Properties of the Mg2+-induced low-affinity nucleotide binding site of (Na++ K+)-activated ATPase

(1) The Mg²⁺-induced low-affinity nucleotide binding by (Na⁺ + K⁺)-ATPase has been further investigated. Both heat treatment (50–65°C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the K_d value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na⁺ + K⁺)-ATPase activity. (2) Thermodynamic parameters (ΔH° and ΔS°) for the apparent affinity in the ATPase reaction (K_m ATP) and for the true affinity in the binding of AdoPP[NH]P (K_d and K_i) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the K_m value, which thus is smaller than the K_d value. (3) Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the K_d value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and AdoPP[NH]P and least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the K_d value. (4) The nucleotide specificity of low-affinity binding site is the same for binding (competition with AdoPP[NH]P) and for the ATPase activity (competition with ATP): AdoPP[NH]P > ATP > ADP > AMP. (5) The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the K_d value is not increased more than by ouabain alone. (6) It is inferred that the low-affinity site is Iocated on the enzyme, more specifically its α-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed.     

Mechanism of inhibition of rat brain (Na +-K +)-stimulated adenosine triphosphatase reaction by cadmium and methyl mercury

The mechanisms of inhibition of rat brain Na ⁺-K ⁺- ATPase by cadmium chloride (CdCl₂) and methylmercuric chloride (CH₃HgCl) were studied in vitro by assessing the effects of these heavy metals on this enzyme and associated component parameters. Both the heavy metals significantly inhibited the overall Na ⁺-K ⁺ -ATPase in a concentration-dependent manner with an estimated median inhibitory concentration (IC-50) of 3.2 × 10^?5M for CdCl₂ and 6 × 10^?6M for CH₃HgCl. Protection of enzyme against heavy metal inhibition by 5 × 10^?5M to 1 × 10^?4 M dithiothreitol (DTT) and glutathione (GSH) or cysteine (CST) indicates that both monothiols and dithiols have the same ability in regenerating sulfhydryl (–SH) groups or chelating the metals. Inhibition of K⁺-p-nitrophenyl phosphatase (K⁺-PNPPase), the component enzyme catalyzing the K⁺-dependent dephosphorylation in the overall Na ⁺-K ⁺ATPase reaction by these heavy metals, indicates that the mechanism of inhibition involves binding to this phosphatase. Reversal of K⁺-PNPPase inhibition by DTT, GSH, and CST suggests sulfhydryl groups as binding sites. Binding of ³H-oubain, a cardiac glycocide and inhibitor of both phosphorylation and dephosphorylation, to brain fraction was significantly decreased by CH₃HgCl, and this inhibition was reversed by the three thiol compounds, suggesting presence of –SH group(s) in the ouabain receptor site. Cadmium chloride failed to inhibit the binding of this receptor, indicating that the mechanics of inhibition of ATPase by CH₃HgCl and CdCl₂ are different from each other. The results suggest that the critical conformational property of enzyme common to both kinase (E₁) and phosphatase (E₂) is susceptible to CH₃HgCl whereas only phosphatase is sensitive to CdCl₂.     

Effect of magnesium ions on the high-affinity binding of eosin to the (Na+ + K+)-ATPase

