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.     

Solvent effects on ouabain binding to the (Na+,K+)-ATPase of rat brain

The effects of the solvents deuterated water (²H₂O) and dimethyl sulfoxide (Me₂SO) on [³H]ouabain binding to (Na⁺,K⁺)-ATPase under different ligand conditions were examined. These solvents inhibited the type I ouabain binding to the enzyme (i.e., in the presence of Mg²⁺+ATP+Na⁺). In contrast, both solvents stimulated type II (i.e., Mg²⁺+P_i-, or Mn²⁺-dependent) binding of the drug. The solvent effects were not due to pH changes in the reaction. However, pH did influence ouabain binding in a differential manner, depending on the ligands present. For example, changes in pH from 7.05 to 7.86 caused a drop in the rate of binding by about 15% in the presence of Mg²⁺+Na⁺+ATP, 75% in the Mg²⁺+P_i system, and in the presence of Mn²⁺ an increase by 24% under similar conditions. Inhibitory or stimulatory effects of solvents were modified as various ligands, and their order of addition, were altered. Thus, ²H₂O inhibition of type I ouabain binding was dependent on Na⁺ concentration in the reaction and was reduced as Na⁺ was elevated. Contact of the enzyme with Me₂SO, prior to ligands for type I binding, resulted in a greater inhibition of ouabain binding than that when enzyme was exposed to Na⁺+ATP first and then to Me₂SO. Likewise, the stimulation of type II binding was greater when appropriate ligands acted on enzyme prior to addition of the solvent. Since Me₂SO and ²H₂O inhibit type I ouabain binding, it is proposed that this reaction is favored under conditions which promote loss of H₂O, and E₁ enzyme conformation; the stimulation of type II ouabain binding in the presence of the solvents suggests that this type of binding is favored under conditions which promote the presence of H₂O at the active enzyme center and E₂ enzyme conformation. This postulation of a role of H₂O in modulating enzyme conformations and ouabain interaction with them is in concordance with previous observations.     

Slow transition of phosphoenzyme from ADP-sensitive to ADP-insensitive forms in solubilized Ca2+, Mg2+-ATPase of sarcoplasmic reticulum: evidence for retarded dissociation of Ca2+ from the phosphoenzyme.

Solubilized Ca²⁺, Mg²⁺-ATPase of sarcoplasmic reticulum was phosphorylated with ATP without added MgCl₂. The phosphoenzyme formed was ADP-sensitive. Ca²⁺ in the medium was chelated after phosphorylation. This induced a slow transition of the phosphoenzyme from ADP-sensitive to ADP-insensitive forms. The ADP-sensitivity was restored by subsequent addition of CaCl₂. These results showed that the transition was caused by dissociation of Ca²⁺ bound to the phosphoenzyme. Further observations indicated that, when Ca²⁺ in the medium was chelated, Ca²⁺ bound to the phosphoenzyme was dissociated much more slowly than Ca²⁺ bound to the dephosphoenzyme. This suggests a possible formation of the occluded form of the Ca²⁺-binding site in the phosphoenzyme.     

Mechanisms of activation of renal (Na+ + K+)-ATPase in the rat Effects of acute and chronic administration of dexamethasone

