Ruthenium red-induced loss of matrix K+ from uncoupled heart mitochondria

Ruthenium red induces the loss of endogenous K⁺ from isolated beef heart mitochondria treated with an uncoupler. This induction of K⁺ loss occurs at the same ruthenium red titer as the inhibition of the Ca²⁺-uniporter. This raises the possibility that ruthenium red may alter the Ca²⁺-uniporter in such a way as to produce a K⁺-conducting channel.     

Adenine nucleotide translocase as a site of regulation by ADP of the rat liver mitochondria permeability to H+ and K+ lons

In the presence of oligomycin ADP inhibits the osmotic swelling of the nonenergized rat liver mitochondria in the NH₄NO₃ medium. With the energized mitochondria ADP enhances contraction of the mitochondria swollen in the NH₄NO₃ medium. Carboxyatractyloside and atractyloside abolish or prevent the effects of ADP. The direct measurements of the proton conductance of rat liver mitochondria shows that the inhibitory action of ADP + oligomycin on the H⁺ permeability does not depend on the energization of mitochondria. In these experiments the local anesthetic nupercaine and ADP additively inhibit the inner membrane conductance for protons, but carboxyatractyloside abolishes only the effect of ADP. In the presence of oligomycin ADP also inhibits the osmotic swelling of the nonenergized liver mitochondria in the KNO₃ medium, and the energy-dependent swelling of rat liver mitochondria in the medium with K⁺ ions and P_i. The inhibition by ADP of the membrane passive permeability for K⁺ is also sensitive to carboxyatractyloside. It is concluded that rat liver mitochondria possess an ADP-regulated channel for H⁺ and K⁺. The properties of this pathway for protons and potassium ions favor the idea that ADP regulates the mitochondrial permeability via adenine nucleotide translocase. It is assumed that the adenine nucleotides carrier should operate according to the “gated pore” mechanism.     

The role of membrane-bound magnesium in the permeability of ghosts to K+

Mg²⁺ is required for restoring a low cation permeability of erythrocyte membranes after osmotic lysis. These ions promote binding of haemoglobin to the ghost membrane. Because the optimal binding occurs at a pH where the K⁺ retention by ghosts is maximal, the possibility exists that Mg²⁺ exerts an indirect effect by facilitating the adsorption of haemoglobin, which may be essential for the maintenance of a low permeability. In order to investigate this possibility, a systematic study was done of the effects of pH and hypotonic washing on the retention of haemoglobin, Mg²⁺ and K⁺ by human erythrocyte ghosts.Between pH 5.5 and 7.5, the retention of haemoglobin paralleled that of K⁺ and Mg²⁺ on resealing immediately after lysis. Such a parallelism was not observed if ghosts were both washed with fresh haemolytic media and resuspended in a medium of lower osmolarity before reversal.No correspondence was found between ghost K⁺ content and membrane-bound haemoglobin, indicating that the latter is not involved in the maintenance of a low permeability. By contrast, Mg²⁺ binding was altered by pH in the same way as ghost K⁺ and a strict relationship between membrane-bound Mg²⁺ and K⁺ retention was obtained.The results suggest that K⁺ permeability is mainly determined by the amount of Mg²⁺ associated with the ghost membrane.     

The participation of pyridine nucleotides redox state and reactive oxygen in the fatty acid-induced permeability transition in rat liver mitochondria

The ability of low concentrations (5-15 microM) of long-chain fatty acids to open the permeability transition pore (PTP) in Ca(2+)-loaded mitochondria has been ascribed to their protonophoric effect mediated by mitochondrial anion carriers, as well as to a direct interaction with the pore assembly [M.R. Wieckowski and L. Wojtczak, FEBS Lett. 423 (1998) 339-342]. Here, we have compared the PTP opening ability of arachidonic acid (AA) with that of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) at concentrations that cause similar quantitative dissipation of the membrane potential (DeltaPsi) in Ca(2+)-loaded rat liver mitochondria respiring on succinate. The initial protonophoric effects of AA and FCCP were only slightly modified by carboxyatractyloside and were followed by PTP opening, as indicated by a second phase of DeltaPsi disruption sensitive to EGTA, ADP, dithiothreitol and cyclosporin A. This second phase of DeltaPsi dissipation could also be prevented by rotenone or NAD(P)H-linked substrates which decrease the pyridine nucleotide (PN) oxidation that follows the stimulation of oxygen consumption induced by AA or FCCP. These results suggest that, under the experimental conditions used here, the PTP opening induced by AA or FCCP was a consequence of PN oxidation. Exogenous catalase also inhibited both AA- and FCCP-induced PTP opening. These results indicate that a condition of oxidative stress associated with the oxidized state of PN underlies membrane protein thiol oxidation and PTP opening.     

A reconstituted Na++K+ pump in liposomes containing purified (Na++K+-ATPase from kidney medulla

Liposomes containing either purified or microsomal (Na⁺,K⁺)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na⁺ and K⁺ with a ratio of approximately 3Na⁺ to 2K⁺, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na⁺,K⁺-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907–5915, 1974), in which liposomes containing purified dog kidney (Na⁺,K⁺)-ATPase did not transport K⁺ but catalyzed ATP-dependent symport of Na⁺ and Cl^?. When purified by our procedure, dog kidney (Na⁺,K⁺)-ATPase showed some ability to transport K⁺ but the ratio of Na⁺ : K⁺ was 5 : 1.     

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.     

K+ transport in mitoplasts

K⁺ transport into mitoplasts, prepared by digitonin disruption and removal of the outer membranes from rat liver mitochondria, has been studied. Unidirectional K⁺ influx has been measured by means of ⁴²K, in the presence of the respiratory substrate succinate. K⁺ influx is inhibited by CN^?, antimycin A and dicyclohexylcarbodiimide, but is insensitive to oligomycin. A linear dependence of the reciprocal of the K⁺-influx rate on the reciprocal of the external K⁺ concentration is observed. Under the conditions studied, the apparent K_m for K⁺ of the transport mechanism is approx. 6 mM, while the V_max of K⁺ influx is approx. 5 μ mol K⁺/g protein per min. The rate of K⁺ influx increases with increasing external pH over the range from 6.8 to 8.0. The observed kinetics, pH dependence and inhibitor sensitivity are essentially similar to previously reported characteristics of K⁺ transport into intact rat liver mitochondria. It is concluded that the outer mitochondrial membrane does not have a role in controlling K⁺ flux into rat liver mitochondria.     

Synthetic peptide K+ carrier with Ca2+ inhibition

Based on a proposed solution conformation of the Ca²⁺ ion complex of the repeat hexapeptide of elastin, l-Val-l-Ala-l-Pro-Gly-l-Val-Gly, it is possible to modify the molecule making it more lipophilic for lipid bilayer permeation while retaining its complexation features. Therefore the two peptides, For-MeVal-Ala-Pro-Sar-Pro-Sar-OMe and For-MeVal-Ala-Pro-Sar-Pro-Sar-OH, were synthesized and evaluated for lipid bilayer activity and cation binding (For, N-formyl; Me, N-methyl; Sar, N-methyl glycine). Both peptides bound Ca²⁺ preferentially but did not exhibit the properties of a Ca²⁺ carrier. They were however active as K⁺ carriers although K⁺ ion titration curves showed a much lower affinity for K⁺ than for Ca²⁺. The addition of Ca²⁺ or Mg²⁺ to the bilayer system inhibited the peptide K⁺ carrier activity. Three possible explanations of this interesting Ca²⁺ inhibition of carrier activity are irreversible complexation of Ca²⁺, mixed ligand complex formation involving Ca²⁺, lipid and peptide, and impermeability of the lipid layer when peptide is complexed with a divalent cation.     

Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells

The K⁺ content and the K⁺ flux were measured in the cell lines ME₂ and MF₂ isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the same K⁺ content and the same K⁺ steady state flux per unit of surface area.In ME₂ cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K⁺ net movement.The (Na⁺ + K⁺)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K⁺ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells.The K⁺ net flux is considerably increased when the intracellular K⁺ level is reduced after preincubation of the cells in a K⁺-free medium. Thus, internal K⁺ seems to regulate the K⁺ influx.     

Regulation of cyclosporin A sensitive mitochondrial permeability transition by the redox state of pyridine nucleotides

The mechanisms involved in the induction of cyclosporine A sensitive mitochondrial swelling by oxidative stress were investigated in isolated guinea pig liver mitochondria. The aim of our study was to investigate, if swelling is inevitably associated with the oxidation of pyridine nucleotides, and if the oxidized pyridine nucleotides have to be hydrolysed for the induction of mitochondrial swelling. Quantitative measurement of oxidized pyridine nucleotides was performed with HPLC. Mitochondrial swelling was recorded by monitoring the decrease in light scattering of the mitochondrial suspension. Reduction and oxidation of pyridine nucleotides were followed by monitoring the changes of the autofluorescence signal of reduced pyridine nucleotides. Qualitative measurement of mitochondrial membrane potential was performed with the fluorescence indicator rhodamine 123. Neither t-butyl hydroperoxide nor the dissipation of the mitochondrial inner membrane potential with FCCP (carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone) induced the opening of the membrane permeability transition pore, unless an extensive oxidation of mitochondrial pyridine nucleotides took place. Mitochondrial swelling induced by our experimental conditions was always sensitive to cyclosporine A and accompanied by a cyclosporine A sensitive release of inner mitochondrial pyridine nucleotides without pyridine nucleotide hydrolysis. Not the cycling of calcium across the mitochondrial inner membrane but the accumulation of calcium inside the mitochondria was a prerequisite for mitochondrial swelling. The mitochondrial membrane permeability transition is neither caused nor accompanied by the hydrolysis of mitochondrial pyridine nucleotides.     

Electrogenic proton ejection coupled to electron transport through the energy-conserving site 2 and K+/H+ exchange in yeast mitochondria

The proton ejection coupled to electron flow from succinate and/or endogenous substrate(s) to cytochrome c using the impermeable electron acceptor ferricyanide is studied in tightly coupled mitochondria isolated from two strains of the yeast Saccharomyces cerevisiae. (1) The observed H⁺ ejection/2e^? ratio approaches an average value of 3 when K⁺ (in the presence of valinomycin) is used as charge-compensating cation. (2) In the presence of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone, an H⁺ ejection/2e^? ratio of 2 is observed. (3) The low stoichiometry of 3H⁺ ejected (instead of 4) per 2e^? and the high rate of H⁺ back-decay (0.1615 $\text{ln}\text{δ-}\text{(}\text{ngatom}\text{)}\text{H}{}^{\mathrm{+}}\text{s}$ and a half-time of 4.6 s for 10 mg protein) into the mitochondrial matrix are related to the presence of an electroneutral K⁺/H⁺ antiporter which is demonstrated by passive swelling experiments in isotonic potassium acetate medium.     

Mechanisms by which Li+ stimulates the (Na++K+)-dependent ATPase

The addition of LiCl stimulated the (Na⁺+K⁺)-dependent ATPase activity of a rat brain enzyme preparation. Stimulation was greatest in high Na⁺/low K⁺ media and at low Mg. ATP concentrations. Apparent affinities for Li⁺ were estimated at the α-sites (moderate-affinity sites for K⁺ demonstrable in terms of activation of the associated K⁺-dependent phosphatase reaction), at the β-sites (high-affinity sites for K⁺ demonstrable in terms of activation of the overall ATPase reaction), and at the Na⁺ sites for activation. The relative efficacy of Li⁺ was estimated in terms of the apparent maximal velocity of the phosphatase and ATPase reactions when Li⁺ was substituted for K⁺, and also in terms of the relative effect of Li⁺ on the apparent K_M for Mg· ATP. With these data, and previously determined values for the apparent affinities of K⁺ and Na⁺ at these same sites, quantitative kinetic models for the stimulation were examined. A composite model is required in which Li⁺ stimulates by relieving inhibition due to K⁺ and Na⁺ (i) by competing with K⁺ for the α-sites on the enzyme through which K⁺ decreases the apparent affinity for Mg·ATP and (ii) by competing with Na⁺ at low-affinity inhibitory sites, which may represent the external sites at which Na⁺ is discharged by the membrane NA⁺/K⁺ pump that this enzyme represents. Both these sites of action for Li⁺ would thus lie, in vivo, on the cell exterior.     