(1) The fluorescence of eosin Y in the presence of (Na⁺ + K⁺)-ATPase is enhanced by Mg²⁺. The enhancement by Mg²⁺ is larger than that obtained with Na⁺ (Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232–240). Mg²⁺ shifts the excitation maximum from 518 to 524 nm, the emission maximum from 538 to 542 nm. Also a shoulder appears at about 490 nm on the excitation curve, as was also observed with Na⁺. (2) The Mg²⁺-dependent enhancement of fluorescence can be reversed by K⁺ as well as by ATP. In the presence of Mg²⁺ + P_i (i.e. under conditions of phosphorylation), the fluorescence enhancement can be reversed by ouabain. With Mg²⁺ and a low concentation of K⁺ (i.e. conditions for vanadate binding), the enhancement of fluorescence can be reversed by vanadate. (3) There is a low-affinity binding of eosin which increases with the Mg²⁺ concentration. This is observed as a slight increase in the fluorescence when the excitation wavelength is above 520 nm. The low-affinity binding is K⁺-, ATP-, ouabain- and vanadate-insensitive. (4) Scatchard analysis of the binding experiments suggests that there are two high-affinity eosin-binding sites per ³²P-labelling site in the presence of 5 mM Mg²⁺ both of which are ouabain-, vanadate- and ATP-sensitive. With 5 M Mg²⁺ + 0.25 P_i, the K_d values are 0.14 μM and 1.3 μM, respectively. With 5 mM Mg²⁺, 150 mM Na⁺, the K_d values are 0.45 μM and 3.2 μM, respectively. With 5 mM Mg²⁺, the addition of K⁺ gives a pronounced decrease in affinity but does not decrease the number of binding sites (which remains at two per ³²P-labelling site). With 5 mM Mg²⁺ + 150 mM K⁺, the affinities of the two binding sites become identical, at a K_d of 17 μM. (5) The rate of conformational transitions was measured using the stopped-flow method. The rate of the transition from the Mg²⁺-form to the K⁺-form is high. Oligomycin has only a small (if any) effect on the rate. Addition of Na⁺ in the presence of Mg²⁺ does not appreciably change the rate of conversion to the K⁺-form, giving a rate constant of about 110 s^?. However, the addition of oligomycin in the presence of Mg²⁺ + Na⁺ had a profound effect: the rate of conversion to the K⁺-form was decreased by a factor of 2000 to about 0.063 s^?1. This suggests that the conformation with Mg²⁺ alone is different from the conformation with Na⁺ alone. (6) The effects of K⁺, ouabain, vanadate and ATP on the high-affinity binding of eosin suggest that the two eosin molecules bound per ³²P-labelling site are bound to ATP sites.     

Insensitivity of lepidopteran tissues to ouabain: Physiological mechanisms for protection from cardiac glycosides

Neuronal tissues from Manduca sexta, the tobacco hornworm, Hyalophora cecropia, the silkmoth and Danaus plexippus, the Monarch Butterfly, contain Na⁺K⁺-ATPase which is sensitive to cardiac glycoside (ouabain). The K_m for K⁺ stimulation of Na⁺K⁺-ATPase in M. sexta and D. plexippus is 2.2 mM and for Na⁺ stimulation in D. plexippus, 6.0 mM. In vitro ouabain concentrations of 1.0 × 10^?5 M and 5.0 × 10^?5 M in the presence of 7.5 mM K⁺ inhibited Na⁺K⁺-ATPase activity in H. cecropia and M. sexta by 50% respectively. Na⁺K⁺-ATPase from D. plexippus was approximately 300 times less sensitive. High concentrations (10^?3 M in haemolymph) of ouabain had no effect on M. sexta in vivo. This is largely explained by haemolymph K⁺ (>; 30 mM) antagonizing the binding of ouabain to Na⁺K⁺-ATPase. As demonstrated in vitro, 30 mM K⁺ totally protects Na⁺K⁺-ATPase from inhibition by 7.5 × 10^?3 M ouabain in D. plexippus and protects the enzyme by 65% in M. sexta. At least part of the physiological burden incurred in utilization of cardiac glycoside ingestion and storage for protection from predation, however, is probably related to the toxic effects of cardiac glycosides on neuronal Na⁺K⁺-ATPase.     

High-affinity Ca2+-Mg2+-ATPase in kidney of euryhaline Gillichthys mirabilis: kinetics,subcellular distribution and effects of salinity

• 1.1. Two components of Ca²⁺-Mg²⁺-ATPase are observed in kidneys of G. mirabilis. The high-affinity component has a K_0.5Ca of 0.23μM; the low-affinity activity K_0.5Ca is 90–110μM. The high-affinity activity requires Mg²⁺, displays Michaelis-Menten kinetics, has peak activity at 1.2 μM Ca²⁺, and is insensitive to ouabain and Na⁺ azide.
• 2.2. In subcellular fractions, the high-affinity component segregates with Na⁺-K⁺-ATPase and is localized predominantly in BLM. The low-affinity component is broadly distributed among membranous organelles, including brush border, and may be equivalent to alkaline phosphatase.
• 3.3. Specific activity of the high-affinity Ca²⁺-Mg²⁺-ATPase is modestly increased following adaptation of fish to FW, but total renal high-affinity activity is greatest in the hypertrophied kidneys of FW-adapted fish and is least in kidneys of fish adapted to 200% SW.
• 4.4. High-affinity Ca²⁺-Mg²⁺-ATPase may be associated with active Ca²⁺ transport or with regulation of intracellular Ca²⁺ concentration of tubular cells.

