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—The ouabain-sensitive K+ uptake and ATPase activities of cultured glioma and neuroblastoma cells were studied. Both cell lines showed ouabain-sensitive K+ uptake which correlated with the level of [Na++ K+]ATPase activity found in the respective total cell homogenate. The glioma cells had a 2.1-fold higher rate of K+ uptake than neuroblastoma cells, and a 2.4-fold higher [Na++ K+]ATPase activity. In the presence of ouabain neuroblastoma cells released K+ and took up Na+ in a 1:1 ratio. These results are compared and contrasted with similar studies on brain tissue and isolated cells. It is suggested that the cultured cell lines may serve as good models for the cation transport properties of their tissue counterparts.

Interactions between the divalent cation ionophore, A23187, and the divalent cations Ca 2+ , Mg 2+ , and Mn 2+ were studied in sarcoplasmic reticulum and mitochondria. Conductance measurements suggest that A23187 facilitates the movement of divalent cations across bilayer membranes via a primarily electroneutral process, although a cationic form of A23187 does carry some current. On the basis of fluorescence excitation spectra, A23187 can form either a 1:1 or 2:1 complex with Ca 2+ in organic solvents. However, in biological membranes, only the 1:1 complexes with Ca 2+ , Mg 2+ , or Mn 2+ are detected. A23187 produces fluorescent transients under conditions of Ca 2+ uptake in sarcoplasmic reticulum, which appear to represent changes in intramembrane Ca 2+ content. Changes in A23187 fluorescence due to mitochondrial Ca 2+ accumulation are much smaller by comparison and fluorescence transients are not detected. Studies of A23187 fluorescence polarization and lifetimes in biological membranes allow a determination of the rotational correlation time (ρh) of the ionophore. In mitochondria at 22 °C, ρh is 11 nsec in the presence of Ca 2+ and Mg 2+ , and less than 2 nsec in the presence of excess EDTA. The present results are consistent with a model of ionophore-mediated cation transport in which free M 2+ binds with A23187 at the membrane surface to form the complex M(A23187) + . Reaction of this complex with another molecule of A23187 at the membrane surfaces results in the formation of electrically neutral M(A23187) 2 , which carries the divalent cation through the membrane. These results are discussed in terms of physical properties of biological membranes in regions in which divalent cation transport occurs.

Human erythrocytes were treated with pronase under conditions which gave complete cleavage of the pronase-sensitive major protein and glycoprotein components of the membrane. 86 Rb + influx of these modified cells did not differ significantly from that of untreated cells, and showed identical sensitivity to inhibition by ouabain. Pronase treatment of porous erythrocyte ghost membranes resulted in complete loss of (Na + + K + )-ATPase activity. These results indicate that the cation-transport complex is not readily accessible from the outer surface of native intact membranes.

The interaction of La3+ with rat liver mitochondria was examined with a wide variety of techniques permitting measurement of respiratory and structural responses. It is concluded that La3+ is accumulated by mitochondria in a process that is at least partially energy-dependent, and is bound with quite high affinity to membrane-associated sites both external and internal to the inner membrane. The relative insensitivity of the accumulation process to respiratory inhibitors and to the permeant anion acetate has interesting implications for the mechanism of active cation transport.

The interactions of Mn2+, inorganic phosphate, and methylphosphonate with membrane-bound sodium plus potassium ion transport adenosine triphosphatase from sheep kidney medulla have been examined by kinetic and magnetic resonance techniques. Electron paramagnetic resonance and water proton relaxation rate studies show that the enzyme, which is 35 to 40 % pure, binds Mn2+ at one tight binding site (Kd = 0.88 µm). Kinetic studies yield an activator constant for Mn2+ of 0.88 µm, identifying the one tight Mn2+-binding site as the active site of the adenosine triphosphatase. Competition studies indicate that Mg2+ binds at this site with a dissociation constant of 1 mm in agreement with its activator constant. Inorganic phosphate and methylphosphonate bind to the enzyme-Mn2+ complex with similar, extremely high affinities and decrease the enhancement, e*, by 26 and 24 %, respectively. Analysis of the frequency dependence of 1/T1 of water indicates that this change in enhancement is due to a decrease from 4 to 3 in the number of rapidly exchanging water protons in the coordination sphere of enzyme-bound Mn2+. The relative effectiveness of Na+ and K+ in facilitating ternary complex formation with Pi was examined as a function of pH. The fraction of the total de-enhancement produced by Pi in the presence of Na+ alone varies from 88 % at pH 6.1 to 7.5 % at pH 7.5 and closely follows the titration curve for the second ionization of Pi. Analogous behavior at a higher pH was observed for methylphosphonate, which has a pKa for its second ionization which is 0.9 pH unit higher than that of Pi. These results indicate that Na+ induces the phosphate monoanion to coordinate to the enzyme-bound Mn2+, while K+ causes the phosphate dianion to coordinate to the enzyme-bound Mn2+. Thus protonation of an enzyme-bound phosphoryl group would convert a K+-binding site to a Na+-binding site. Dissociation constants for K+ and Na+ estimated from our nuclear magnetic resonance titrations agreed with kinetically determined activator constants of these ions consistent with binding to the active site. A mechanism is proposed for the ATPase and cation transport in which the ionization state of phosphate at a single enzyme active site controls the binding and transport of Na+ or K+. This model also accounts for the order of magnitude weaker binding of Na+ compared to K+, as well as for all the observed effects of ATP on the enzyme. The Mn2+-binding studies also indicate a large number of additional Mn2+ binding sites, 60 intermediate sites and 170 to 290 weak binding sites depending on the ionic strength. Increased ionic strength exposes an additional 120 weak sites and reduces the affinity of Mn2+ for all classes of sites. The number and affinity of the intermediate and weak Mn2+ sites, which do not activate the enzyme but may be inhibitory, are consistent with coordination to the lipid phosphoryl groups in the preparation.

