Significance of the β-Subunit in the Biogenesis of Na+/K+-ATPase

This review summarizes our experiments on the significance of the -subunit in the functional expression of Na⁺/K⁺-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na⁺/K⁺-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH₂-terminal domain to the extracellular COOH-terminus: the NH₂-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na⁺/K⁺-ATPase by Na⁺/K⁺- and H⁺/K⁺-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na⁺/K⁺-ATPase -subunit and from Ile89 to the COOH–terminus of the H⁺/K⁺-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na⁺/K⁺-ATPase.     

Phosphorylation of the α-subunit of (Na+ + K+)-ATPase by carbachol in tissue slices and the role of phosphoproteins in stimulus-secretion coupling

This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses (M_r) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22 000 and 96 000. The M_r 96 000 protein precipitated at 120 000 × g while most of the M_r 22 000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the M_r 22 000 and 96 000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the M_r 22 000 protein consisted of a rapid incrase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphoryaltion of the M_r 96 000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the M_r 96 000 protein was phosphorylated by ATP in the presence of Na⁺ and Mg²⁺. It was dephosphorylated by K⁺. This proves that the M_r 96 000 dalton protein is the α-subunit of the (Na⁺ + K⁺)-ATPase.     

Variability of the K+−Na+ discrimination of beauvericin in mitochondrial membranes

In intact mitochondria supplemented with succinate or -hydroxybutyrate, the rates of oxygen consumption induced by beauvericin followed the ionic selectivity pattern: Na⁺>Rb⁺, Cs⁺, K⁺, Li⁺.When the respiratory substrate is glutamate plus malate in the absence of phosphate, the selectivity pattern is: K⁺>Rb⁺>Cs⁺>Li⁺>Na⁺.When the media are supplemented with phosphate, the Na⁺/K⁺ discrimination of beauvericin is considerably modified with all the respiratory substrates, being K⁺>Na⁺ with succinate and Na⁺>K⁺ with glutamate plus malate, whereas no significant ionic selectivity differences were obtained with -hydroxybutyrate.The respiratory control induced by oligomycin in submitochondrial particles is released by beauvericin only in the presence of a nigericin-like carboxylic antibiotic and an alkali metal cation, being far more effective in K⁺ than in Na⁺.This selectivity is maintained regardless of whether NADH or succinate is used as a respiratory substrate.Release of respiratory control can also be obtained with a combination of beauvericin and NH₄Cl.This information indicates that the ionic selectivity pattern obtained with beauvericin in mitochondrial membranes is an intrinsic property of the antibiotic which, however, can be significantly modified by factors such as the nature of the translocatable substrate anion or other anionic species, as well as the possible operation of a Na⁺/H⁺ antiporter existent in the membrane.     

Role of Na+–H+ exchange in the modulation of L-type Ca2+ current during fluid pressure in rat ventricular myocytes

Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca²⁺ current through both enhancement of Ca²⁺ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na⁺–H⁺ exchange (NHE), we examined the possible role of NHE in the regulation of I_Ca during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16 dyn/cm²) fluid was applied onto single rat ventricular myocytes, and the I_Ca was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the I_Ca (IC₅₀ = 0.97 ± 0.12 μM) without altering current–voltage relationships and inactivation time constants. In contrast, the I_Ca in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the I_Ca under FP was not altered by intracellular high Ca²⁺ buffering. Replacement of external Cl⁻ with aspartate to inhibit the Cl⁻-dependent acid loader eliminated the inhibitory effect of HOE642 on I_Ca. These results suggest that NHE may attenuate FP-induced I_Ca suppression by preventing intracellular H⁺ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca²⁺-dependent inhibition of the I_Ca during FP exposure.     

Application of DFT methods to the study of the coordination environment of the VO2+ ion in V?proteins