     

The effect of sodium on inorganic phosphate- and p-nitrophenyl phosphate-facilitated ouabain binding to (Na+ + K+)-activated ATPase

The effect of the hydrolysis product P_i and the artificial substrate p-nitrophenyl phosphate (p-nitrophenyl-P) on ouabain binding to (Na⁺ + K⁺)-activated ATPase was investigated.The hypothesis that (Mg²⁺ + p-nitrophenyl-P)-supported ouabain binding might be due to P_i release and thus (Mg²⁺ + P_i)-supported could not be confirmed.The enzyme · ouabain complexes obtained with different substrates were characterized according to their dissociation rates after removal of the ligands facilitating binding. The character of the enzyme · ouabain complex is determined primarily by the monovalent ion present during ouabain binding, but, qualitatively at least, it is immaterial whether binding was obtained with p-nitrophenyl phosphate or P_i.The presence or absence of Na⁺ during binding has a special influence upon the character of the enzyme · ouabain complex. Without Na⁺ and in the presence of Tris ions the complex obtained with (Mg²⁺ + P_i) and that obtained with (Mg²⁺ + p-nitrophenyl-P) behaved in a nearly identical manner, both exhibiting a slow decay. High Na⁺ concentration diminished the level of P_i-supported ouabain binding, having almost no effect on p-nitrophenyl phosphate-supported binding. Both enzyme · ouabain complexes, however, now resembled the form obtained with (Na⁺ + ATP), as judged from their dissociation rates and the K⁺ sensitivity of their decay. The complexes obtained at a high Na⁺ concentration underwent a very fast decay which could be slowed considerably after adding a low concentration of K⁺ to the resuspension medium. The most stable enzyme · ouabain complex was obtained in the presence of Tris ions only, irrespective of whether p-nitrophenyl phosphate or P_i facilitated complex formation. The presence of K⁺ gave rise to a complex whose dissociation rate was intermediate between those of the complexes obtained in the presence of Tris and a high Na⁺ concentration.It is proposed that the different ouabain dissociation rates reflect different reactive state of the enzyme. The resemblance between the observations obtained in phosphorylation and ouabain binding experiments is pointed out.     

Interactions of cardiac glycosides with cultured cardiac cells: II. Biochemical and electron microscopic studies on the effects of ouabain on muscle and non-muscle cells

Electron microscopic and biochemical studies revealed a salient difference in the response to toxic doses of ouabain by cultured cardiac muscle and non-muscle cells from neonatal rats. Progressive cellular injury in myocytes incubated with 1 · 10^?4–1 · 10^?3 M ouabain ultimately leads to swelling and necrosis. The morphological damage in myocytes was accompanied by a drastic decrease in ¹⁴CO₂ formation from ¹⁴C-labeled stearate or acetate but not glucose. Neither morphological nor biochemical impairments were observed in non-muscle cells. The interaction between ouabain and the cultured cells, using therapeutic doses of ouabain (i.e., <1 · 10^?7 M), was characterized. Two binding sites were described in both classes of cells, one site is a saturable K⁺-sensitive site whereas the other is non-saturable and K⁺-insensitive. The complexes formed between the sarcolemma receptor(s) and ouabain, at low concentrations of the drug (e.g., 7.52 · 10^?9 M), had K_d values of 8.9 · 10^?8 and 2.3 · 10^?8 M for muscle and non-muscle cells, respectively. The formation and dissociation of the complexes were affected by temperature and potassium ions.     

