Energy-dependence exchange of K+ in heart mitochondria. K+ efflux.

The efflux ⁴²K⁺ from isolated beef heart mitochondria under conditions of near steadystate K⁺ is increased by repiration and is sensitive to uncouplers and to exogenous Mg² The respiration-dependent efflux is strongly activated by inorganic phosphate in the presence of external K⁺, but not Na⁺, and is inhibited by oxidative phosphorylation. Low concentrations of mersalyl also activate respiration-dependent efflux of ⁴²K⁺ in the absence of net alteration in matrix K⁺. Acetate in the presence of mersalyl brings about net accumulation of K⁺ with retention of internal ⁴²K⁺. The results are consistent with a model in which nearly constant matrix K⁺ is maintained by the regulated interplay between a K⁺ uniport (which is responsive to membrane potential and which is the pathway for K⁺ influx) and a $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchanger (which responds to the transmembrane pH differential and which is the pathway for net K⁺ efflux).     

Energy-dependent exchange of K+ in heart mitochondria. K+ influx.

The tuberculostatic and carcinogenic drug isonicotinic acid hydrazide (“isoniazid”) is oxidized to pyridine-4-carboxaldehyde by the horseradish peroxidase/Mn²⁺/O₂ system. Eosin and related sensitizers greatly accelerate the reaction and generate light detectable with the liquid scintillation counter or with the photon counter. If the isoniazid concentration is raised, the rate and extent of O₂ uptake are increased, but above a certain concentration of isoniazid, emission is reduced and even suppressed. The strong quencher of triplet eosin, potassium ferricyanide, abolished both effects of eosin, that is, catalysis and light emission. Superoxide dismutase at high concentrations partially suppressed the emission and almost totally removed the catalytic effect. It is tentatively proposed that the isoniazid/peroxidase/Mn²⁺/O₂ system efficiently produces the aldehyde in the triplet state, which in turn transfers energy to eosin. Because of the presence of oxygen, only a small yield of triplet eosin is obtained and only a small fraction of these triplet eosin molecules is able to react with isoniazid. Nevertheless, it will contribute efficiently to a cyclic process of oxidation of the isoniazid.     

Cation flux in the ehrlich ascites tumor cell. Evidence for Na+-for-Na+ and K+-for-K+ exchange diffusion.

In a previous study, evidence was presented for an external Na+-dependent, ouabain-insensitive component of Na+ efflux and an external K+-dependent component of K+ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na+ and K+ they represent Na+-for-Na+ and K-+for-K+ exchange mechanisms. Using 86Rb to monitor K+ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb+ influx and a component of Rb+ efflux, both representing approx. 30 percent of the total flux. Inhibition of Rb+ efflux is greatly reduced by removal of extracellular K+. Furosemide does not alter steady-state levels of intracellular K+ and it does not prevent cells depleted of K+ by incubation in the cold from regaining K+ upon warming. Using 22Na to monitor Na+ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na+ efflux which represents approx. 22 percent of total Na+ efflux. Furosemide does not alter steady-state levels of intracellular Na+ and does not prevent removal of intracellular Na+ upon warming from cells loaded with Na+ by preincubation in the cold. The ability of furosemide to affect unidirectional Na+ and K+ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of alpha-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na+-dependent amino acid transport system A.     

Amiloride-sensitive Na+-H+ antiporter in Escherichia coli.

In everted vesicles of Escherichia coli, delta pH caused by H+ efflux through the Na+/H+ antiporter was measured by using a fluorescent dye. Amiloride inhibited the activity of the Na+/H+ antiporter. Kinetic studies showed that amiloride competed with Na+. The inhibition constant of 40 microM was obtained.     

Compound d+y+ particles of Australia antigen.

Australia antigen (HB8 Ag) particles vary in their antigenic structure. They are generally found to carry either determinant d or determinant y, but not both. This report describes seven sera which contain Australia antigen carrying both d and y on the same particle.     

The effect of Na+ and K+ on the thermal denaturation of Na+ and + K+-dependent ATPase.

To increase our understanding of the physical nature of the Na+ and K+ forms of the Na+ + K+-dependent ATPase, thermal-denaturation studies were conducted in different types of ionic media. Thermal-denaturation measurements were performed by measuring the regeneration of ATPase activity after slow pulse exposure to elevated temperatures. Two types of experiments were performed. First, the dependence of the thermal-denaturation rate on Na+ and K+ concentrations was examined. It was found that both cations stabilized the pump protein. Also, K+ was a more effective stabilizer of the native state than was Na+. Secondly, a set of thermodynamic parameters was obtained by measuring the temperature-dependence of the thermal-denaturation rate under three ionic conditions: 60 mM-K+, 150 mM-Na+ and no Na+ or K+. It was found that ion-mediated stabilization of the pump protein was accompanied by substantial increases in activation enthalpy and entropy, the net effect being a less-pronounced increase in activation free energy.     