The turnover numbers of the ionophores valinomycin, the macrolide actins, enniatin B and dicyclohexyl 18-crown-6 for translocation of cations through the mitochondrial membrane have been compared quantitatively. The rank order of decreasing maximum turnover number for K+ transport calculated on the basis of the ionophore concentration within the membrane is trinactin>dinactin∼monactin∼ valinomycin>nonactin>18-crown-6>enniatin B. The strength of binding of the ionophores to the mitochondria has the following rank order: valinomycin>macrolide actins>enniatin B>18-crown-6.

Phosphatidylcholine vesicles acquire a negative surface charge in the presence of furosemide or ethacrynic acid. In addition, these same vesicles also display a higher efflux rate for 22Na and 86Rb when exposed to these two diuretics. The experiments reported in this study indicate that the increased cation permeability is due to the creation of this negative surface charge. Cations will accumulate in the electrical double layer adjacent to the interface, resulting in a larger trans-membrane flux. In addition, ethacrynic acid also facilitates H+ transport in these vesicles, a property not shared by furosemide. Although the explanation for the enhanced H+ permeability is unclear, the experiments indicate that it occurs through a different mechanism than that by which alkali-metal cation movement is increased.

1. 1. Arrhenius plots of the increase in oxygen uptake by rat or mouse liver mitochondria following a sub-maximal dose of gramicidin D or valinomycin show clear-cut inflections at about 16°C. 2. 2. With the mammalian preparations activation energies above the inflection are low for gramicidin but significantly higher for valinomycin. Over the lower part of the range gramicidin shows greater temperature dependence than valinomycin. There is no indication of a non-intersecting discontinuity in the response to valinomycin at the transition temperature. 3. 3. Arrhenius plots for rainbow trout liver mitochondria are linear over the whole experimental temperature range (5–30°C) for both antibiotics. The activation energy for gramicidin is significantly greater than that for valinomycin. 4. 4. These results are discussed in relation to other work on phase transitions in artificial lipid systems and the temperature dependence of membrane-bound enzyme activities. 5. 5. The possible effect of configurational changes in the mitochondrial membranes is considered in conjunction with the differing modes of action of these two antibiotics.

Using refinements of quantitative histochemistry, i.e. oil-well technique and enzymaticPianalysis combined with an enzymatic cycling system, the activity of Na K ATPase (E.C. 3.6.1.3), an enzyme which is integrated in the transmembrane cation transport, was measured in single dissected segments of the proximal and distal convolution from the superficial nephron of the rat kidney following uninephrectomy. In the distal convolution an acute rise in Na K ATPase activity was apparent already during the first day after uninephrectomy, whereas in the proximal convolution the first significant increase occurred seven days after surgery. These data indicate a rapid adaptation of Na K ATPase which is limited to the distal tubule. The difference between the proximal and distal tubule confirms the functional correlations to uninephrectomy with more reabsorptive work in the distal tubule per unit length. Na K ATPase seems to have a special functional meaning for the distal tubule in regulating cation transport.

The marked sensitivity of phytohemagglutinin (PHA)-activated pig lymphocytes to ouabain suggested that antibody-forming cells may also be sensitive to this glycoside. Preincubation of sensitized pig and rabbit splenic lymphoid cells with ouabain led to inhibition of plaque formation to sheep red blood cells (SRBC). The degree of inhibition was dependent on the concentration of the glycoside and on the duration of exposure, and was diminished when the concentration of potassium in the medium was increased. Species differences in PFC sensitivity to ouabain were similar to those previously observed for the inhibition of lymphocyte blastogenesis by the glycoside.