Effect of inotropic agents on the calcium binding to isolated cardiac sarcolemma

Ca²⁺ binding to fragmented sarcolemma isolated from canine heart was measured by an ultracentrifugation technique. Two classes of binding site with dissociation constants of 2.0 · 10^?5 and 1.2 · 10^?3 M were identified. The capacities of the high- and low-affinity sites were 15 and 452 nmol/mg, respectively. These sites were not affected by treatment with neuraminidase. The effects of various cations and drugs on Ca²⁺ binding were studied. All cations tested inhibited Ca²⁺ binding with the following order of potency: trivalent > divalent > monovalent cations. The order of potency for the monovalent ions was: Na⁺ > K⁺ > Li⁺ ? Cs⁺ and for the divalent and trivalent ions: La³⁺ ? Mn²⁺ > Sr²⁺ ? Ba²⁺ > Mg²⁺. 1 · 10^?3 M caffeine and 1 · 10^?8 M ouabain increased the capacity of the low-affinity sites to 1531 and 837 nmol/mg, respectively. 1 · 10^?7 M verapamil, acidosis (pH 6.4), 1^?10^?5 M Mn²⁺ and 1 · 10^?4 M ouabain depressed the capacity of the low-affinity sites to a range of 154–291 nmol/mg. The dissociation constants of the high- and low-affinity sites and the capacity of the high-affinity sites were not affected by these agents.     

Properties of an ATP-dependent Ca2+ pump and (Ca2+ + Mg2+)-ATPase in brain synaptosome membrane vesicles from the bertha armyworm,Mamestra configurata Wlk

Biochemical and kinetic properties under identical substrate and reaction conditions were obtained for an ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase in synaptosome membrane vesicles prepared from the brain of the moth, Mamestra configurata. Both the ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase had single, high-affinity binding sites for ATP (K_m = 14 and 116 μM, respectively), Ca²⁺_free (K_m = 0.13 nM and 0.072 nM, respectively), and Mg²⁺ (K_m = 1.1 mM and 0.07 mM, respectively). Both systems were relatively little affected by K⁺ and were insensitive to ouabain, an inhibitor of (Na⁺ + K⁺)-ATPase. The results indicate that the ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase are functionally coupled in synaptic membranes and constitute a mechanism for Ca²⁺ transport in the brain of M. configurata. Although moth brain (Ca²⁺ + Mg²⁺)-ATPase is maximally active at nanomolar concentrations of free calcium ion, the enzyme retains at least one-half of its maximal activity at micromolar calcium concentrations, indicating either that the enzyme has two binding sites for calcium (a high-affinity site at nanomolar Ca²⁺_free and a low-affinity site at micromolar Ca²⁺_free), or that there are two enzymes with high and low affinity for calcium, respectively. Calcium extrusion from brain neurones of M. configurata may operate in a two-stage, concentration-dependent process in which a first stage, low-affinity pump reduces intraneuronal calcium to a concentration at which a second stage, high-affinity pump becomes activated.     

Some in vivo and in vitro effects of ethacrynic acid on renal Na+,K+ -ATPase

Binding of [¹⁴C]ethaerynic acid [EA]at concentrations of EA from 10^?4m to 10^?2m to a membrane preparation containing Na⁺,K⁺-ATPase activity in vitro occurred in a nonsaturable manner; binding was stimulated by Na⁺ or K⁺, but was not affected by Mg²⁺ and/or ATP. [¹⁴C]EA significantly bound to a microsomal preparation with low Na⁺,K⁺-ATPase activity as well as to a heat-denatured enzyme; this binding reaction was not stimulated by Na⁺. These observations suggest that EA binds non-specifically or to nonspecific sites on membrane preparations. Nonselective binding of [¹⁴C]EA to subcellular particles after fractionation of slices also suggested the presence of nonspecific EA binding sites in vivo. In vitro [³H]ouabain binding to medullary and cortical Na⁺,K⁺-ATPase preparations was partially reduced by pretreatment with EA. On the other hand, [¹⁴C]EA binding to Na⁺,K⁺-ATPase was not affected by pretreatment of the preparation with ouabain (10^?6m to 5 × 10^?4m). EA reduced the sensitivity of [³H]ouabain binding to the enzyme preparation to Na⁴ and K⁺.EA was infused (0.1, 1.0, and 10 mg/min) into one renal artery of hydropenic dogs. A prompt natriuresis in the infused kidney occurred. Similar changes were observed in the contralateral kidney 20 min after starting the infusion. Both kidneys were removed 30 min after the beginning of the infusion, and Na⁺,K⁺-ATPase was isolated from the cortex and the medulla. Enzyme activity from cortex and medulla of either kidney was not significantly different from enzyme activity from cortex and medulla of control, uninfused dogs, regardless of dose of EA or method of enzyme isolation. Furthermore, in vitro binding of [³H]ouabain to Na⁺,K⁺-ATPase membrane preparations from cortex and medulla was the same for experimental and control kidneys. In vitro incubation of 2 × 10^?3m EA with a membrane preparation caused the same inhibition of ATPase activity when the enzyme was isolated either from control or EA-infused dogs. The inhibition could not be reversed by recentrifugation or rehomogenization of the enzyme. Our results do not support the concept that Na⁺,K⁺-ATPase is a pharmacological receptor for ethacrynic acid.     

