Specific modification of gastric K+-stimulated ATPase activity by thimerosal

Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K+-ATPase and the K+-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K+-stimulated ATPase, while K+-p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K+-p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K+-p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K+-p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; Km for p-nitrophenylphosphate unchanged for Ka for K+ increased. ATP, which is a potent inhibitor of K+-p-nitrophenylphosphatase activity in native membranes (KI approximately 200 microM). These data suggest that there are multiple SH groups which differentially influence the gastric K+-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K+-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H+-transport reaction.     

Kinetic studies on the (Na+ + K+)-dependent ATPase. Evidence for coexisting sites for Na+, K+ and Mg2+

Na⁺-ATPase activity of a dog kidney (Na⁺ + K⁺)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl₂, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl₂. The $\text{K}{}_{0.5}$ for the effect of MgCl₂ was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na⁺-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl₂. Similar thimerosal treatment reduced (Na⁺ + K⁺)-ATPase activity by half but did not appreciably affect the $\text{K}{}_{0.5}$ for activation by either Na⁺ or K⁺, although it reduced inhibition by high Na⁺ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na⁺: Na⁺-discharge sites at which Na⁺-binding can drive E₂-P back to E₁-P, thereby inhibiting Na⁺-ATPase activity, and sites activating E₂-P hydrolysis and thereby stimulating Na⁺-ATPase activity, corresponding to the K⁺-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na⁺-discharge and K⁺-acceptance sites. Mg²⁺ may stimulate Na⁺-ATPase activity by favoring E₂-P over E₁-P, through occupying intracellular sites distinct from the phosphorylation site or Na⁺-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na⁺-ATPase reaction and lessen Na⁺-inhibition of the (Na⁺ + K⁺)-ATPase reaction by increasing the efficacy of Na⁺ in activating E₂-P hydrolysis.     

Endogenous protease mediated manifestation of a Ca2+-stimulated ATPase in purified dog gastric microsomes

An endogenous soluble protease has been demonstrated to unmask a Ca²⁺-stimulated ATPase activity in purified dog gastric microsomes. The presence of ATP during protease treatment appears essential for the manifestation of the gastric Ca²⁺-stimulated ATPase activity. The endogenous protease appears to have trypsin-like activity, since soybean trypsin inhibitor completely blocks the protease effect. Manifestation of the Ca²⁺-stimulated ATPase occurs without affecting the microsomal (H⁺ +K⁺)-ATPase activity and associated H⁺ uptake ability. The unmasked Ca²⁺-stimulated ATPase appears insensitive to calmodulin. Possible roles of the enzyme in the regulation of gastric H⁺ transport have been discussed.     

Possible role of sulphatide in the K+-activated phosphatase activity

A microsomal fraction rich in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has been isolated from the outer medulla of pig kidney. $\text{(}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{activated}$ ouabain-sensitive phosphatase activity was studied in this preparation treated with arylsulphatase, an enzyme that specifically hydrolyzes ceramide galactose-3-sulphate. The activity of phosphatase was inactivated in proportion to the amount of sulphatide hydrolyzed. A maximum inactivation of ouabain-sensitive activity was obtained with 60% of the sulphatide content hydrolyzed. The inactivation caused by arylsulphatase was partially reversed by the sole addition of sulphatide. The evidence offered in this paper about sulphatide function in the sodium pump mechanism supports the idea that sulphatides are involved in the K⁺-activated phosphatase, a partial reaction of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$.     

Protective effect of EGTA on rat gastric membranes enriched in (H+-K+)-ATPase

Rat gastric membranes enriched in (H⁺-K⁺)-ATPase, when prepared in the presence of 1 mM ethyleneglycol-bis-(β-aminoethyl ether)N,N′-tetraacetic acid, showed the ability to accumulate H⁺ ions upon addition of ATP, KCl, and valinomycin. The membranes were largely impermeable to K⁺ and Cl^?. In contrast, the rat membranes prepared without the Ca²⁺ chelator lost the ability to develop a pH gradient because of the membrane leakiness to H⁺. A majority of these membrane vesicles became also permeable to K⁺. We suggest that the calcium chelator preserved the gastric membrane permeability barrier during isolation by inhibiting various Ca²⁺-dependent phospholipases in rat gastric mucosa.     