S-adenosyl-L-methionine modulates Na+ + K+-ATPase activity in rat colonic basolateral membranes.

Rat colonic basolateral membranes were incubated with S-adenosyl-L-[methyl-3H]methionine (0.3 mM) at 37 degrees C for 2 h at pH 9.0. This resulted in an increase in the specific activity of Na+ + K+-ATPase by 60%. Kinetic parameter analysis revealed a 2-fold increase in the Vmax. of this enzymatic activity, whereas the Km for ATP was unchanged. The methylation inhibitor S-adenosyl-L-homocysteine (2 mM) significantly reduced these S-adenosyl-L-methionine-stimulated increases in specific activity and the Vmax. of Na+ + K+-ATPase. S-Adenosyl-L-methionine treatment of basolateral membranes was also found to significantly increase the fluidity of these preparations, as assessed by steady-state fluorescence polarization techniques using the fluorophore 1,6-diphenyl-1,3,5-hexatriene; S-adenosyl-L-homocysteine (2 mM) again markedly reduced this S-adenosyl-L-methionine-induced increase in fluidity. While transmethylation reactions involving phospholipids, non-polar lipids and proteins were all found to exist in rat colonic basolateral membranes, based on a number of observations, the results of the present studies suggest that transmethylation of membrane phospholipids, but not membrane non-polar lipids or proteins, influenced the fluidity of basolateral membranes which, in turn, modified Na+ + K+-ATPase activity in these membranes.     

Activation of Na+-K+-adenosine triphosphatase by spermine.

Spermine activated Na⁺-K⁺-ATPase when the concentrations of K⁺ and ATP were low, whereas it inhibited K⁺-dependent and ouabain-inhibitable monophosphatase. The activating effect of sperimine was not due to the substitution for K⁺ or Na⁺. Excess K⁺ inhibited Na⁺-K⁺-ATPase partially, and reduced the spermine activation. When 1 mM ATP was used, spermine at higher concentrations inhibited Na⁺-K⁺-ATPase, and did not activate at all. It is suggested that the K⁺-sites essential to Na⁺-K⁺-ATPase and the K⁺-phosphatase co-exist at different places of the enzyme.     

Active K+ transport in Mycoplasma mycoides var. Capri. Net and unidirectional K+ movements.

Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K+ concentration (Ki: 200--300 mM) which greatly exceeds that of the growth medium, and a low Na+ concentration (Na+i: 20 mM). Unlike Na+i,K+i varies with cell aging. The K+ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K+ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, K+i decreases and is partially compensated by a gain in Na+. This substitution completely reverses when metabolic substrate is added (K+ reaccumulation process). Kinetic analysis of K+ movement in cells with steady K+ level shows that most of K+ influx is mediated by an autologous K+-K+ exchange mechanism. On the other hand, during K+ reaccumulation by K+-depleted cells, a different mechanism (a K+ uptake mechanism) with higher transport capacity and affinity drives the net K+ influx. Both mechanisms are energy-dependent. Ouabain and anoxia have no effect on K+ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg2+ -dependent ATPase activity.     

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.     

Cation transport by gastric H+:K+ ATPase.

A vesicular microsomal fraction isolated from hog fundic mucosa demonstrates the capacity to take up equal amounts of RB+ and Cl-. The amount of the Rb+ uptake is sensitive to the extravesicular osmolarity, and rate of uptake is sensitive to temperature. 86Rb+ efflux is dependent upon the cation composition of the diluting solution. ATP, but not beta-gamma methylene ATP, induces a reversible efflux of 86Rb+ from loaded vesicles, and this is dependent upon a functional K+-ATPase. The ATP induced efflux is not affected by CCCP (carbonyl cyanide m-chlorophenylhydrazone) or TCS (tetrachlorosalicylanilide) nor by lipid soluble ions or valinomycin. Nigericin inhibits the efflux by 40%. Uptake of the lipid soluble ion 14C-SCN- has been demonstrated and is enhanced by ATP only in the presence of valinomycin. The results are consistent with a neutral or isopotential exchange of H+ for Rb+ mediated by K+-ATPase.     

Discrimination between Rb+ and K+ by Escherichia coli.

1. The K+ requirment of Escherichia coli is only partially fulfilled by Rb+. The molar growth yield on Rb+ was about 5% of that on K+ and the growth rate in Rb+-supplemented media is lower thatn in K+ influx by any of the four K+ transport systems of E. coli. The high-affinity Kdp system (Km = 2 micron) is poorly traced by 86Rb+. It discriminates against a 86Rb+ tracer at least 1000-fold. The two moderate affinity systems, the high-rate TrkA system (Km = 1.5 mM) and the moderate rate TrkD system (Km = 0.5 mM), discriminate against a 86Rb+ tracer by approximately 10-fold and 25-fold, respectively. 86Rb+ is preferred by the low-rate TrkF system and overestimates its K+ influx by 40%.     