Studies on (NA + K)-activated ATPase XLV. Magnesium induces two low-affinity non-phosphorylating nucleotide binding sites per molecule

ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na⁺ + K⁺)-ATPase in the absence of Mg²⁺ (EDTA present) with a homogeneous but 15-fold different affinity, the K_d values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na⁺ + K⁺)-ATPase, respectively. The K_d value for ATP is equal to the K_m for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na⁺ + K⁺)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg²⁺, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The K_d and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg²⁺ concentration. The K_d increases from 0.06 mM at 0.5 mM Mg²⁺ to a maximum of 0.26 mM at 2 mM Mg²⁺ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg²⁺ to 3.3 nmol/mg protein at 4 mM Mg²⁺. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg²⁺ concentration yields a maximal binding capacity at infinite Mg²⁺ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na⁺ + K⁺)-ATPase. The K_d for Mg²⁺ at the sites, where it exerts this effect, is 0.8 mM. The K_d for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg²⁺ to a maximum of 4.2 μM at 2 mM Mg²⁺ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg²⁺ concentration. Hence, Mg²⁺ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na⁺ + K⁺)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.     

Equilibrium constants under physiological conditions for the reactions of the nonphosphorylated pathway of L-serine biosynthesis

The observed equilibrium constants (K_obs) for the reactions of d-2-phosphoglycerate phosphatase, d-2-Phosphoglycerate^3? + H₂O → d-glycerate^? + HPO₄^2?; d-glycerate dehydrogenase (EC 1.1.1.29), d-Glycerate^? + NAD⁺ → NADH + hydroxypyruvate^? + H⁺; and l-serine:pyruvate aminotransferase (EC 2.6.1.51), Hydroxypyruvate^? + l-H · alanine^± → pyruvate^? + l-H · serine^±; have been determined, directly and indirectly, at 38 °C and under conditions of physiological ionic strength (0.25 m) and physiological ranges of pH and magnesium concentrations. From these observed constants and the acid dissociation and metal-binding constants of the substrates, an ionic equilibrium constant (K) also has been calculated for each reaction. The value of K for the d-2-phosphoglycerate phosphatase reaction is 4.00 × 10³m [ΔG⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)]([H₂0] = 1). Values of K_obs for this reaction at 38 °C, [K⁺] = 0.2 m, I = 0.25 M, and pH 7.0 include 3.39 × 10³m (free [Mg²⁺] = 0), 3.23 × 10³m (free [Mg²⁺] = 10^?3m), and 2.32 × 10³m (free [Mg²⁺] = 10^?2m). The value of K for the d-glycerate dehydrogenase reaction has been determined to be 4.36 ± 0.13 × 10^?13m (38 °C, I = 0.25 M) [ΔG⁰ = 73.6 kJ/mol (17.6 kcal/mol)]. This constant is relatively insensitive to free magnesium concentrations but is affected by changes in temperature [ΔH⁰ = 46.9 kJ/mol (11.2 kcal/mol)]. The value of K for the serine:pyruvate aminotransferase reaction is 5.41 ± 0.11 [ΔG⁰ = ?4.37 kJ/mol (?1.04 kcal/mol)] at 38 °C (I = 0.25 M) and shows a small temperature effect [ΔH⁰ = 16.3 kJ/ mol (3.9 kcal/mol)]. The constant showed no significant effect of ionic strength (0.06–1.0 m) and a response to the hydrogen ion concentration only above pH 8.5. The value of K_obs is 5.50 ± 0.11 at pH 7.0 (38 °C, [K⁺] = 0.2 m, [Mg²⁺] = 0, I = 0.25 M). The results have also allowed the value of K for the d-glycerate kinase reaction (EC 2.7.1.31), d-Glycerate^? + ATP^4? → d-2-phosphoglycerate^3? + ADP^3? + H⁺, to be calculated to be 32.5 m (38 °C, I = 0.25 M). Values for K_obs for this reaction under these conditions and at pH 7.0 include 236 (free [Mg²⁺] = 0) and 50.8 (free [Mg²⁺] = 10^?3m).     