Stability constants of sodium and potassium complexes of valinomycin in some alcohols and water—organic solvent mixtures have been determined by titration, using circular dichroism to monitor complex formation. Constants range from 10 1 to 10 6 M −1 . Stability of the potassium and sodium complexes increases with decreasing dielectric constant, but the ratio of the constants remains about 10 3 –10 4 . As others have shown, a similar selectivity for K + is observed in a number of other types of measurements involving valinomycin. These include the permeability and conductance ratios which characterize the selectivity of cation transport through membranes and the ratio of salt extraction equilibrium constants. On the basis of data presented here, and elsewhere, it is suggested that conformational constraints within the depsipeptide part of the complexes aid ion selectivity and that differences in cation solvation and carbonyl ligand binding energies make an important, roughly equal, contribution.

1. 1. Cation transport in relation to cell metabolism in the high Na+, low K+ seal erythrocyte was investigated. Under steady-state conditions at 40°C, Na+ efflux was32 ± 4·0 (S.D.) m-equiv. × kg RBC H2O−1 × hr−1and K+ influx was 1·1 ± 0·38m-equiv. × kg RBC H2O−1 × hr−1. 2. 2. Both fluxes were insensitive to ouabain (10−4 M) and an inhibitor of anaerobic metabolism, monoiodoacetate (10−4 M). 3. 3. Ethanol (5 × 10−1 M) decreased Na+ efflux in the seal erythrocyte and the high Na+, low K+ cat erythrocyte but did not affect Na+ transport in the low Na+, high K+ human erythrocyte. 4. 4. Valinomycin (10−5 M) produced increased permeability to K+ without effect on Na+ transport. 5. 5. Lactate production (2·4 ± 0·29 mM×1.RBC H2O−1 × hr−1) was not decreased significantly by ouabain. 6. 6. Adenosinetriphosphate (ATP) concentrations (0·4 ± 0·03 (S.D.)mM/5·0 mM Hb) and 2,3 diphosphoglycerate (2,3 DPG) concentrations (6·5 ± 0·53 (S.D.) mM/5·0 mM Hb) provide evidence that this cell possesses an active glycolytic mechanism for high energy phosphate production. 7. 7. Quantitatively, the level of energy transduction is similar in both high Na+, low K+ seal erythrocytes and low Na+, high K+ human erythrocytes despite the small calculated minimal work cost of cation transport in the former.

OVER the past decade, evidence has accumulated that fragments of cellular membranes exhibit ATPase activity which is dependent on sodium ions and inhibited by ouabain, and which is probably involved in the active transport of sodium and potassium ions1. Another type of cation transport, characterized by an insensitivity to ouabain and coupled directly to the energy transfer system of the respiratory chain, has been demonstrated in isolated mitochondria. Interest in this system has been concerned primarily with the transport of divalent cations, and their exchange with hydrogen ions2, but it is now obvious that a similar mechanism can operate for the transport of monovalent cations, provided that the mitochondria are treated with certain polypeptide antibiotics. Valinomycin specifically promoted the uptake of potassium, rubidium and caesium ions3,4, whereas gramicidin also activated uptake of sodium and lithium ions by isolated mitochondria5.

This investigation was undertaken to examine whether the rat, like other mammals that have been studied, maintains a stable concentration of Mg2+ in cerebrospinal fluid (CSF) when the plasma Mg2+ concentration is changed. CSF was sampled from the cisterna magna in adult rats made hypomagnesemic by being fed a magnesium-free diet. The [Mg2+] in CSF did not decrease significantly during hypomagnesia, in disagreement with previous findings in the rat. The disagreement could have been due to a difference in the age of the animals when they were studied, cation transport processes possibly being incompletely developed in young rats, but we found that rats 3 to 4 weeks old also maintained a stable [Mg2+] in CSF during hypo-niagnesemia. The effect of 4 hrs of hypercapnia on the [Mg2+] in CSF was examined in normal hypomagnesemic adult rats. Hypercapnia did not affect the [Mg2+] in CSF in either group.

Net K+ uptake by wild-type Neurospora shows a complex dependence on the extracellular pH. From pH 4 to 6, uptake follows standard Michaelis Menten kinetics as a function of external potassium but shifts to sigmoid kinetics at pH 8. Net flux measurements have now been carried out on mutant strain R2449, isolated by virtue of an abnormally high potassium requirement for growth, and previously shown to have an elevated K 1 2 for steady-state K+ exchange at pH 5.8. The results indicate that R2449 is abnormal in net K+ uptake at pH 4, 5.8 and 8, consistent with the notion that a single cation transport system exists in Neurospora, capable of functioning over the entire pH range. Two models are presented, an allosteric model and a 2-site model, which can account quantitatively for the shift from Michaelis-Menten kinetics at low pH to sigmoid kinetics at high pH and for the defect in R2449. Further experiments will be needed to distinguish between the models.

The sodium, potassium, and magnesium ion contents of Serratia marcescens and those of its salt-tolerant relative, S. marinoruba , were determined by atomic-absorption spectrometry. The intracellular K + and Mg 2+ contents of both microorganisms were found to be dependent on the ionic strength of the growth or suspending medium. The Mg 2+ content of S. marinoruba was generally greater than that of S. marcescens . The Na + content of the cells was normally low and did not increase as the cells aged or when the cells were grown in media of high ionic strength. The transport of K + by resting cells suspended in hypertonic solution was studied by chemical and light-scattering techniques and was found to be more rapid in S. marcescens than in S. marinorubra . The slower rate of K + transport in S. marinorubra is probably related to the lower glycogen reserves found in resting cells of this microorganism. K + transport was found to have a p H optimum of 5.5 to 6.1 for S. marcescens , and the K m for K + was approximately 1.6 m m . Na + and Mg 2+ were not taken up by the cells, although the presence of Mg 2+ tended to decrease rates of K + uptake. Tris-(hydroxymethyl)aminomethane, routinely used for resuspending the cells, was apparently taken up by the cells at p H >7.

A transition in the temperature dependences of Ca2+ accumulation and ATPase activity occurs at 20 ° C in Sarcoplasmic reticulum membranes. The transition is characterized by an abrupt change in the activation energies for the cation transport process and the associated enzyme activities. The difference in activation energies below and above 20 °C appears to be due to changes in the entropy of activation rather than in the free energy of activation. Also, the temperature dependences of spectral parameters of lipophilic spin-labeled probes and protein-bound spin labels exhibit different behaviors on either side of this temperature. Above 20 °C the lipid matrix probed by the labels exhibits a large increase in molecular motion and a decrease in the apparent ordering of lipid alkyl chains. In addition, labels covalently bound to enzymic reactive sites indicate that the motion of protein side-chains is sensitive to this transition. The results are consistent with an order-disorder transition involving the lipid alkyl chains of the Sarcoplasmic membrane, and with a model in which molecular motion, Ca2+ transport and enzyme activity are limited by local viscosity of hydrophobic regions at temperatures below the transition. Another modification of the Sarcoplasmic reticulum membrane occurs between 37 and 40 °C. It appears that at this temperature the processes governing Ca2+ accumulation and ATPase activity are uncoupled, and Ca2+ accumulation is inhibited, while ATPase activity and passive Ca2+ efflux proceed at rapid rates. Parallel transitions of spectroscopic parameters originating from spin labels, covalently bound to the Sarcoplasmic reticulum ATPase, indicate that the uncoupling is due to a thermally-induced protein conformational change.

1. Aerobic lactic acid production in ascites tumor cells was not inhibited by rutamycin, indicating that the ADP and Pi required to sustain glycolysis were not supplied by mitochondrial ATPase. In contrast, the marked stimulation of lactic acid production induced by dinitrophenol was eliminated by rutamycin. 2. Parallel studies on the inhibition of lactic acid production and monovalent cation transport by ouabain revealed a close relationship between the two processes. About 1 mole of lactic acid was formed per mole of monovalent cation translocated. 3. The depression of glycolysis by ouabain was shown to result from the inhibition of (Na+, K+)-ATPase rather than from a lack of K+ as indicated by the high rate of aerobic glycolysis induced by dinitrophenol in the presence of ouabain. In the absence of dinitrophenol both Na+ and K+ were required for optimal glycolysis. 4. Evidence for a sodium-linked transport of Pi was obtained. 5. Dextran sulfate and other related compounds were found to have a profound effect on the permeability properties of ascites tumor cells. Glycolysis was markedly inhibited by dextran sulfate, and loss of intracellular ions, adenine nucleotides, and protein was demonstrated. In the presence of excess adenine nucleotides and Pi in the medium, dextran sulfate did not inhibit; in fact, it consistently stimulated glycolysis. It was shown that under these conditions AMP entered the cells and was converted into ATP. 6. The regulation of glycolysis in tumor cells by energy-linked ion transport is discussed.