The Ca2+-induced membrane transition in mitochondria. III. Transitional Ca2+ release.

The efflux of Ca²⁺ from mitochondria respiring at steady state, and much of uncoupler-induced Ca²⁺ efflux, is shown to be a consequence of the Ca²⁺-induced membrane transition (the Ca²⁺-induced transition is the Ca²⁺-dependent sudden increase in the nonspecific permeability of the mitochondrial inner membrane which occurs spontaneously when mitochondria are incubated under a variety of conditions (D. R. Hunter, R. A. Haworth, and J. H. Southard, 1976, J. Biol. Chem.251, 5069–5077)). Ca²⁺ release from mitochondria respiring at steady state is shown to be transitional by four criteria: (1) Ca²⁺ release is inhibited by Mg²⁺, ADP, and bovine serum albumin (BSA), all inhibitors of the transition; (2) release is selective for Ca²⁺ over Sr²⁺, a selectivity also found for the transition; (3) the time course of Ca²⁺ release is identical to the time course of the change in the mitochondrial population from the aggregated to the orthodox configuration; and (4) from kinetics, Ca²⁺ release from individual mitochondria is shown to occur suddenly, following a lag period during which no release occurs. Ca²⁺ release induced by uncoupler is shown to be mostly by a transitional mechanism, as judged by four criteria: (1) release of Ca²⁺ is ruthenium red-insensitive and is an order of magnitude faster than Sr²⁺ release which is ruthenium red-sensitive; (2) release of Ca²⁺ is strongly inhibited by keeping the mitochondrial NAD⁺ reduced; (3) the kinetics of Ca²⁺ release indicates a transitional release mechanism; and (4) uncoupler addition triggers the aggregated to orthodox configurational transition which, at higher levels of Ca²⁺ uptake, occurs in the whole mitochondrial population at a rate equal to the rate of Ca²⁺ release. Na²⁺-induced Ca²⁺ release was not accompanied by a configurational change; we therefore conclude that it is not mediated by the Ca²⁺-induced transition.     

A role of H+ flux in active Ca2+ transport into sarcoplasmic reticulum vesicles. II. H+ ejection during Ca2+ uptake

Proton efflux during Ca2+ transport into sarcoplasmic reticulum vesicles was examined. Although a rapid H+ ejection was observed during the initial phase of Ca2+ uptake and the amount of the liberated H+ was more than that due to hydrolysis of ATP, generation of a pH difference as a result of the H+ efflux could not be detected by direct pH measurement with a pH meter. Alkalinization of the inside of the vesicles during Ca2+ uptake was more precisely examined by flow dialysis assay and a significant uptake of acetate or salicylate into the vesicles was found, suggesting the generation of a small pH difference across the SR membrane. From these results, it was concluded that counter-transport of H+ was operative in Ca2+ uptake but that only a relatively small pH difference was generated as a result of the H+ efflux. The intrinsic buffering capacity of sarcoplasmic reticulum vesicles was measured and a relatively large value (130 nmol H+/pH unit/mg at pH 6.2) was obtained.     

Effects of detergents on Na+ + K+-dependent ATPase activity in plasma-membrane fractions prepared from frog muscles. Studies of insulin action on Na+ and K+ transport.

The increase in Na+/K+ transport activity in skeletal muscles exposed to insulin was analysed. Plasma-membrane fractions were prepared from frog (Rana catesbeiana) skeletal muscles, and examination of the Na,K-ATPase (Na+ + K+-dependent ATPase) activity showed that it was insensitive to ouabain. In contrast, plasma-membrane fractions prepared from ouabain-pretreated muscles, by the same procedures, showed extremely low Na,K-ATPase activity. On adding saponin to the membrane suspension, the Na,K-ATPase activity increased, according to the detergent concentration. The maximum activity was about twice the control value, at 0.33 mg of saponin/mg of protein. Thus saponin makes vesicle membranes leaky, allowing ouabain in assay solutions to reach receptors on the inner surface of vesicles. Addition of insulin to saponin-treated membrane suspensions had no effect on the Na,K-ATPase activity, whereas the maximum activity of Na,K-ATPase in whole muscles was stimulated by exposure to insulin. The results show that the stimulation of Na+/K+ transport by insulin is not directly due to insulin binding to receptors on the cell surface, but rather support the view that the increase in the Na,K-ATPase induced by insulin requires an alteration of intracellular events.