Conversion of high affinity acetylcholine receptor from Torpedo californica electroplax to an altered form.

Most Triton extracts of fresh electroplax membranes showed a single kind of acetylcholine binding, of high affinity, with K_d = 17 nM. Fresh membrane preparations without Triton also showed only high-affinity binding (K_d = 14 nM), but membrane preparations from electroplax stored for 5 months in liquid nitrogen gave equal amounts of low-affinity binding material (K_d = 0.2 μM). Heating Triton extracts at 40 °C for 40 min or treating with 10^?4mp-chloromercuribenzoate or 5,5′-dithiobis(2-nitrobenzoic acid) converted the preparation to more than 90% low-affinity binding. It is suggested that the high-affinity is the native form and the low-affinity is an oxidation product.     

K+-stimulated ATPase in purified microsomes of bullfrog oxyntic cells

Differential centrifugation of oxyntic cell homogenates yielded microsomal fractions which contained large amounts of mitochondrial membrane. The presence of marker enzymes (succinate dehydrogenase and cytochrome c oxidase) indicated that mitochondrial contamination of crude microsomes ranged from 20 to 60% in different preparations. A discontinuous sucrose density gradient procedure was developed for the routine preparation of purified oxyntic cell microsomes. A K⁺-stimulated, Mg²⁺-requiring ATPase was localized in these purified membranes and coincided with the presence of a K⁺-stimulated p-nitrophenylphosphatase. Na⁺ and ouabain had no effect on the K⁺ stimulation of the microsomal ATPase. The apparent activation constant for K⁺ was approximately 1 mM at pH 7.5, the optimal pH for stimulation.An anion-sensitive ATPase has been widely studied in gastric microsomal preparations. We found that the basal microsomal ATPase (i.e. without K⁺) and the mitochondrial ATPase were inhibited by SCN^? and enhanced by HCO₃^?, however, the K⁺-stimulated component of the microsomal ATPase was virtually unaffected by these anions.     

Ouabain receptor interactions in (Na + K)-ATPase preparations. III. On the stability of the ouabain receptor against physical treatment, hydrolases and SH reagents

The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant (K_D) of 13 nM was found in the presence of (Mg²⁺ + P_i) and (Na⁺ + Mg²⁺ + ATP). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg²⁺, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K⁺ and its congeners, by Na⁺ in the presence of (Mg²⁺ + P_i), and by ATP analogs (ADP-C-P, ATP-OCH₃). Ca²⁺ antagonized the action of K⁺ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (R_α) and a high affinity conformational state (R_β). The equilibrium between both states is influenced by ligands of (Na⁺ + K⁺)-ATPase. With 3 mM Mg²⁺ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot).     

Two types of cyclic GMP binding site associated with the cyclic AMP-dependent protein kinase from lymphocytes

Low- and high-affinity binding sites for cyclic GMP were found to be associated with the cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from human tonsillar lymphocytes, but neither of them was identical with the cyclic AMP binding site.The enzyme activated by cyclic GMP phosphorylated the same site of calf thymus H2b histone as the cyclic AMP activated enzyme; however, more complex kinetics of activation were found with cyclic GMP.Two classes of cyclic GMP binding site were demonstrated by kinetic analysis of cyclic [³H]GMP binding in the enzyme preparations eluted by 0.1 M potassium phosphate (pH 7.0) from DEAE cellulose. The high-affinity cyclic GMP binding site (K_d about 44 · 10^?8 M belonged to some complex form of the protein kinase, as evidenced by the mutual inhibition of cyclic AMP binding and high affinity cyclic GMP binding. However, the high-affinity cyclic GMP binding site disappeared on Sephadex G-100 gel chromatography of the enzyme preparation, whereas the cyclic AMP binding activity was recovered quantitively as separate fractions. The low-affinity cyclic GMP binding site (K_d 2–5 · 10^?6 M) was demonstrated by the inhibitory effect of 10^?5 M cyclic GMP on cyclic AMP binding in each cyclic AMP binding fraction obtained by gel chromatography. However, cyclic AMP did not inhibit the binding of cyclic GMP to the low-affinity binding site.     