Density functional theory (DFT) methods were used to simulate the environment of vanadium in several V proteins, such as vanadyl-substituted carboxypeptidase (sites A and B), vanadyl-substituted chloroplast F(1)-ATPase (CF(1); site 3), the reduced inactive form of vanadium bromoperoxidase (VBrPO; low- and high-pH sites), and vanadyl-substituted imidazole glycerol phosphate dehydratase (IGPD; sites α, β, and γ). Structural, electron paramagnetic resonance, and electron spin echo envelope modulation parameters were calculated and compared with the experimental values. All the simulations were performed in water within the framework of the polarizable continuum model. The angular dependence of [Formula: see text] and [Formula: see text] on the dihedral angle θ between the V=O and N-C bonds and on the angle φ between the V=O and V-N bonds, where N is the coordinated aromatic nitrogen atom, was also found. From the results it emerges that it is possible to model the active site of a vanadium protein through DFT methods and determine its structure through the comparison between the calculated and experimental spectroscopic parameters. The calculations confirm that the donor sets of sites B and A of vanadyl-substituted carboxypeptidase are [[Formula: see text], H(2)O, H(2)O, H(2)O] and [N(His)(||), N(His)(⊥), [Formula: see text], H(2)O], and that the donor set of site 3 of CF(1)-ATPase is [[Formula: see text], OH(Thr), H(2)O, H(2)O, [Formula: see text]]. For VBrPO, the coordination modes [N(His)(||), N(His)(∠), OH(Ser), H(2)O, H(2)O(ax)] for the low-pH site and [N(His)(||), N(His)(∠), OH(Ser), OH(-), H(2)O(ax)] or [N(His)(||), N(His)(∠), [Formula: see text], H(2)O] for the high-pH site, with an imidazole ring of histidine strongly displaced from the equatorial plane, can be proposed. Finally, for sites α, β, and γ of IGPD, the subsequent deprotonation of one, two, and three imidazole rings of histidine and the participation of a carboxylate group of a glutamate residue ([N(His)(||), [Formula: see text], H(2)O, H(2)O], [N(His)(||), N(His)(||), [Formula: see text], H(2)O], and [N(His)(||), N(His)(||), [Formula: see text], OH(-), [Formula: see text]], respectively) seems to be the most plausible hypothesis.     

Possible role for Ca2+ in the pathophysiology of the prion protein?

Transmissible spongiform encephalopathies, or prion diseases, are lethal neurodegenerative disorders caused by the infectious agent named prion, whose main constituent is an aberrant conformational isoform of the cellular prion protein, PrP(C) . The mechanisms of prion-associated neurodegeneration and the physiologic function of PrP(C) are still unclear, although it is now increasingly acknowledged that PrP(C) plays a role in cell differentiation and survival. PrP(C) thus exhibits dichotomic attributes, as it can switch from a benign function under normal conditions to the triggering of neuronal death during disease. By reviewing data from models of prion infection and PrP-knockout paradigms, here we discuss the possibility that Ca(2+) is the hidden factor behind the multifaceted behavior of PrP(C) . By featuring in almost all processes of cell signaling, Ca(2+) might explain diverse aspects of PrP(C) pathophysiology, including the recently proposed one in which PrP(C) acts as a mediator of synaptic degeneration in Alzheimer's disease.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO ₃ ^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO ₃ ^– /Na⁺ exit on the serosal side of the tissue.     

Mineralocorticoids modulate the expression of the β-3 subunit of the Na+, K+-ATPase in the renal collecting duct

Renal sodium reabsorption depends on the activity of the Na⁺,K⁺-ATPase α/β heterodimer. Four α (α_1–4) and 3 β (β_1–3) subunit isoforms have been described. It is accepted that renal tubule cells express α₁/β₁ dimers. Aldosterone stimulates Na⁺,K⁺-ATPase activity and may modulate α₁/β₁ expression. However, some studies suggest the presence of β₃ in the kidney. We hypothesized that the β₃ isoform of the Na⁺,K⁺-ATPase is expressed in tubular cells of the distal nephron, and modulated by mineralocorticoids. We found that β₃ is highly expressed in collecting duct of rodents, and that mineralocorticoids decreased the expression of β₃. Thus, we describe a novel molecular mechanism of sodium pump modulation that may contribute to the effects of mineralocorticoids on sodium reabsorption.     

Role of the Na+,K+-ATPase α2 isoform in the positive inotropic effect of ouabain and marinobufagenin in the rat diaphragm

It was found that ouabain and marinobufagenin, specific inhibitors of Na⁺,K⁺-ATPase, increased the contraction of the isolated rat diaphragm by ～15% (positive inotropic effect) at EC₅₀ = 1.2 ± 0.3 nM and 0.3 ± 0.1 nM, respectively, which was indicative of the participation of the ouabain-sensitive Na⁺,K⁺-ATPase α2 isoform. Analysis of the dose-response curves for the effect of ouabain on the resting membrane potential of muscle fibers in the absence and in the presence of 100 nM acetylcholine (hyperpolarizing the membrane) showed the presence of two pools of Na⁺,K⁺-ATPase α2 that differed in affinity for ouabain. Only the high-affinity pool (IC₅₀ ～ 9 nM) mediates the hyperpolarizing effect of nanomolar concentrations of acetylcholine. Most likely, it is this pool of that is involved in the positive inotropic effect of ouabain, which can be a mechanism of regulation of the muscle function by circulating endogenous inhibitors of Na⁺,K⁺-ATPase.     

