified Ca-ATPases of sarcoplasmic reticulum (SR) at low [gamma-32P]ATP (4-10 microM), 0.1 M KCl, pH 7.0, and 0 degrees C. When the phosphorylation reaction was stopped by adding an excess of EDTA over Ca and Mg, two components of EP, EPfast (rate constant, kfast = 15-20 min-1), and EPslow (kslow = 0.3-0.4 min-1), were recognized in the time course of EP decomposition. These two rates did not depend on the Ca2+ or Mg2+ concentration in the medium during the phosphorylation reaction, although the proportions of EPfast and EPslow essentially depended on the concentrations of MgATP and CaATP in the phosphorylation reaction medium. The proportion of EPfast increased with increasing [MgATP]/[CaATP] in the medium, whereas that of EPslow decreased. The rate of EPslow hydrolysis in the presence of excess EDTA was basically the same as that of EP formed from CaATP. These results suggest that EPfast and EPslow are derived from MgATP and CaATP, respectively, and EPfast is a reaction intermediate with Mg bound at the substrate site (MgEP), while the main EPslow is a reaction intermediate with Ca bound at the substrate site (CaEP) which is readily converted to metal-free EP by EDTA addition (Shigekawa et al., (1983) J. Biol. Chem. 258, 8698-8707). Mg2+ added outside SR vesicles stimulated the conversion of CaEP to MgEP and inhibited the hydrolysis of MgEP in the relatively high concentration range (K(Mg) = 7.9 mM). Ca2+ added outside SR vesicles stimulated the conversion of MgEP to CaEP and inhibited the conversion of CaEP to MgEP by Mg2+ addition. The Ca2+ outside SR vesicles did not essentially affect the hydrolysis of MgEP. These results suggest that the interconversion between MgEP and CaEP takes place during the reaction by exchange of the divalent cation on the substrate site. The following scheme is proposed. (formula: see text)