Studies on (Na++K+)-activated ATPase: XXXVII. Stabilization by cations of the enzyme-ouabain complex formed with Mg2+ and inorganic phosphate

Dissociation of the (Na⁺+K⁺)-ATPase ouabain complex, formed presence of Mg²⁺ and inorganic phosphate (Complex II), is inhibited by Mg²⁺ (21–45%) and the alkali cations Na⁺ (25–59%) and K⁺ (27–75%) when kidney cortex tissue (bovine, rabbit, guinea pig) is the enzyme source. Choline chloride at 200 mM, equivalent to the highest concentration of NaCl tested, does not inhibit. Dissociation of Complex II from brain cortex (bovine, rat, rabbit) or heart muscle (rabbit) is much less inhibited: 0–11% by Na⁺ and 11–19% by K⁺. The degree of inhibition is not directly related to the size of the dissociation rate constant ($\text{k}{}^{\mathrm{?}}$) of the various complexes, but rather to the extent of interaction between the cation and ouabain binding sites for these tissues.Inhibition curves for Na⁺ and K⁺ are sigmoidal. Half-maximal inhibition for rabbit brain and kidney cortex is at 30–40 mM Na⁺ and 6–10 mM K⁺, and the maximally inhibitory concentrations are 50–150 and 15–20 mM, respectively. Maximal inhibition by Na⁺ or K⁺ for these tissues is the same. For guinea pig kidney cortex Na⁺ and K⁺ are almost equally effective, but 150 mM K⁺ or 200 mM Na⁺ are still not saturating, and inhibition curves indicate high- and low-affinity binding sites for the alkali cations.The inhibition curve for Mg²⁺ is not sigmoidal. In the kidney preparations Mg²⁺ inhibits half-maximally at 0.4-0.5 mM, maximally at 1–3 mM. Maximal inhibition by Mg²⁺ is higher than by Na⁺ or K⁺ for rabbit cortex and lower for guinea pig kidney cortex.There is no competition or additivity among the cations, indicating the existence of different binding sites for Mg²⁺ and the alkali cations.Complex II differs in stability, in the extent of inhibition, in the dependence of inhibition on the cation concentration and in the absence of antagonism between Na⁺ and K⁺, from the ouabain complex formed via phosphorylation by ATP (Complex I). This indicates that the phosphorylation states for the complexes are clearly different.     

The mode of activation by potassium of potassium-dependent and ouabain-sensitive phosphatase activity.

A new simple procedure has been developed for the purification of plasma membranes from rabbit kidney microsomes which yields a three- to fourfold increase in the specific activity of Na⁺-K⁺-adenosine triphosphatase (ATPase). The procedure differs from previous methods with deoxycholate or other detergents and does not change the molecular activity of the ATPase. The K⁺-dependent p-nitrophenylphosphatase activity of the native Na⁺-K⁺-ATPase is controlled more effectively by Mg²⁺ in the presence of K⁺ at concentrations higher than that of Mg²⁺, and by K⁺ in the presence of Mg²⁺ at concentrations higher than that of K⁺. The enzyme in its Mg²⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 1, is less sensitive to ouabain (I_0.5 = 90 μM) and corresponds to the enzyme conformation reported previously which is inhibited by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin (I_0.5 is the midpoint of the saturation curve). The enzyme in its K⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 2, is more sensitive to ouabain inhibition (I₀₅ = 8 μM) and corresponds to the enzyme conformation which is stimulated by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin. There appear to be two conformations of the enzyme that are regulated by Mg²⁺ binding on the inhibitory sites of the enzyme.     

Regulation of high- and low-affinity epidermal growth factor receptors by glucocorticoids

It is reported that receptors for epidermal growth factor (EGF) in HeLa S₃ cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 1.5}\text{nm}$), which contains approximately 6 × 10⁴ binding sites per cell, and a second, high-affinity class of receptors ($\text{K}{}_{\mathrm{d}}\text{? 0.16}\text{nm}$) containing approximately 5 × 10³ binding sites per cell. Exposure of HeLa S₃ cells to 10^?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10^?11 to 10^?6m. EGF binding is half-maximal near 2–4 × 10^?9m, a concentration equal to the K_d of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S₃ cells are exposed to 10^?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10^?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc.