K(+)- and Mg2(+)-dependent hydrolysis of acetyl phosphate catalyzed by the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

The (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg2+ and EGTA and is stimulated by Ca2+. The Mg2(+)-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5 mM MgCl2, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca2+ causes no further increase in the rate of hydrolysis and Ca2+ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca2+ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by Pi unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect Pi inhibition of the Mg2(+)-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a Pi-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and Pi. In this conformation the enzyme is transformed from a Ca2(+)- and Mg2(+)-dependent ATPase into a (K+ + Mg2+)-ATPase.     

Ca2+-stimulated,Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m  0.25 μM, V_max  24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.     

Fast reversal of the initial reaction steps of the plasma membrane (Ca2+ + Mg2+)-ATPase

(1) Calmodulin-depleted red cell membranes catalyse a Ca²⁺, Mg²⁺-dependent ATP-[³H]ADP exchange at 37° C. The Ca²⁺, Mg²⁺-dependent exchange, measured at 20 μM CaCl₂, 1.5 mM MgCl₂, 1.5 mM ADP and 1.5 mM ATP, is comparable to the (Ca²⁺ + Mg²⁺)-ATPase activity, between 0.3 and 0.8 mmol/litre original cells per h. (2) EDTA-washed membranes present a Ca²⁺-dependent ATP-ADP exchange whose rate is not more than 7% of that found in a Mg²⁺-containing medium, while their Ca²⁺-dependent ATPase is essentially zero. Addition of 1.5 mM MgCl₂ to the medium restores both activities to the levels found with membranes not treated with EDTA. (3) Calmodulin (16 μg/ml) produces an eight-fold stimulation of the Ca²⁺-dependent ATP-ADP exchange, slightly less than it stimulates the Ca²⁺-dependent ATP hydrolysis. The effect of 1.5 mM MgCl₂ on the exchange is greater in the presence than in the absence of calmodulin. (4) It is proposed that the reversal of the initial phosphorylation of the Ca²⁺ pump, occurring at a fast rate at 37° C, involves a conformational change in the phosphoenzyme. Thus, it would be an ADP-liganded phosphoenzyme of the form EP(ADP) that would experience the fast conformational transition at 37° C. The great difficulty in producing an overall reversal of the Ca²⁺ pump should then be due to one or more reaction steps later than and including Ca²⁺ release and dephosphorylation.     

Phospholipid requirement of Ca2+-stimulated,Mg2+-dependent ATP hydrolysis in rat brain synaptic membranes

The phospholipid requirement for Ca²⁺-stimulated, Mg²⁺-dependent ATP hydrolysis (Ca²⁺/Mg²⁺-ATPase) and Mg²⁺-stimulated ATP hydrolysis (Mg²⁺-ATPase) in rat brain synaptosomal membranes was studied employing partial delipidation of the membranes with phospholipase A₂ (Hog pancreas), phospholipase C (Bacillus cereus) and phospholipase D (cabbage). Treatment with phospholipase A₂ caused an increase in the activities of both Ca²⁺/Mg²⁺-ATPase and Mg²⁺-ATPase whereas with phospholipase C treatment both the enzyme activities were inhibited. Phospholipase D treatment had no effect on Ca²⁺/Mg²⁺-ATPase but Mg²⁺-ATPase activity was inhibited. Inhibition of Mg²⁺-ATPase activity after phospholipase C treatment was relieved with the addition of phosphatidylinositol-4,5-bisphosphate (PIP₂) and to a lesser extent with phosphatidylinositol-4-phosphate (PIP) and phosphatidylcholine (PC). Phosphatidylserine (PS), phosphatidic acid (PA), PIP and PIP₂ brought about the reactivation of Ca²⁺/Mg²⁺-ATPase. Phosphatidylinositol (PI) and PA inhibited Mg²⁺-ATPase activity.K _ms for Ca²⁺ (0.47 M) and Mg²⁺ (60 M) of the enzyme were found to be unaffected after treatment with the phospholipases.     

Properties of (Mg2+ + Ca2+)-ATPase of erythrocyte membranes prepared by different procedures: Influence of Mg2+, Ca2+, ATP,and protein activator

Erythrocyte membranes prepared by three different procedures showed (Mg²⁺ + Ca²⁺)-ATPase activities differing in specific activity and in affinity for Ca²⁺. The (Mg²⁺ + Ca²⁺)-ATPase activity of the three preparations was stimulated to different extents by a Ca²⁺-dependent protein activator isolated from hemolystes. The Ca²⁺ affinity of the two most active preparations was decreased as the ATP concentration in the assay medium was increased. Lowering the ATP concentration from 2 mM to 2–200 μM or lowering the Mg:ATP ratio to less than one shifted the (Mg²⁺ + Ca²⁺)-ATPase activity in stepwise hemolysis membranes from mixed “high” and “low” affinity to a single high Ca²⁺ affinity. Membranes from which soluble proteins were extracted by EDTA (0.1 mM) in low ionic strengh, or membranes prepared by the EDTA (1–10 mM) procedure, did not undergo the shift in the Ca²⁺ affinity with changes in ATP and MgCl₂ concentrations. The EDTA-wash membranes were only weakly activated by the protein activator. It is suggested that the differences in properties of the (Mg²⁺ + Ca²⁺)-ATPase prepared by these three procedures reflect differences determined in part by the degree of association of the membrane with a soluble protein activator and changes in the state of the enzyme to a less activatable form.     

Identification and characterization of a Mg+2-dependent and an independent Ca+2-ATPase in microsomal membranes of rat testis

Rat testicular microsomal membrane fraction contains both Mg⁺²-dependent and Mg⁺²-independent Ca⁺²-ATPase activity. The latter activity is about two times higher than the former. Calcium ion required for maximum activation of Mg⁺²-independent Ca⁺²-ATPase in 3.0 mM, whereas for the dependent one it is 2.5 mM. Both the enzymes are resistant to cold shock upto seven days. Histidine and imidazole buffers are found to be the most suitable for dependent and independent enzyme activities, respectively. The pH optima for dependent one is 7.5, whereas for the independent one it is 8.5. Temperature optima for the former is 37°C and for latter one it is 40°C. Among all the nuclestides tested, ATP is found to be the best substrate for both the enzymes. The optimum concentration of ATP for dependent and independent enzyme activities are 3.0 mM and 1.5 mM respectively. Divalent metal ions like Zn⁺², Ba⁺² and Mn⁺² have been found to inhibit Mg⁺²-dependent Ca⁺²-ATPase activity whereas Mg⁺²-independent Ca⁺²-ATPase activity is inhibited by the divalent ions except zinc which is found to stimulate the enzyme activity. Both the enzymes are inhibited by vanadata, EDTA and EGTA. I₅₀, for vanadate is 0.05 and 0.125 mM for dependent and independent activities, respectively. Sulfhydral groups modifying agents e.g., NEM, DTNB and chlorpromazine are found to affect the enzyme activities in different ways. Thus NEM and chlorpromazine are found to inhibit and DTNB stimulate the enzyme activities in both the cases.     

The thermodynamic efficiency of the Ca2+-Mg2+-ATPase is one hundred percent

The thermodynamic efficiency of the Ca²⁺-Mg²⁺-ATPase of skeletal sarcoplasmic reticulum has been evaluated by comparing the Ca²⁺ gradient established with the ATP/(ADP*P_i) ratio. The evaluation was made at an external Ca²⁺ level (4.7 × 10^–8 M) which is below theK _m value of 7 × 10^–8 M. The Mg-ATP and phosphate concentrations were held constant (0.1 mM) and the ADP concentration was varied. Maximal uptake to an internal free Ca²⁺ concentration of 17 mM was observed at infinite ATP/(ADP*P_i) ratio (absence of ADP). This corresponds to a [Ca²⁺]_i/[Ca²⁺]₀ gradient of 3.6 × 10⁵. A Ca²⁺ gradient one-half as large was observed at an ATP/(ADP*P_i) ratio of 3.5 × 10³ M^–1. The square of the Ca²⁺ gradient is shown to be proportional to the ATP/(ADP*P_i) ratio, for finite values of the latter. The proportionality constant is identical to the equilibrium constant for hydrolysis of ATP (9.02 × 10⁶ M) under these conditions (0.1 mM Mg²⁺, 30°C). The intrinsic thermodynamic efficiency of the pump is shown to be 100%, with a maximal uncertainty of 3%. The efficiency is lower under less optimal conditions, when the pump is inhibited and passive leak processes compete.Dedicated to Prof. Philip George, University of Pennsylvania, whose instruction, research, and example made this contribution possible.     

Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary In reconstituted rabbit skeletal muscle (Ca²⁺ + Mg²⁺)-ATPase proteoliposomes, Ca²⁺-uptake is decreased by more than 90% with T₂ cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T₃ cleavage, which represents the cleavage of A₁ fragment to A_1a + A_1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca²⁺ efflux between control SR and tryptically digested SR in the absence of Mg⁺ ruthenium red or in the presence of ATP. However, the passive Ca²⁺ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg²⁺ + ruthenium red. These results show that the Ca²⁺ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca²⁺ channel inhibitors, Mg²⁺ and ruthenium red. In the reconstituted ATPase proteoliposomes, the Ca²⁺ efflux rates are the same regardless of digestion (T₂); also, efflux is not affected by the presence or absence of Mg²⁺ + ruthenium red. These results indicate that T₂ cleavage causes uncoupling of the Ca²⁺-pump from ATP hydrolytic activity.A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca²⁺ + Mg²⁺)-ATPase polypeptide with the loss of Ca²⁺-transport. Fits of the theoretical equations to the data are consistent with that Ca²⁺-transport system appears to require a dimer of the polypeptide (Ca²⁺ + Mg²⁺)-ATPase.     

Disulfiram lowers Ca2+, Mg2+-ATPase activity of rat brain synaptosomes

The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase activity. In vitro studies indicated that DS (ID₅₀=20 M) produced a dose-dependent inhibition of Ca²⁺, Mg²⁺-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca²⁺, Mg²⁺-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID₅₀=20 M) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase specific activity was observed when treated with DS (30 M) and EGTA (100 M) respectively. The remaining 25–30% of total activity is suggested to be of Mg²⁺-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70–75% of total Ca²⁺, Mg²⁺-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca²⁺, Mg²⁺-ATPase activity could affect its function of maintaining intracellular Ca²⁺ concentration. This could contribute to the deleterious effects on CNS.     

Efflux of magnesium and potassium ions from liver mitochondria induced by inorganic phosphate and by diamide

Addition to rat liver mitochondria of 2 mM inorganic phosphate or 0.15 mM diamide, a thiol-oxidizing agent, induced an efflux of endogenous Mg²⁺ linear with time and dependent on coupled respiration. No net Ca²⁺ release occurred under these conditions, while a concomitant release of K⁺ was observed. Mg²⁺ efflux mediated either by P_i or low concentrations of diamide was completely prevented by EGTA, Ruthenium red, and NEM. These reagents also inhibited the increased rate of state 4 respiration induced both by P_i and diamide. At higher concentrations (0.4 mM), diamide induced an efflux of Mg²⁺ which was associated also with a release of endogenous Ca²⁺. Under these conditions EGTA completely prevented Mg²⁺ and K⁺ effluxes, while they were only partially inhibited by Ruthenium red and NEM. It is assumed that Mg²⁺ efflux, occurring at low diamide concentrations or in the presence of phosphate, is dependent on a cyclic in-and-out movement of Ca²⁺ across the inner mitochondrial membrane, in which the passive efflux is compensated by a continuous energy linked reuptake. This explains the dependence of Mg²⁺ efflux on coupled respiration, as well as the increased rate of state 4 respiration. The dependence of Mg²⁺ efflux on phosphate transport is explained by the phosphate requirement for Ca²⁺ movement.Abbreviations Diamide diazenedicarboxylic acidbis-dimethylamide - FCCP p-trifluoromethoxyphenylhydrazone - EGTA ethylene glycol-bis-(2-amino ethyl ether)-N,N-tetracetic acid - P_i inorganic phosphate - Ruthenium red Ru₂(OH)₂Cl₄ · 7NH₃ · 3H₂O - state 4 controlled state of respiration in the presence of substrate - RCI respiratory control index - NEM N-ethyl maleimide A partial and preliminary report of these results has been published inBiochem. Biophys. Res. Comm.,78 (1977) 23.     

A comparative study of the rat heart sarcolemmal Ca2+-dependent ATPase and myosin ATPase

In order to gain some information regarding Ca²⁺-dependent ATPase, the enzyme was purified from cardiac sarcolemma and its properties were compared with Ca²⁺-ATPase activity of myosin purified from rat heart. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by Ca²⁺ but the maximal activation of Ca²⁺-dependent ATPase required 4 mM Ca²⁺ whereas that of myosin ATPase required 10 mM Ca²⁺. These ATPases were also activated by other divalent cations in the order of Ca²⁺ > Mn²⁺ > Sr²⁺ > Br²⁺ > Mg²⁺; however, there was a marked difference in the pattern of their activation by these cations. Unlike the myosin ATPase, the ATP hydrolysis by Ca²⁺-dependent ATPase was not activated by actin. The pH optima of Ca²⁺-dependent ATPase and myosin ATPase were 9.5 and 6.5 respectively. Na⁺ markedly inhibited Ca²⁺-dependent ATPase but had no effect on the myosin ATPase activity. N-ethylmaleimide inhibited Ca²⁺-dependent ATPase more than myosin ATPase whereas the inhibitory effect of vanadate was more on myosin ATPase than Ca²⁺-dependent ATPase. Both Ca²⁺-dependent ATPase and myosin ATPase were stimulated by K-EDTA and NH₄-EDTA. When myofibrils were treated with trypsin and passed through columns similar to those used for purifying Ca²⁺-ATPase from sarcolemma, an enzyme with ATPase activity was obtained. This myofibrillar ATPase was maximally activated at 3–4 mM Ca²⁺ and 3 to 4 mM ATP like sarcolemmal Ca²⁺-dependent ATPase. K⁺ stimulated both ATPase activities in the absence of Ca²⁺ and inhibited in the presence of Ca²⁺. Both enzymes were inhibited by Na⁺, Mg²⁺, La³⁺, and azide similarly. However, Ca²⁺ ATPase from myofibrils showed three peptide bands in SDS polyacrylamide gel electrophoresis whereas Ca²⁺ ATPase from sarcolemma contained only two bands. Sarcolemmal Ca²⁺-ATPase had two affinity sites for ATP (0.012 mM and 0.23 mM) while myofibrillar Ca²⁺-ATPase had only one affinity site (0.34 mM). Myofibrillar Ca²⁺-ATPase was more sensitive to maleic anhydride and iodoacetamide than sarcolemmal Ca²⁺-ATPase. These observations suggest that Ca²⁺-dependent ATPase may be a myosin like protein in the heart sarcolemma and is unlikely to be a tryptic fragment of myosin present in the myofibrils.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

ATP-dependent calcium transport and its correlation with Ca2+-ATPase activity in basolateral plasma membranes of rat duodenum

Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca²⁺-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH₂)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent K_m of 0.2 μM Ca²⁺ and a V_max of 5.3 nmol Ca²⁺/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca²⁺-ATPase are: K_m = 0.25 μM Ca²⁺ and V_max = 19.2 nmol P_i/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the V_max of ATP-dependent calcium uptake amounts to 25 nmol Ca²⁺/min per mg protein and of the Ca²⁺-ATPase to 23 nmol P_i/min per mg protein. The close correlation between kinetic parameters of Ca²⁺-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca²⁺-pump located in duodenal basolateral plasma membranes.     

Fluoride inhibition of yeast enolase: Crystal structure of the enolase–Mg2+–F−–Pi complex at 2.6 Å resolution

Enolase in the presence of its physiological cofactor Mg²⁺ is inhibited by fluoride and phosphate ions in a strongly cooperative manner (Nowak, T, Maurer, P. Biochemistry 20:6901, 1981). The structure of the quaternary complex yeast enolase–Mg²⁺–F^?–P_i has been determined by X-ray diffraction and refined to an R = 16.9% for those data with F/σ(F) ≥ 3 to 2.6 Å resolution with a good geometry of the model. The movable loops of Pro-35-Ala-45, Val-153-Phe-lo9, and Asp-255-Asn-266 are in the closed conformation found previously in the precatalytic substrate–enzyme complex. Calculations of molecular electrostatic potential show that this conformation stabilizes binding of negatively charged ligands at the Mg²⁺ ion more strongly than the open conformation observed in the native enolase. This closed conformation is complementary to the transition state, which also has a negatively charged ion, hydroxide, at Mg²⁺. The synergism of inhibition by F^? and P_i most probably is due to the requirement of P_i, for the closed conformation. It is possible that other Mg²⁺-dependent enzymes that have OH^? ions bound to the metalion in the transition state also will be inhibited by fluoride ions. © Wiley-Liss, Inc.     

The rate of Ca2+ translocation by sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase measured with intravesicular arsenazo III

Release of Ca²⁺ from the (Ca²⁺ + Mg²⁺)-ATPase into the interior of intact sarcoplasmic reticulum vesicles was measured using arsenazo III, a metallochromic indicator of Ca²⁺. Arsenazo III was placed inside the sarcoplasmic reticulum vesicles by making the vesicles transiently leaky with an osmotic gradient in the presence of arsenazo III. External arsenazo III was then removed by centrifugation. Addition of ATP to the (Ca²⁺ + Mg²⁺)-ATPase in the presence of Ca²⁺ causes the rapid phosphorylation of the enzyme at which time the bound Ca²⁺ becomes inaccessible to external EGTA. The release of Ca²⁺ from the (Ca²⁺ + Mg²⁺)-ATPase to the interior of the vesicle measured with intravesicular arsenazo III was much slower indicating that there is an occluded from the Ca²⁺-binding site which precedes the release of Ca²⁺ into the vesicle. The rate of Ca²⁺ accumulation by sarcoplasmic reticulum vesicles is increased by K⁺ (5–100 mM) and ATP (50–1000 μM) but the initial rate of Ca²⁺ translocation measured after the simultaneous addition of ATP and EGTA to vesicles that were preincubated in Ca²⁺ was not influenced by these concentrations of K⁺ and ATP.     

The calmodulin-activated form of the Ca2+-pumping ATPase of the cardiac sarcolemmal membrane produces Ca2+ gradients with a thermodynamic efficiency of 100%

The thermodynamic efficiency of the calmodulin-activated form of the Ca²⁺-pumping ATPase of the bovine cardiac sarcolemma (SL) was evaluated in sealed vesicles under reversible conditions. The free internal Ca²⁺ concentration ([Ca²⁺]_i) established in the SL vesicle lumen by action of the ATPase was determined as a function of the [ATP]/([ADP][P_i]) ratio for the following experimental conditions: 250mM sucrose, 100mM KCI, 0.1mM Mg²⁺, 25mM HEPES, 25mM Tris, pH 7.40, at 37°C, [Ca²⁺]_o=50nM (1mM Ca/EGTA buffer), 0.75mM Mg-ATP, 0.1mM P_i, variable [ADP]. Under these conditions, with the pump working near itsK _m of 64nM, the [Ca²⁺]_i achieved was 18mM, decreasing with increasing [ADP] for [ADP] 0.84mM. A plot of the square of the [Ca²⁺]_i/[Ca²⁺]_o ratio against [ATP]/([ADP][P_i]) gave a straight line with a slope of 1.5×10⁷M. This was in agreement, within the experimental error, with the equilibrium constant for ATP hydrolysis under these conditions (1.09×10⁷M). These results demonstrate (1) tight coupling between Ca²⁺ transport and ATP hydrolysis with a stoichiometry of 2 Ca²⁺ moved per ATP split and (2) a low degree of passive leakage. Analysis at low [ADP] (<0.83mM) showed the unexpected result that ADP increases the rate of theforward reaction of the pump. The maximal effect on the initial rate is a 96±5% increase, with an EC₅₀ of approximately 0.4mM (ADP). Similar but lesser stimulation was observed with CDP. The implications of the above results for the energetics of the pump and for its physiological function in the beating heart are discussed.     

Calmodulin stimulation of calcium uptake and (Ca2+-Mg2+)-ATPase activities in microsomes from canine tracheal smooth muscle

The role of calmodulin in the regulation of microsomal ⁴⁵Ca²⁺ transport in canine tracheal smooth muscle was studied. Calmodulin stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities in microsomes treated with 0.5 mM EDTA and 0.5 mM EGTA. Oxalate also stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities and the stimulation was additive to the effects of calmodulin. The (Ca²⁺Mg²⁺)-ATPase and ATP-dependent ⁴⁵Ca²⁺ uptake activities are probably related as they exhibited similar [Ca²⁺]_free- and [calmodulin]-dependencies. These results indicate that calmodulin may play a role in the control of the cytosolic [Ca²⁺]_free in canine tracheal smooth muscle.     

Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg+ by (Na+ + K+)-activated ATPase

(1) Contrary to what has usually been assumed, (Na⁺ + K⁺)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na⁺ + Mg²⁺ to ADP-NH₂ and P_i. The activity is ouabain-sensitive and is not detected in the absence of either Mg²⁺ or Na²⁺. The specific activity of the Na⁺ + Mg²⁺ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K⁺. The activity is not stimulated by K⁺, nor can K⁺ replace Na⁺ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na⁺ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The K_m value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH₂]P are not detectably hydrolysed by (Na⁺ + K⁺)-ATPase in the presence of Na⁺ + Mg²⁺. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h^?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg²⁺ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h^?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na⁺ + Mg²⁺ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na⁺ + K⁺)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C.     

A Ca2+-ATPase and a Mg2+/H+-antiporter are present on tonoplast membranes from roots of Zea mays L.

The primary or secondary energized transport of Ca²⁺, Mg²⁺ and H⁺ into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca²⁺ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca²⁺ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Ca²⁺-pumping and tonoplast H⁺-pyrophosphatase (PP_iase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca²⁺-pumping activity could be detected. The ATP-dependent Ca²⁺ uptake into tonoplast vesicles (using Ca²⁺ concentrations from 0.8–1 μM) was completely inhibited by the Ca²⁺ ionophore ionomycin (1 μM) whereas the protonophore nigericin (1 μM) which eliminates ATP-dependent intravesicular H⁺ accumulation had no effect. Vanadate (IC₅₀ = 43 μM) and diethylstilbesterol (IC₅₀ = 5.2 μM) were potent inhibitors of this type of Ca²⁺ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%–50% of the ATP-dependent activity (K _m = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca²⁺ transport mechanism is the only functioning Ca²⁺ transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca²⁺ concentration. In contrast, the secondary energized Ca²⁺-transport mechanism of the tonoplast, the low-affinity Ca²⁺/H⁺-antiporter, which was reported to allow the uptake of Ca²⁺ in exchange for H⁺, functions chiefly as an Mg²⁺ transporter under physiological conditions because cytosolic Mg²⁺ is several orders of magnitude higher than the Ca²⁺ concentration. This conclusion was deduced from experiments showing that Mg²⁺ ions in a concentration range of 0.01 to 1 mM triggered a fast efflux of H⁺ from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H⁺-ATPase and H⁺-PP_iase was found to be influenced by Ca²⁺ differently from and independently of the Mg²⁺ concentration. Calcium was a strong inhibitor for the H⁺-PP_iase (IC₅₀ = 18 μM, Hill coefficient nH = 1.7) but a weak one for the H⁺-ATPase (IC₅₀ = 330 μM, nH = 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca²⁺-and Mg²⁺-regulated H⁺-PP_iase, (ii) the newly described high-affinity Ca²⁺-AT-Pase, (iii) the low-affinity Mg²⁺(Ca²⁺)/H⁺-antiporter and (iv) the H²⁺-ATPase.