The mechanisms of activation of renal (Na⁺ + K⁺)-ATPase by administration of the synthetic glucocorticoid hormone, dexamethasone, have been investigated in adrenalectomized rats. Chronic treatment with dexamethasone (1–5 mg/100 g body wt. daily for 5 days) stimulated (Na⁺ + K⁺)-ATPase specific activity in crude homogenated and microsomal fractions of renal cortex (by approx. 100–150%) and renal medulla (by approx. 100%). Acute treatment with dexamethasone (0.5–10 mg/100 g body wt.) also stimulated enzyme activity in crude homogenates and microsomal fractions of renal cortex and medulla (by approx. 40–50%). Stimulation was dose dependent and occurred within 2h after hormone treatment. In vitro addition of dexamethasone (10^?4–10^?8 M) to microsomal fractions did not modify the specific activity of (Na⁺ + K⁺)-ATPase. Stimulation of (Na⁺ + K⁺)-ATPase activity by acute and chronic administration of the hormone was demonstrated whether specific activities were expressed as a function of cellular protein or cellular DNA. Dexamethasone treatment increased the ratios protein:DNA and, to a lesser extent, the ratios RNA:DNA. However, these effects were mainly due to a reduction in the renal contents of DNA, which suggests that the observed enzyme activation is not due to an action of the hormone on renal hypertrophy. Dexamethasone also reduced cellular DNA contents in the liver. The characteristics of the activation process were essentially similar after treatment with single or multiple doses of the hormone. There were increases in the value for Na⁺ (approx. 100%), K⁺ (approx. 40%) and ATP (approx. 160%). The K_m values for Na⁺ (approx. 17 mM) and K⁺ (approx. 1.8 mM) were unchanged and there was a small increase in the K_m value for ATP (0.7 mM as against 1.7 mM). There was no difference in the Hill coefficients for the three substrates. The levels of the high-energy P_i intermediate of the (Na⁺ + K⁺)-ATPase reaction were augmented by dexamethasone treatment and the increased levels were quantitatively correlated with the observed stimulation of (Na⁺ + K⁺)-ATPase specific activity. The apparent turnover numbers of the reaction remained unchanged. The specific activity of the ouabain-sensitive p-nitrophenylphosphatase increased proportionally to the increase in (Na⁺ + K⁺)-ATPase specific activity. Enzyme activation by acute dexamethasone treatment occurred in the absence of changes in glomerular filtration rate and tubular Na⁺ excretion.These results indicate that (Na⁺ + K⁺)-ATPase activation by acute and chronic dexamethasone treatment represents an increase in the number of enzyme units with little or no change in the kinetic properties (affinity, cooperativity) of the enzyme. In addition, the information presented suggests a direct regulatory effect of glucocorticoid hormones on the activity of renal (Na⁺ + K⁺)-ATPase and is inconsistent with the concept that changes in Na⁺ loads mediate the effects of these hormones on enzyme activity. Instead, the results suggests a primary role for glucocorticoid hormones in the renal regulation of Na⁺ homeostasis.     

Insulin action on cardiac glucose transport Studies on the role of the Na+/K+ pump

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na⁺/K⁺ pump. ⁸⁶Rb⁺-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40–60 min, and was used to monitor the activity of the Na⁺/K⁺ pump. Ouabain (10^?3 mol/I) inhibited the steady-state uptake of ⁸⁶Rb⁺ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive ⁸⁶Rb⁺-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. ⁸⁶Rb⁺-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na⁺/K⁺ pump activity by ouabain and a concomitant shift in the intracellular Na⁺:K⁺ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na⁺/K⁺ pump and the glucose transport system.     

A comparison of the ouabain-sensitive (Na+ + K+)-ATPase of normal and dystrophic skeletal muscle

An NaI-extraction procedure was modified to prepare muscle fiber segments with Mg²⁺-dependent, ouabain-sensitive (Na⁺ + K⁺)-ATPase activity. This enzyme was assayed in preparations of skeletal muscle from normal and dystrophic mice. The ouabain-sensitive (Na⁺ + K⁺)-ATPase activity of dystrophic muscle preparations was found to be significantly lower than that of control preparations.     

Na+-dependence of 45Ca2+ uptake in adult rat isolated cardiac cells

We developed a technique that yields isolated adult rat myocytes, 70% of which are elongated and morphologically similar to intact tissue. Electrophysiological studies showed most of these cells were quiescent, Ca²⁺-tolerant and exhibited normal action potentials accompanied by contractions. We analyzed ⁴⁵Ca²⁺ uptake data in terms of instantaneous, fast and slow compartments. 69% of total exchangeable Ca²⁺ was found in the slow compartment; the rest was almost equally divided between the instantaneous and fast compartments. Replacement of extracellular Na⁺ by Li⁺ or Tris increased ⁴⁵Ca²⁺ uptake by the fast compartment; high [K⁺]_o increased this uptake further. These increases appeared to be related also to internal concentrations of Na⁺. This conclusion was supported by experiments with digitonin-treated cells. Our results indicate that the way Na⁺-dependent ⁴⁵Ca²⁺ uptake is affected by [Na⁺]_o, [Na⁺]_i and [K⁺]_o is compatible with the Na⁺-Ca²⁺ exchange mechanism. Our preparation should prove useful in studies of regulation of Ca²⁺ transport in cardiac muscles.     

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.     

Exchange between inorganic phosphate and adenosine triphosphate in (Na+,K+)-ATPase

(Na⁺,K⁺)-ATPase is able to catalyze a continuous ATP?P_i exchange in the presence of Na⁺ and in the absence of a transmembrane ionic gradient. At pH 7.6 the Na⁺ concentration required for half-maximal activity is 85 mM and at pH 5.1 it is 340 mM. In the presence of optimal Na⁺ concentration, the rate of exchange is maximal at pH 6.0 and varies with ADP and P_i concentration in the assay medium. ATP?P_i exchange is inhibited by K⁺ and by ouabain.     