Showdomycin,a nucleotide-site-directed inhibitor of (Na+ + K+)-ATPase

Showdomycin [2-(β-d-ribofuranosyl)maleimide] is a nucleoside antibiotic containing a maleimide ring and which is structurally related to uridine. Showdomycin inhibited rat brain (Na⁺ + K⁺)-ATPase irreversibly by an apparently bimolecular reaction with a rate constant of about 11.01·mol^?1·min^?1. Micromolar concentrations of ATP protected against this inhibition but uridine triphosphate or uridine were much less effective. In the presence of K⁺, 100 μM ATP was unable to protect against inhibition by showdomycin. These observations show that showdomycin inhibits (Na⁺ + K⁺)-ATPase by reacting with a specific chemical group or groups at the nucleotide-binding site on this enzyme. Inhibition by showdomycin appears to be more selective for this site than that due to tetrathionate or N-ethylmaleimide. Since tetrathionate is a specific reactant for sulfhydryl groups it appears likely that the reactive groups are sulfhydryl groups. The data thus show that showdomycin is a relatively selective nucleotide-site-directed inhibitor of (Na⁺ + K⁺)-ATPase and inhibition is likely due to the reaction of showdomycin with sulfhydryl group(s) at the nucleotide-binding site on this enzyme.     

Greater sensitivity to vanadate of rat renal relative to cardiac (Na++K+)-ATPase

Vanadate (VO₄^?3) produces a positive inotropic effect in rats and also promotes diuresis as well as natriuresis. Although the mechanism(s) of these effects is uncertain, in the kidney, VO₄^?3 may act through inhibition of (Na⁺+K⁺)-ATPase activity, whereas in the heart, other or additional mechanisms are likely. Under the assay conditions used in the present study, microsomal (Na⁺+K⁺)-ATPase activities from rat kidney cortex and medulla were inhibited to a greater extent than was left ventricular (Na⁺+K⁺)-ATPase activity over a range of VO₄^?3 concentrations. The apparent dissociation constant for left ventricular (Na⁺+K⁺)-ATPase (10.95 ± 1.26 × 10^?7M VO₄^?3) was significantly greater than that of (Na⁺+K⁺)-ATPase from the cortex (3.46±0.96×10^?7 M VO₄^?3) or the medulla (3.32±0.7×10^?7M VO₄^?3, N=6, P<.05) whereas there were no significant differences between the effects of VO₄^?3 on (Na⁺+K⁺)-ATPase from the cortex and medulla. The greater inhibition by VO₄, of (Na⁺+K⁺)-ATPase from the cortex relative to that of the left ventricle, occurred over a range of Na⁺ and K⁺ concentrations, and K⁺ enhanced the inhibition by VO₄^?3 to a greater extent for (Na⁺+K⁺)-ATPase from the cortex than the left ventricle. These results suggest that renal (Na⁺+K⁺)-ATPase is more sensitive than left ventricular (Na⁺+K⁺)-ATPase to inhibition by VO₄^?3 and would, therefore, be more likely to be modulated $\text{in}$$\text{vivo.}$     

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.     

短期NaCl胁迫对不同小麦品种幼苗K+吸收和Na+、K+积累的影响

为研究盐胁迫下小麦幼苗生长及Na⁺、K⁺的吸收和积累规律,以中国春、洲元9369和长武134等3种耐盐性不同小麦品种为材料,采用非损伤微测技术检测盐胁迫2 d后的根系K⁺离子流变化,并对植株体内的Na⁺、K⁺含量进行测定。结果表明:短期(2d)盐胁迫对小麦生长有抑制作用,且对根系的抑制大于地上部,耐盐品种下降幅度小于盐敏感品种。盐胁迫下,小麦根际的 K⁺大量外流,盐敏感品种中国春K⁺流速显著高于耐盐品种长武134,最高可达15倍。小麦幼苗地上部分和根系均表现为Na⁺积累增加,K⁺积累减少,Na⁺/K⁺比随盐浓度增加而上升。中国春限Na⁺能力显著低于长武134,Na⁺/K⁺则显著高于长武134。综上所述,盐胁迫下造成小麦组织器官中Na⁺/K⁺比上升的主要原因是根系K⁺大量外流和Na⁺的过量积累,耐盐性不同的小麦品种间差异显著,并认为根系对K⁺的保有能力可能是作物耐盐性评价的一个重要指标。     

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.     

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.