Enthalpy, entropy and heat capacity changes induced by binding of calcium ions to cardiac troponin C

Microcalorimetric titrations have been used to study the binding of Ca2+ to cardiac troponin C. Measurements were made both in the presence and in the absence of Mg2+, and at temperatures of 5 degrees, 15 degrees and 25 degrees C. Changes in enthalpy, entropy and heat capacity of troponin C associated with Ca binding have been determined. Cardiac troponin C exhibited a decrease in enthalpy and an increase in entropy associated with Ca binding. Enthalpy changes increased linearly with temperature, indicating that the Ca binding causes negative changes in the heat capacity of troponin C. These results show that the Ca binding causes a strong hydrophobic effect and a tightening of the molecular structure of cardiac troponin C.     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

Ca2+-dependent K+ permeability of heart sarcolemmal vesicles. Modulation by cAMP-dependent protein kinase activity and by calmodulin

The Ca2+-dependent K+ permeability of heart sarcolemma vesicles was measured by following the transmembrane movement of the charge compensating tetraphenylborate anion. The increase in vesicles permeability induced by Ca2+ is lost when membrane proteins are dephosphorylated by an endogenous protein phosphatase and is restored by a phosphorylation process catalysed by a cAMP-dependent protein kinase. The calmodulin antagonist R 24571 lowers the Ca2+-dependent K+ permeability by decreasing the Ca2+ affinity of the K+ transporting system.     

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⁺.     

The K+- and HCO3−-stimulated phosphatase activities in renal microsomes of rats

The K⁺-stimulated phosphatase activity of microsomes from rat kidney was not inhibited by l-phenylalanine, but the HCO₃^?-stimulated phosphatase activity was markedly inhibited by l-phenylalanine. Valinomycin enhanced the HCO₃^?-stimulated phosphatase activity, but did not enhance the K⁺-stimulated phosphatase activity. Ouabain did not inhibit the HCO₃^?-stimulated phosphatase activity, but inhibited the K⁺-stimulated phosphatase activity.The renal K⁺-stimulated phosphatase activity was suppressed to 40% of the control values by adrenalectomy, but the renal HCO₃^?-stimulated phosphatase activity was little suppressed by adrenalectomy. The renal K⁺-stimulated phosphatase activity in intact and adrenalectomized rats was found to be significantly elevated, in a manner similar to the elevation of the renal (Na⁺ + K⁺)-ATPase activity by aldosterone treatment (P < 0.02).     

Effect of external ATP on the plasma membrane permeability and (Na++K+)-ATPase activity of mouse neuroblastoma cells

1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na⁺ relative to K⁺, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K⁺ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na⁺. Under these conditions the pumping activity of the Neuro-2A (Na⁺+K⁺)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na⁺+K⁺)-ATPase for ouabain, as measured by direct [³H]ouabain-binding studies and by inhibition studies of active K⁺ uptake. In the presence of 3.5 mM ATP and the absence of external K⁺ both techniques indicate an apparent dissociation constant for ouabain of 2·10^?6 M. Neuro-2A cells contain (3.5±0.7)·10⁵ ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7±0.4)·10^?20 mol K⁺/min per copy of (Na⁺+K⁺)-ATPase at room temperature.     

Studies of gastric Ca2+-stimulated adenosine triphosphatase I. characterization and general properties

Gastric microsomes do not contain any significant Ca²⁺-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in a significant (2–3-fold) increase in the basal (with Mg²⁺ as the only cation) ATPase activity, with virtual elimination of the K⁺-stimulated component. Such treatment causes unmaksing of a latent Mg²⁺-dependent Ca²⁺-stimulated ATPase. Other divalent cations such as Sr²⁺, Ba²⁺, Zn²⁺ and Mn²⁺ were found ineffective as a substitute for Ca²⁺. Moreover, those divalent cations acted as inhibitors of the Ca²⁺-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 70 μM for ATP and the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ values for Mg²⁺ and Ca²⁺ are about $\text{4 · 10}{}^{\mathrm{?4}}\text{M}$ and 10^?7 M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H⁺ transport have been discussed.     

A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia

Deciliation of Paramecium tetraurelia by a Ca²⁺ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca²⁺-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca²⁺, Sr²⁺, or Ba²⁺, but not by Mg²⁺ or by monovalent cations. The crude enzyme has a specific activity of 2–3 μmol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50°C, and which has a sedimentation coefficient of 8–10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.     

The K+-induced apparent heterogeneity of high-affinity nucleotide-binding sites in (Na+ + K+)-ATPase can only be due to the oligomeric structure of the enzyme

K⁺ induces an apparent heterogeneity among an otherwise homogeneous population of nucleotide-binding sites in (Na⁺ + K⁺)-ATPase preparations from pig kidney. With the help of ouabain we show that this heterogeneity cannot be due to a mixture of different and independent sites and conclude that each enzyme molecule must contain two nucleotide site-containing units that show interaction. Na⁺ induces an apparent heterogeneity among an otherwise homogeneous population of ouabain-binding sites. The argument is, therefore, extended to include one ouabain site on each of the structural units that bind nucleotide. All these structural units are shown to hydrolyse substrate at identical rates. Using the presently available molecular weight data, it is concluded that the enzyme is composed of two subunits each possessing one nucleotide-binding site, one ouabain-binding site, one α-peptide and the capacity for hydrolysing ATP and $\text{p-}\text{nitrophenyl}$ phosphate.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca²⁺ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl₂, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca²⁺ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca²⁺ uptake, a second phase of Ca²⁺ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca²⁺ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca²⁺ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca²⁺ influx of sarcoplasmic reticulum near steady state of Ca²⁺ uptake was measured by pulse labeling with ⁴⁵Ca²⁺. The Ca²⁺ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca²⁺ uptake in normal medium, Ca²⁺ influx was balanced by Ca²⁺ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca²⁺ exchange rate at the first plateau of Ca²⁺ uptake was about half of that in normal medium. When the second phase of Ca²⁺ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca²⁺ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca²⁺ uptake was also observed. These data suggest that the Ca²⁺ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca²⁺ efflux, which subsequently stimulates Ca²⁺ exchange.     

The influence of methanol on the interactions of calcium with the pyridine nucleotides

The interactions of calcium with NAD+, NADH, NADP+ and NADPH in a 50% (by volume) methanol/water mixture (pH 7, 25 degrees C) were studied by calorimetry. The association constants for 1:1 complex formation were found to be 6.6 +/- 0.2, 270 +/- 76, 18 +/- 3 and 98 +/- 10 for NAD+, NADH, NADP+ and NADPH, respectively. Comparing these to the association constants for an aqueous system reveals that as the polarity of the solvent system is decreased the interactions involving NAD+, NADP+ and NADPH are all decreased. In contrast, the interaction involving NADH is markedly increased. All the interactions were found to be endothermic.     

Solubilization of active (H+ + K+)-ATPase from gastric membrane

(H⁺ + K⁺)-ATPase-enriched membranes were prepared from hog gastric mucosa by sucrose gradient centrifugation. These membranes contained Mg²⁺-ATPase and p-nitrophenylphosphatase activities (68 ± 9 μmol P_i and 2.9 ± 0.6 μmol p-nitrophenol/mg protein per h) which were insensitive to ouabain and markedly stimulated by 20 mM KCl (respectively, 2.2- and 14.8-fold). Furthermore, the membranes autophosphorylated in the absence of K⁺ (up to 0.69 ± 0.09 nmol P_i incorporated/mg protein) and dephosphorylated by 85% in the presence of this ion. Membrane proteins were extracted by 1–2% (w/v) n-octylglucoside into a soluble form, i.e., which did not sediment in a 100 000 × g × 1 h centrifugation. This soluble form precipitated upon further dilution in detergent-free buffer. Extracted ATPase represented 32% (soluble form) and 68% (precipitated) of native enzyme and it displayed the same characteristic properties in terms of K⁺-stimulated ATPase and p-nitrophenylphosphatase activities and K⁺-sensitive phosphorylation: Mg²⁺-ATPase (μmol P_i/mg protein per h) 32 ± 9 (basal) and 86 ± 20 (K⁺-stimulated); Mg²⁺-p-nitrophenylphosphatase (μmol p-nitrophenol/mg protein per h) 2.6 ± 0.5 (basal) and 22.2 ± 3.2 (K⁺-stimulated); Mg²⁺-phosphorylation (nmol P_i/mg protein) 0.214 ± 0.041 (basal) and 0.057 ± 0.004 (in the presence of K⁺). In glycerol gradient centrifugation, extracted enzyme equilibrated as a single peak corresponding to an apparent 390 000 molecular weight. These findings provide the first evidence for the solubilization of (H⁺ + K⁺)-ATPase in a still active structure.