Kinetic properties of Na+/Ca2+ exchange in basolateral plasma membranes of rat small intestine

The presence of an Na⁺/Ca²⁺ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na⁺ gradient-dependent Ca²⁺ uptake and the inhibition of ATP-dependent Ca²⁺ accumulation by Na⁺. The presence of 75 mM Na⁺ in the uptake solution reduces ATP-dependent Ca²⁺ transport by 45%, despite the fact that Na⁺ does not affect Ca²⁺-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na⁺ inhibition of ATP-dependent Ca²⁺ uptake to 20%, apparently by preventing accumulation of Na⁺ in the vesicles realized by the Na⁺-pump. It was concluded that high intravesicular Na⁺ competes with Ca²⁺ for intravesicular Ca²⁺ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca²⁺ transport shows a sigmoidal dependence on the Na⁺ concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na⁺ is between 15 and 20 mM. Uptake of Ca²⁺ in the absence of ATP can be enhanced by an Na⁺ gradient (Na⁺ inside > Na⁺ outside). This Na⁺ gradient-dependent Ca²⁺ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca²⁺ of this system is below 1 μM. In contrast to the ATP-dependent Ca²⁺ transport, there is no significant difference in Na⁺ gradient-dependent Ca²⁺ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca²⁺ uptake is 5-times greater than the Na⁺/Ca²⁺ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na⁺/Ca²⁺ exchange mechanism is not subject to regulation by 1α,25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na⁺/Ca²⁺ exchange system while it doubles the ATP-driven Ca²⁺ pump activity.     

Lack of stimulation of renal (Na+ + K+)-ATPase by thyroid hormones in the rabbit

The effect of l-3,5,3′-triiodothyronine (T₃) and thyroxine (T₄) on (Na⁺ + K⁺)-ATPase activities was examined in rabbit kidneys because in this tissue almost 80% of the metabolism is connected to active sodium transport. T₃-receptor concentrations were estimated as 0.62 and 0.80 pmol/mg per DNA in the cortex and outer medulla, respectively. A dose of 0.5 mg T₃/kg body weight for 3 days increased basal metabolic rate by almost 60%, and the mitochondrial 1-α-glycerophosphate dehydrogenase activity was increased by 50% in both the cortex and medulla. (Na⁺ + K⁺)-ATPase activity in the liver was raised by almost 50%. However, no changes in (Na⁺ + K⁺)-ATPase activities or binding sites for [³H]ouabain in either the kidney cortex or medulla could be observed. T₄ at 16 mg/kg daily for 14 days was also without effect on renal (Na⁺ + K⁺)-ATPase activities. Furthermore, the response to T₃ was absent at high sodium excretion rates induced by unilateral nephrectomy and extracellular volume expansion. Thus, despite stimulation of basal metabolic rate and renal 1-α-glycerophosphate dehydrogenase activity by T₃ and T₄, the (Na⁺ + K⁺)-ATPase activity in the rabbit kidney is identical in euthyroid and hyperthyroid states. However, thyroid hormones prevent the normal natriuretic response to extracellular volume expansion.     

Estimation of ouabian specifically bound to dog renal (Na+ + K+)-ATPase in vivo

It is not known whether ouabain injected into the kidney in vivo is bound exclusively to the (Na⁺ + K⁺)-ATPase and whether the reduction of sodium pumping capacity is large enough to account for the reduction in sodium reabsorption. In the present study on dogs the total amount of parenchymal ouabain was therefore estimated and the specific renal binding compared to the reduction in (Na⁺ + K⁺)-ATPase activity. Ouabain, 120 nmol/kg body weight, was injected into the renal artery in vivo reducing the (Na⁺ + K⁺)-ATPase activity by 3lmost 80%. After nephrectomy, tissue ouabain could be quantified by radioimmunoassay after heating the homogenate to 70°C for 30 min; negligible amounts were detectable without heating. No correlation between ouabain binding and tissue volume, protein content, DNA content or Mg²⁺-ATPase content could be found when comparing the following four fractions of the kidney: outer cortex, inner cortex, outer medulla and papilla. For the whole kidney, mean parenchymal tissue concentration of ouabain equalled 0.58 ± 0.03 μmol/100 g wet tissue. Only 21.3 ± 1.2% of the ouabain was confined to the outer medulla corresponding to 54 ± 4 nmol giving a tissue concentration of 1.08 ± 0.05 μmol/100 g wet tissue. The renal ouabain concentrations were highly correlated to the reduction in (Na⁺ + K⁺)-ATPase activity, giving a ratio between the reduction in hydrolysis rate and bound ouabain (turnover number) of 6105 min^?1 which is close to the value of 7180 min^?1 found by in vitro Scatchard analysis. No ouabain seems to be bound to other tissue components than the (Na⁺ + K⁺)-ATPase and the present method is therefore a simple way of measuring the number of inhibited (Na⁺ + K⁺)-ATPase molecules after in vivo injection of ouabain.     

Variations in mitochondrial monoamine oxidase during progressive starvation in the brain of developing rats

Effects of progressive starvation of 12, 24, 48 and 60 h upon brain mitochondrial monoamine oxidase activity were studied. The enzyme activity was determined by three different substrates: ¹⁴C-labeled tryptamine, dopamine and kynuramine. With dopamin as substrate, the enzyme activity showed decline during 24 and 48 h starvation. Monoamine oxidase when determined by tryptamine as the substrate, showed a decreased after 60 h of starvation. The use of kynuramine as substrate also produced a decrease in enzyme activity after 48 and 60 h of starvation. Refeeding the 60-h-starved rats for the following 24 h resulted in further decrease of monoamine oxidase activity of brain mitochondria from the 60 h starved values. The results suggest that oxidative deamination of biogenic amines is greatly inhibited during progressive starvation and remains low even after feeding the 60 h starved rats for 24 h.     

Purification and characterization of an (Na++K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain

Highly purified lamb kidney (Na⁺+K⁺)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na⁺+K⁺)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (γ1 and γ2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that γ1 is an aggregated form of γ2. The total yield was 0.8–1.5 mol of γ component per mol of catalytic subunit recovered. This indicates that the γ component is present in stoichiometric amounts in the (Na⁺+K⁺)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.     

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.     

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.     

Disassembly of microtubules by the action of calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues

Microtubules assembled by the incubation of GTP at 37 °C were disassembled by the action of calmodulin-dependent protein kinase (Kinase II) which occrs only in the brain tissues. This disassembly required the presence of ATP and physiological concentrations of Ca²⁺ and calmodulin.     

Interaction of fluorescein isothiocyanate with the (H+ + K+)-ATPase

Fluorescein isothiocyanate was used to covalently label the gastric (H⁺ + K⁺)-ATPase. FITC treatment of the enzyme inhibited the ATPase activity while largely sparing partial reactions such as the associated $\text{p-}\text{nitrophenylphosphatase}$ activity. ATP protected against inhibition suggesting the ligand binds at or near an ATP binding site. At 100% inhibition the stoichiometry of binding was 1.5 nmol FITC per mg Lowry protein a value corresponding to maximal phosphoenzyme formation. Binding occurred largely to a peptide of 6.2 isoelectric point, although minor labelling of a peptide of $\text{p}\text{I}$ 5.6 was also noted. Fluorescence was quenched by K⁺, Rb⁺ and Tl⁺ in a dose-dependent manner, and the $\text{K}{}_{0.5}$ values of 0.28, 0.83 and 0.025 mM correspond rather well to the values required for dephosphorylation at a luminal site. Vanadate, a known inhibitor of the gastric ATPase produced a slow Mg²⁺-dependent fluorescent quench. Ca²⁺ reversed the K⁺-dependent loss of fluorescence and inhibited it when added prior to K⁺. This may relate to the slow phosphorylation in the presence of ATP found when Ca²⁺ was substituted for Mg²⁺ and the absence of K⁺-dependent dephosphorylation. The results with FITC-modified gastric ATPase provide evidence for a conformational change with K⁺ binding to the enzyme.     

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na⁺ + K⁺)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca²⁺ transport. The Ca²⁺ pump was stimulated 1.7- and 2.1-fold by external Na⁺ and K⁺, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca²⁺ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca²⁺ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca²⁺ in a medium containing 160 mM NaCl. Ca²⁺ movements were also studied in the absence of ATP and Mg²⁺. Vesicles containing an outwardly directed Na⁺ gradient showed the highest Ca²⁺ uptake activity. These findings suggested the operation of a Ca²⁺/Na⁺ antiporter in addition to the active Ca²⁺ pump in these sarcolemmal vesicles. A valinomycin-induced inward K⁺-diffusion potential stimulated the Na⁺- Ca²⁺ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg²⁺ the transmembrane Na_i⁺/Na_o⁺ gradient exceeded 160/15 mM concentrations, Ca²⁺ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na⁺ gradient-induced Ca²⁺ uptake to be a translocation of Ca²⁺ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na⁺-Ca²⁺ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.