Ca2+-antagonists inhibit the N-methyltransferase‐dependent synthesis of phosphatidylcholine in the heart

Evidence indicates that, in addition to the Ltype Ca²⁺ channel blockade, Ca²⁺antagonists target other functions including the Ca²⁺pumps. This study was conducted to test the possibility that the reported inhibition of heart sarcolemmal (SL) and sarcoplasmic reticular (SR) Ca²⁺pumps by verapamil and diltiazem could be due to druginduced depression of phosphatidylethanolamine (PE) Nmethylation which modulates these Ca²⁺transport systems. Three catalytic sites individually responsible for the synthesis of PE monomethyl (site I), dimethyl (site II) and trimethyl (phosphatidylcholine (PC), site III) derivates were examined in SL and SR membranes by employing different concentrations of SadenosylLmethionine (AdoMet). Total methyl group incorporation into SL PE, in vitro, was significantly depressed by 10^–6–10^–3 M verapamil or diltiazem at site III. The catalytic activity of site I was inhibited by 10^–3 M verapamil only, whereas the site II activity was not affected by these drugs. The inhibition induced by verapamil or diltiazem (10^–5 M) was associated with a depression of the V_max value without any change in the apparent affinity for AdoMet. Both drugs decreased the SR as well as mitochondrial PE Nmethylation at site III. A selective depression of site III activity was also observed in SL isolated from hearts of rats treated with verapamil in vivo. Furthermore, administration of [³H-methyl]methionine following the treatment of animals with verapamil, reduced the synthesis of PC by Nmethyltransferase. Verapamil also depressed the N-methylation-dependent positive inotropic effect induced by methionine in the isolated Langendorff heart. Both agents depressed the SL Ca²⁺pump and although diltiazem also inhibited the SR Ca²⁺pump, verapamil exerted a stimulatory effect. In addition, verapamil decreased SR Ca²⁺-release. These results suggest that verapamil and diltiazem alter the cardiac PE Nmethyltransferase system. This action is apparently additional to the drugs' effect on Ltype Ca²⁺ channels and may serve as a biochemical mechanism for the drugs' inhibition of the cardiac Ca²⁺pumps and altered cardiac function.     

A reappraisal of the role of Zn2+ in TGF-β1-induced apoptosis in primary hepatocytes

There is now a wealth of information regarding the apoptotic mode of cell death and its importance in toxicological studies in many mammalian organs including the liver. In this study, we investigated the modulatory effects of the heavy metal Zn²⁺ on transforming growth factor-β₁ (TGF-β₁)-induced apoptosis in primary rat hepatocytes. Apoptosis induced by TGF-β₁ (1 ng/ml) in hepatocytes was accompanied by nuclear condensation as assessed morphologically by staining with Hoechst 33258 and DNA cleavage as detected biochemically by in situ end-labeling, field inversion and conventional gel electrophoresis. Pretreatment with 100 μmol/L Zn²⁺ abrogated the nuclear condensation, in situ end-labeling, and DNA laddering in TGF-β₁-treated hepatocytes. Surprisingly, Zn²⁺ did not inhibit the formation of high-molecular-weight DNA fragments (30–50 kbp to 250–300 kbp). These data provide evidence that Zn²⁺ exerts its effects on the endonucleases that act downstream in the execution phase of TGF-β₁-induced apoptosis in hepatocytes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of terminal dipole charges in aggregation of α-helix pair in the voltage gated K+ channel

The voltage sensor domain (VSD) of the potassium ion channel KvAP is comprised of four (S1–S4) α-helix proteins, which are encompassed by several charged residues. Apart from these charges, each peptide α-helix having two inherent equal and opposite terminal dipolar charges behave like a macrodipole. The activity of voltage gated ion channel is electrostatic, where all the charges (charged residues and dipolar terminal charges) interact with each other and with the transmembrane potential. There are evidences that the role of the charged residues dominate the stabilization of the conformation and the gating process of the ion channel, but the role of the terminal dipolar charges are never considered in such analysis. Here, using electrostatic theory, we have studied the role of the dipolar terminal charges in aggregation of the S3b–S4 helix pair of KvAP in the absence of any external field (V = 0). A system attains stability, when its potential energy reaches minimum values. We have shown that the presence of terminal dipole charges (1) change the total potential energy of the charges on S3b–S4, affecting the stabilization of the α-helix pair within the bilayer lipid membrane and (2) the C- and the N-termini of the α-helices favor a different dielectric medium for enhanced stability. Thus, the dipolar terminal charges play a significant role in the aggregation of the two neighboring α-helices.     

Defining the roles of Ca2+ — permeable channels in sperm

Ion channels exert a vital role in the dialogue between male and female gametes and thus in the generation of new individuals in many species. Intracellular Ca²⁺ is possibly the key messenger between gametes. Different Ca²⁺-permeable channels have been detected in the plasma membrane and in the organelle-like acrosome membrane of sperm, which play vital roles in determining sperm fertilizing ability. Recent reports from several laboratories have adequately documented that the Ca²⁺-permeable channels of a sperm control a variety of functions ranging from motility to the acrosome reaction. In this article, we have reviewed the data from our and other laboratories, and have documented the mechanisms of different Ca²⁺-permeable channels involved in the fertilization event.     

Partial characterization of the trait for enhanced K+−Na+ discrimination in the D genome of wheat

The long arm of chromosome 4D of wheat (Triticum aestivum L.) contains a gene (or genes) which influences the ability of wheat plants to discriminate between Na⁺ and K⁺. This discrimination most obviously affects transport from the roots to the shoots, in which less Na⁺ and more K⁺ accumulate in those plants which contain the long arm of chromosome 4D. Concentrations of Na⁺ and K⁺ in the roots, and Cl⁻ concentrations in the roots and shoots, are not significantly affected by this trait, but Na⁺, K⁺ and Cl⁻ contents of the grain are reduced. The trait operates over a wide range of salinities and appears to be constitutive. At the moment it is not possible to determine accurately the effect of this trait on growth or grain yield because the aneuploid lines which are available are much less vigorous and less fertile than their euploid parents.     

Adsorption of phage λ to Salmonella typhimurium lamB + requires the presence of lipopolysaccharide in the outer membrane

Summary Two S. typhimurium strains TA1534 (rfa ⁺) and TA1538 (rfaE) were transformed with the lamB expression plasmid pAMH70. Transposition events with placMu55 hybrid phage were successful only with TA1534/pAMH70 strain. Using SDS-PAGE, the LamB protein was present in the total cell proteins but not in the outer membrane proteins of the TA1538/pAMH70 strain. The LamB protein must linked to the LPS of the outer membrane to allow adsorption of phage in S. typhimurium.     

Degradation of DNA into 5′-Monodeoxyribonucleotides in the Presence of Mn2+ Ions

DNA is known to be aggregated by metal ions including Mn²⁺ ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn²⁺ ions ([Mn]/[P] = 46.3) at 70 °C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol–chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, and Cd²⁺, as transition element metal ions, were effective as to the degradation into dNMP. Mg²⁺, Ca²⁺, Sr²⁺, and Ba²⁺, as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl^?, CH₃OO^?, and NO₃ ^? were found to increase the degradation rate. Sixty μg of the 120 μg of the starting DNA in 450 μl was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn²⁺ ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH^? ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5′-NMP, based on the H¹NMR spectra. This prosess should prove to be a new process for the production of 5′-dNMP in addtion to the exonuclease.     

Both Intra- and Extracellular Ca2+ Participate in the Regulation of the Lateral Diffusion of the PDGF-β2 Receptor

When the receptors for platelet-derived growth factor (PDGF) are activatedthey aggregate, become tyrosine-phosphorylated and elicit a cascade ofdown-stream signals, including mobilization of Ca²⁺ from intra- andextracellular stores. Receptor mobility in the plane of the membrane isa prerequisite for receptor aggregation and further signalling. Using humanforeskin fibroblasts (AG 1523) and fluorescence recovery afterphotobleaching (FRAP), we therefore assessed the lateral mobilitycharacteristics of PDGF-2 receptors by their diffusioncoefficient (D), and fraction of mobile receptors (R). This was done oncells stimulated with either normal human serum (NHS) or PDGF underdifferent Ca²⁺-conditions.The results suggest that both intra- and extracellular free Ca²⁺influence the mobility characteristics of the PDGF-2receptor. Interestingly, the extracellular Ca²⁺ seems to imposegeneral restrictions on the mobility of receptors, since R increased whenextracellular Ca²⁺ was quenched with EGTA, whereas intracellularclamping of Ca²⁺ transients with MABTAM (BAPT/AM) primarily affectedD. When both intra- and extracellular Ca²⁺ were quenced, D remainedlow and R high, further supporting the proposition that they achievedistinct effects. Inhibition of tyrosine phosphorylation with Erbstatin,partly inhibited the NHS effects and released PDGF-induced receptorimmobilization. Ratio imaging with Fura-2 displayed that both NHS and PDGFinduced changes in intracellular free [Ca²⁺]. In view of the presentdata it might have important effects on the state of the receptor in themembrane, for instance by regulating its lateral mobility, communicationwith other receptors and signalling functions in the membrane.     

Asymmetric orientation of amino groups in the α-subunit and the β-subunit of (Na+ + K+)-ATPase in tight right-side-out vesicles of basolateral membranes from outer medulla

The orientation of amino groups in the membrane in the α- and β-subunits of (Na⁺ + K⁺)-ATPase was examined by labeling with Boldon-Hunter reagent, N-succinimidyl 3-(4-hydroxy,5-[¹²⁵I]iodophenyl)propionate), in right-side-out vesicles or in open membrane fragments from the thick ascending limbs of the Henles loop of pig kidney. Sealed right-side-out vesicles of basolateral membranes were separated from open membrane fragments by centrifugation in a linear metrizamide density gradient. After labeling, (Na⁺ + K⁺)-ATPase was purified using a micro-scale version of the ATP-SDS procedure. Distribution of label was analyzed after SDS-gel electrophoresis of α-subunit, β-subunit and proteolytic fragments of α-subunit. Both the α- and the β-subunit of (Na⁺ + K⁺)-ATPase are uniformly labeled, but the distribution of labeled residues on the two membrane surfaces differs markedly. All the labeled residues in the β-subunit are located on the extracellular surface. In the α-subunit, 65–80% of modified groups are localized to the cytoplasmic surface and 20–35% to the extracellular membrane surface. Proteolytic cleavage provides evidence for the random distribution of ¹²⁵I-labeling within the α-subunit. The preservation of (Na⁺ + K⁺)-ATPase activity and the observation of distinct proteolytic cleavage patterns of the E₁- and E₂-forms of the α-subunit show that the native enzyme structure is unaffected by labeling with Bolton-Hunter reagent. Bolton-Hunter reagent was shown not to permeate into sheep erythrocytes under the conditions of the labeling experiment. The data therefore allow the conclusion that the mass distribution is asymmetric, with all the labeled amino groups in the β-subunit being on the extracellular surface, while the α-subunit exposes 2.6-fold more amino groups on the cytoplasmic than on the extracellular surface.     

Do H+ ions obscure electrogenic Na+ and K+ binding in the E1 state of the Na,K-ATPase?

In contrast to other P-type ATPases, the Na,K-ATPase binding and release of ions on the cytoplasmic side, to the state called E1, is not electrogenic with the exception of the third Na+. Since the high-resolution structure of the closely related SR Ca-ATPase in state E1 revealed the ion-binding sites deep inside the transmembrane part of the protein, the missing electrogenicity in state E1 can be explained by an obscuring counter-movement of H+ ions. Evidence for such a mechanism is presented by analysis of pH effects on Na+ and K+ binding and by electrogenic H+ movements in the E1 conformation of the Na,K-ATPase.     

Evidence for the involvement of (Cu-ATP)2− in the inhibition of human erythrocyte (Ca2+ + Mg2+-ATPase by copper

The effects of copper on the activity of erythrocyte $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ have been tested on membranes stripped of endogenous calmodulin or recombined with purified calmodulin. The interactions of copper with Ca²⁺, calmodulin and (Mg-ATP)^2? were determined by kinetic studies. The most striking result is the potent competitive inhibition exerted by (Cu-ATP)^2? against (Mg-ATP)^2?$\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 2.8 μ}\text{M}$), while free copper gives no characteristic inhibition. Our results also demonstrate that copper does not compete with calcium either on the enzyme or on calmodulin. The fixation of calmodulin on the enzyme is not altered in the presence of copper as shown by the fact that the dissociation constant remains unaffected. It may be speculated that (Cu-ATP)^2? is the active form of copper, which could plausibly be at the origin of some of the pathological features of erythrocytes observed in conditions associated with excess copper.