Differentiation between Ca2+ transport and ATP-induced Ca2+ binding by sarcoplasmic reticulum

The Ca²⁺ actively accumulated by sarcoplasmic reticulum isolated from skeletal muscle is composed of two fractions; one represented by intravesicular free Ca²⁺ and another represented by Ca²⁺ selectively bound to the membranes. Both of these Ca²⁺ fractions depend on ATP, although it is not clear whether ATP hydrolysis is essential for accumulation of the second Ca²⁺ fraction. The existence of the membrane-bound Ca²⁺ induced by ATP is clearly shown in experiments in which the Ca²⁺ retention by sarcoplasmic reticulum is measured in the presence and in the absence of X-537A, a Ca²⁺ ionophore, which makes the membrane permeable to Ca²⁺. Thus, in the presence of X-537A all Ca²⁺ accumulated due to ATP is bound to the membranes. This membrane-bound Ca²⁺ represents about 30 nmol/mg protein in the range of external $\text{p}\text{Ca}$ values of 7 to 3.5. The magnitude of this Ca²⁺ fraction is slightly higher whether or not the experiments are performed in the presence of oxalate, which greatly increased the intravesicular Ca²⁺ accumulation. Furthermore, taking advantage of the impermeability of sarcoplasmic reticulum to EGTA, it is possible to show the existence of the membrane-bound Ca²⁺ as a distinct fraction from that which exists intravesicularly.     

The role of calcium in transferrin and iron uptake by reticulocytes

The possible role of calcium in the uptake of transferrin and iron by rabbit reticulocytes was investigated by altering cellular calcium levels through the use of the chelating agents EDTA and $\text{ethyleneglycol-bis}\text{-(3-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid (EGTA) and the ionophores, A23187 and X537A. Incubation of reticuloyctes with EDTA or EGTA at 4°C had no effect on transferrin and iron uptake but incubation at 37°C resulted in an irreversible inhibition associated with decreased adsorption of transferrin to the cells and evidence of inactivation or loss of the transferrin receptors. Transferrin and iron uptake were also inhibited when the cells were incubated with A23187 or X537A. In the case of A23187 the action was primarily exerted on the temperature-sensitive stage of transferrin uptake and was associated with loss of cellular K⁺ and decrease in cell size. The effect was greater when Ca²⁺ was added to the incubation medium than its absence. X537A produced relatively greater inhibition of iron uptake than of transferrin uptake, associated with a reduction in cellular ATP concentratio. The action of X537A was unaffected by the presence of Ca²⁺ in the incubation medium.The results obtained with EDTA and EGTA indicate that cell membrane Ca²⁺ is required for the integrity or binding of transferrin receptors to the reticulocyte membrane. No evidence was obtained from the experiments with ionophores that an increase of cellular Ca²⁺ affects transferrin and iron uptake directly. The inhibition caused by A23187 was mainly due to a reduction in cell size resulting from increased membrane permeability to K⁺ and that caused by X537A appeared to result from an inhibition of energy metabolism and ATP production.     

The kinetics of ionophore X-537A-mediated transport of manganese through dipalmitoylphosphatidylcholine vesicles A 1H-NMR study

We have studied the kinetics of ionophore X-537A-mediated transport of manganese ions into small unilamellar vesicles formed from dipalmitoylphosphatidylcholine. To follow the transport we used the paramagnetic effect of manganese on the ¹H-NMR signal from choline trimethylammonium groups on the inner phospholipid monolayer. The transport of only one manganese ion produces an intravesicular concentration which is high enough (approx. 1 mM) to substantially broaden this signal. The observed signal thus arises predominantly from those vesicles which contain no manganese. Therefore, as manganese is transported into the vesicles the observed signal decreases in intensity, but does not broaden. The initial time-dependence of the intensity of the signal, $\text{S(t)}$, can be approximated by the simple first-order rate law: $\text{S(t) = S}\text{(O)}\text{exp}\text{(?K′t)}$, where $\text{K′}$ is the probability per unit time for the transport of a manganese ion from the external medium to the intravesicular space. From the dependence of $\text{K′}$ on the ionophore X-537A concentration we conclude that manganese is transported into the vesicles via both 1 : 1 and 2 : 1 complexes with ionophore X-537A. At low ratios of ionophore X-537A to vesicles transport via the 1 : 1 complex predominates; at high ratios transport via the 2 : 1 complex predominates. From the dependence of $\text{K′}$ on manganese concentration we determined that under our conditions the equilibration of ionophore X-537A between vesicles is much faster than the transport of manganese through the vesicles. Lastly, from the dependence of $\text{K′}$ on temperature, we conclude that the ionophore X-537A-mediated transport of manganese into the dipalmitoylphosphatidylcholine vesicles is very sensitive to the gel-liquid crystalline phase transition.     

Effects of the ionophores,X-537A and A-23187 on catecholamine release from the in vitro frog adrenal

1. The ionophore X-537A increases the rate of catecholamine release from the $\text{in vitro}$ frog adrenal.2. The ratio of epinephrine/norepinephrine measured during X-537A stimulation was the same as that during spontaneous release.3. Even when Ca⁺⁺ was removed from the Ringer, X-537A stimulated catecholamine release, but depolarization by elevated extra-cellular K⁺ was no longer effective.4. X-537A also increases the release of dopamine β-hydroxylase, suggesting that the ionophore acts, at least in part, by stimulating the exocytosis of the chrommaffin granule contents.5. Therefore, it is questionable whether the release of catecholamines by X-537A is owing to its action as a Ca⁺⁺- ionophore.6. The divalent cation ionophore, A-23187 (50μM), did not affect the rate of catecholamine release.     

Transport of alpha-methyl glucoside in mutants of Escherichia coli K12 deficient in Ca2+, Mg2+-activated adenosine triphosphatase.

The initial rate of uptake of methyl α-D-glucopyranoside by $\text{Escherichia coli}$ is inhibited by respiration. The inhibition is more pronounced in mutant strains which cannot use the energy-rich state of the membrane to form ATP because of a defective Ca²⁺, Mg²⁺-activated ATPase. In both mutant and normal strains, the inhibition of glucoside uptake is not accompanied by an increase of the ATP content of the cells and is abolished by carbonyl cyanide $\text{m}$-chlorophenylhydrazone, a drug which dissipates membrane energy. It appears, therefore, that the inhibitory effect of respiration is mediated by the energy-rich state of the membrane and that ATP does not participate in the inhibition.     

Utilization of X-537A to distinguish between intravesicular and membrane-bound calcium ions in sarcoplasmic reticulum

The Ca²⁺ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca²⁺ and surface membrane-bound Ca²⁺ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca²⁺ in the absence of ATP, 10–12 h are necessary for measurable amounts of Ca²⁺ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca²⁺ from ‘loaded’ vesicles only after this period of incubation. A fraction of Ca²⁺ of 50–60nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg²⁺ and K⁺ in the medium and is readily released by ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca²⁺ (30–40 nmol/mg protein) is rapidly released by X-537A. The results indicate that most of the Ca²⁺ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca²⁺ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.     

31P NMR lineshapes of β-P (ATP) in the presence of mg2+ and ca2+ : estimate of exchange rates

The ³¹ P NMR chemical shift of β-P of adenosine triphosphate (ATP) undergoes a substantial change (2̃–3 ppm) upon chelation of divalent ions such as Mg²⁺ or Ca²⁺. In the presence of nonsaturating amounts of Mg²⁺ or Ca²⁺, the lineshape of this resonance depends on the characteristic association and dissociation rates of these metal-ATP complexes. A procedure for computer simulation of this lineshape is outlined. A comparison of computer-simulated lineshapes with the experimental lineshapes obtained at 121 MHz was used to determine the following dissociation rate of Mg²⁺ and Ca²⁺ from their ATP complexes at 20°C and pH 8.0: Ca²⁺, > 3 × 10⁵ s^?1 (Hepes buffer); Mg²⁺, 1200 s^-1 (no buffer), 1000 s^-1 (Tris buffer) and 2100 s^?1 (Hepes buffer). The limits of error are ± 10% in these values. For the Mg²⁺ complexes, the rates were determined as a function of temperature to obtain activation energies (with a maximum deviation of 10% in the least-squares fit): 8.1 $\text{Kcal}\text{mole}$ (no buffer and Hepes buffer) and 6.8 $\text{kcal}\text{mole}$ (Tris buffer). Lineshapes of the β-Presonance simulated as a function of Mg²⁺ concentration, using 2100 s^?1 for the dissociation rate, are also presented. The computer simulation of lineshapes offers a reliable and straightforward method for the determination of exchange rates of diamagnetic cations from their ATP complexes, under a variety of sample conditions.     

Phosphoglycerate mutase in developing forespores of Bacillus megaterium may be regulated by the intrasporal level of free manganous ion.

When young intact forespores of Bacillus megaterium were incubated with either Mn⁺⁺ or the ionophore X-537A, the pool of 3-phosphoglyceric acid (3-PGA) was stable. However, incubation of forespores with Mn⁺⁺ plus the ionophore X-537A resulted in rapid and complete utilization of the 3-PGA. This effect was not seen with Ca⁺⁺ or Mg⁺⁺, and was also not observed with older forespores or fresh dormant spores. Since the phosphoglycerate mutase of $\text{B.}\text{megaterium}$ has an absolute and specific requirement for Mn⁺⁺, it is possible that phosphoglycerate mutase in developing forespores may be inactive because of a low intrasporal level of free Mn⁺⁺.     

The relation between fatty acid mobilization and contractility in the isolated,perfused rat heart

The contractility of hearts from normal fed rats is decreased by 70% during perfusion with 50 μM chloroquine, which is a potent inhibitor of endogenous lipolysis. In triacylglycerol-rich hearts, obtained by feeding rats rapeseed-oil, chloroquine depresses lipolysis much less, while contractility was found to be inhibited only 30%. In both groups of hearts the effect of chloroquine was decreased by adding fatty acids, prostaglandin E₁, the $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ionophore X-537A or more Ca²⁺ to the perfusion fluid. Norepinephrine and glucagon also stimulate chloroquine-depressed hearts. The conclusion is therefore reached that fatty acids act as Ca²⁺-vehicles in heart cells and that chloroquine, by inhibiting lipolysis, decreases Ca²⁺-transport by lowering unesterified fatty acid levels.     

Purification of a water-soluble Mg2+-ATPase from human erythrocyte membranes

A water-soluble Mg²⁺-ATPase previously reported (White, M.D. and Ralston, G.B. (1976) Biochim. Biophys. Acta 436, 567–576) has been purified from human erythrocyte membranes. The purified enzyme has a molecular weight of 575 000; the apparent minimum molecular weight was 100 000, corresponding to a soluble protein of the component 3 region. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP was 1 mM and apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was 3.6 mM. By means of histochemical activity staining in acrylamide gels it was shown that the purified ATPase preparation could be inhibited by Cd²⁺ and Zn²⁺ salts, $\text{p-}\text{chloromercuribenzoate}$ and $\text{N-}\text{ethylmaleimide}$, known inhibitors of membrane endocytosis.     

Synthesis of phosphatidylglycerol by chloroplasts from leaves of Spinacia oleracea L. (spinach)

Purified chloroplasts from leaves of Spinacia oleracea L. (spinach) incorporated glycerol 3-phosphate into diacylglycerol, monoacylglycerol, phosphatidylglycerol, phosphatidic acid, and lysophosphatidic acid. The omission of ATP or CTP, CoA or illumination decreased the incorporation markedly. The fraction of incorporated glycerol 3-phosphate found in phosphatidylglycerol was greatly reduced by the omission of bicarbonate, acetate, and ATP, or in darkness, low-osmolarity medium, or high magnesium ion concentration (10 mM). Incorporation of glycerol 3-phosphate into lipid and specifically into phosphatidylglycerol was optimal at a $\text{Mg}{}^{\mathrm{2+}}\text{CTP}$ ratio of 1, whereas the optimal ratio for $\text{Mg}{}^{\mathrm{2+}}\text{ATP}$ was closer to 2. The $\text{Mg}{}^{\mathrm{2+}}\text{CTP}$ gave lower total incorporation but a higher fraction of incorporation in phosphatidylglycerol. Triton X-100 inhibited incorporation of glycerol 3-phosphate into lipid, especially into phosphatidylglycerol.     

Conversion of the Ca2+-ATPase from Rhodospirillum rubrum into a Mg2+-dependent enzyme by 1,N6-etheno ATP

Nucleoside triphosphate hydrolysis of $\text{R.}\text{rubrum}$ ATPase complexes can be changed from Ca²⁺-dependence to Mg²⁺-dependence by replacing ATP with 1,N⁶-etheno ATP. Four ATPase complexes which have been prepared by different procedures hydrolyze ATP and 1,N⁶-etheno ATP at different rates in dependence on the added metal ions. These differences allow an easy distinction of the various enzyme forms.     

Effects of lanthanum on calcium-dependent phenomena in human red cells

Lanthanum (0.25 mM) does not penetrate into fresh or Mg²⁺-depleted cells, whereas it does into ATP-depleted or $\text{ATP + 2,3-diphosphoglycerate-depleted cells}$, into cells containing more than 3 mM calcium, or cells stored for more than 4 weeks in acid/citrate/dextrose solution. In fresh cells loaded with calcium, extracellular lanthanum blocks the active Ca²⁺-efflux completely and inhibits $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) activity to about 50%. In Mg²⁺-depleted cells Ca²⁺-Ca²⁺ exchange is inhibited by lanthanum. Ca²⁺-leak is unaffected by lanthanum up to 0.25 mM concentration; higher lanthanum concentrations reduce leak rate. In NaCl medium Ca²⁺-leak ± S.D. amounts to $\text{0.28 ± 0.08 μ}\text{mol/l}$ of cells per min, whereas in KCl medium to $\text{0.15 ± 0.04 μ}\text{mol/l}$ of cells per min at 2.5 mM [Ca²⁺]_e and 0.25 mM [La³⁺]_e pH 7.1.Lanthanum inhibits Ca²⁺-dependent rapid K⁺ transport in ATP-depleted and propranolol-treated red cells, i.e. whenever intracellular calcium is below a critical level. The inhibition of the rapid K⁺ transport can be attributed to protein-lanthanum interactions on the cell surface, since lanthanum is effectively detached from the membrane lipids by propranolol.Lanthanum at 0.2–0.25 mM concentration has no direct effect on the morphology of red cells. The shape regeneration of Ca²⁺-loaded cells, however, is blocked by lanthanum owing to Ca²⁺-pump inhibition. Using lanthanum the transition in cell shape can be quantitatively correlated to intracellular Ca²⁺ concentrations.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg²⁺, the plasma membrane preparation exhibits a Ca²⁺-dependent ATPase activity of 2 μmol P_i per h per mg protein. It is suggested that this $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ activity is related to the measured Ca²⁺ transport which was characterized by $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for ATP and Ca²⁺ of $\text{44 ± 9 μ}\text{M}$ and $\text{0.25 ± 0.10 μ}\text{M}$, respectively. Phosphorylation of plasma membranes with [γ-³²P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca²⁺-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and from the catalytic subunit of $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca²⁺-pump in plasma membranes isolated from nucleated cells.     

Calcium transport and Ca2+-ATPase activity in ram spermatozoa plasma membrane vesicles

Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca²⁺-activated Mg²⁺-ATPase and Ca²⁺ transport activities. Membrane vesicles that were exposed to oxalate as a Ca²⁺-trapping agent accumulated Ca²⁺ in the presence of Mg²⁺ and ATP. The $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for Ca²⁺ uptake was 33 nmol/mg protein per h, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for Ca²⁺ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca²⁺ ionophore A23187, added initially, completely inhibited net Ca²⁺ uptake and, if added later, caused the release of Ca²⁺ previously accumulated. A Ca²⁺-activated ATPase was present in the same membrane vesicles which had a $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 1.5 μmol/mg protein per h at free Ca²⁺ concentration of 10 μM. This Ca²⁺-ATPase had $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 4.5 μM and 110 μM for Ca²⁺ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca²⁺ by the vesicles. The Ca²⁺-ATPase activity was insensitive to ouabain. Both Ca²⁺ transport and Ca²⁺-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca²⁺ transport activity and a Ca²⁺-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin.     

Protein-bound histidine,as well as protein-bound serine,residues are sites of phosphorylation in the synaptic plasma membrane

When synaptic plasma membrane fragments are incubated with ATP in the presence of Mg²⁺, phosphate is transferred, not only to protein-bound serine, but also to protein-bound histidine. The phosphorylation of protein-bound serine is stimulated by cyclic AMP and has a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP of about 0.12 mM, both in the presence and absence of cyclic AMP. By contrast, the phosphorylation of protein-bound histidine is unaffected by cyclic AMP and does not follow Michaelis-Menton kinetics since a non-linear double reciprocal plot is given when activity is measured at various ATP concentrations.     

ATP/ADP exchange activity of gastric (H+ + K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the $\text{(}\text{H}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to $\text{P}{}^1$, $\text{P}{}^5$ -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg²⁺ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration ($\text{K}{}_{0.5}\text{= 116 μ}\text{M}$) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg²⁺ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K⁺ as a function of pH and Mg²⁺.     

Multiple ion-stimulated adenosine triphosphatase activities associated with membranes of the diatom Nitzschia alba.

At least ten distinct ATP-hydrolyzing activities are associated with mitochondria, endoplasmic reticulum-, Golgi-, and plasma membrane-enriched fractions from the marine diatom, Nitzschia alba. These activities are divided into four groups: Ca²⁺-dependent, Mg²⁺-dependent monovalent cation-stimulated, Mg²⁺-anion-stimulated ATPases, and Mg²⁺-dependent nucleotidases.The Mg²⁺-dependent activities hydrolyze nucleoside triphosphates and, in some membranes, nucleoside diphosphates. Molar ratios of 1:2 $\text{ATP}\text{Mg}{}^{\mathrm{2+}}$ are preferred. However, their divalent cation requirements are not specific, and they can effectively utilize Ca²⁺, Mn²⁺, Mg²⁺, or Zn²⁺. The most effective inhibitors of the Mg²⁺-dependent activities are oligomycin, NaN₃, and NaF.Optimal activity of the Mg²⁺-dependent monovalent cation-stimulated ATPase is obtained at Na⁺, or Na⁺ plus K⁺ concentrations of 100–300 mm. Under these high salt conditions, ATP is hydrolyzed almost exclusively, and Mg²⁺ is specifically required for activation. Preference is for a molar ratio of $\text{ATP}\text{Mg}{}^{\mathrm{2+}}\text{≧ 2}$, and the sulfhydryl-blocking agents, p-chloromecuribenzoate, N-ethylmaleimide, and iodoacetamide strongly or completely inhibit ATP hyrolysis.     

Activation of heart sarcolemmal Ca2+/Mg2+ ATPase by cyclic AMP-dependent protein kinase.

The sarcolemmal membrane obtained from rat heart by hypotonic shock-LiBr treatment method was found to incorporate ³²P from [γ-³²P] ATP in the absence and presence of cyclic AMP and protein kinase. The phosphorylated membrane showed an increase in Ca²⁺ ATPase and Mg²⁺ ATPase activities without any changes in Na⁺K⁺ ATPase activity. The observed increase in $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ ATPase activity was found to be associated with an increase in V_max value of the reaction whereas K_a value for $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}$ was not altered. These results provide information concerning biochemical mechanism for increased calcium entry due to hormones which are known to elevate cyclic AMP levels in myocardium and produce a positive inotropic effect.     

Use of an ionophore to maintain repeated caffeine contractures in holothurian muscle

When treated with caffeine (10 mM) isolated longitudinal retractor muscles of $\text{Isostichopus badionotus}$ show a contracture which diminishes progressively with repeated doses. Brief treatment with X-537A (10^?5 M) restores contractility for the duration of the exposure. However, after longer treatment with X-537A, restored contractility persists even after X-537A has been washed off. The persistent contractility may indicate that X-537A has partitioned into muscle cell membranes, thus speeding the time course of refilling of calcium stores.