Replica and transmission electron microscopy were used to study the ordered topography of the amorphous oxide film on electropolished aluminum. The symmetry, periodicity, and amplitude of the oxide film thickness variation constituting the regular topography were investigated as a function of substrate orientation. The results are consistent with previously reported data and have enabled the dislocation model proposed by Doherty and Davis for the oxide—aluminum interface to be amplified and the nature of the interface to be more precisely defined. The model postulates that the first oxide to form has long‐range lattice symmetry identical to that of the substrate, resulting in a regular network of interfacial dislocations. The amorphous oxide subsequently grown by cation transport is thinnest over the dislocations, as the interface is the rate‐controlling barrier. No correlation was observed between the existence of ordered topography and epitaxy of the high‐temperature crystalline oxide which nucleates heterogeneously on the ordered oxide surface originally in contact with the metal. It is concluded that, for the range of orientations over which ordered topography exists, the dislocation cores are wide, i.e., almost a pure vernier. The observed defects in the long‐range order of the various topographies may be accounted for by corresponding defects in the ordered oxide adjacent to the interface. The dependence of the existence and periodicity of the topographies on electropolishing temperature and voltage was investigated and may be explained by the difference in thermal expansion of the substrate and the ordered oxide at the interface. It is concluded from these data that the lattice parameter of the ordered overgrowth is 6.0% larger than that of the substrate, and that, when the first layer of oxide is formed, the temperature of the metal surface may be considerably greater than the electrolyte temperature. The absence of ordered topography for surface treatments other than electropolishing suggests that the minimum surface temperature for the formation of interfacial dislocations is greater than room temperature.

The unidirectional fluxes of Na+ and Cl- were measured across the isolated gastric mucosa of the bullfrog (R. catesbiana). The addition of strophanthidin, a cardiac aglycone, resulted in marked reductions of the spontaneous potential and short-circuit current. Associated with these changes, the isolated gastric mucosa ceased secreting chloride and hydrogen ion. Although the active component of chloride transfer was inhibited, the exchange diffusion component seemed to increase. No significant changes in membrane conductance or sodium flux were noted. Possible mechanisms of strophanthidin inhibition were discussed in view of its effect on chloride transport across the gastric mucosa and on sodium and potassium transfer in other tissues. It was concluded that the cardiac glycosides may not be specific inhibitors of sodium and potassium transport. This non-specific inhibition suggests that active chloride transport is affected by strophanthidin directly and/or anion secretion is dependent upon normal functioning of cation transport systems in the tissue.

The resuspension of K-poor, Na-rich stationary phase E. coli in fresh medium at pH 7.0 results in a rapid uptake of K and extrusion of Na by the cells. In all experiments net K uptake exceeded net Na extrusion. An investigation of the uptake of glucose, PO4, and Mg and the secretion of H by these cells indicates that the excess K uptake is not balanced by the simultaneous uptake of anions but must be accompanied by the extrusion of cations from the cell. The kinetics of net K uptake are consistent with the existence of two parallel influx processes. The first is rapid, of brief duration, and accounts for approximately 60 per cent of the total net K uptake. This process is a function of the extracellular K concentration, is inhibited in acid media, and appears to be a 1 for 1 exchange of extracellular K for intracellular H. The second influx process has a half-time of approximately 12 minutes, and is not affected by acid media. This process is a function of the intracellular Na concentration, is dependent upon the presence of K in the medium, and may be ascribed to a 1 for 1 exchange of extracellular K for intracellular Na.

The effects of the general anaesthetics ether and chloroform on the active cation transport in human erythrocytes are reported. The rate of active K + and Na + transport is enhanced by the presence of anaesthetics. The level of activation of the Na + pump depends on the internal Na + and the external K + concentrations and by varying these concentrations it was shown that the anaesthetics act by increasing the degree of activation of the active transport process. Anaesthetics do not affect the maximum possible pump rate.

studied in detail (Deul and McIlwain, 1961; Schwartz, Bachelard, and McI1wain, 1962; Skou, 1962; Aldridge, 1962; Jirnefelt, 1962, 1964; Whittam and Blond, 1964; Tanaka and Strickland, 1965; Albers, Arnaiz, and De Robertis, 1965; Davrainville and Gayet, 1966; Fujita et al., 1966; Palmer, Lasseter, and Melvin, 1966; Rendina, 1966; Robinson, 1967; Nakamaru, Kosakai, and Konishi, 1967). The two enzyme fractions may be differentially extracted (Nakao et al., 1963; Nakao et al., 1965) and so may represent separate enzyme systems (Bowler and Duncan, 1967). We have recently presented a detailed study of the effect of temperature on these two ATPase components from a rat brain microsomal preparation (Bowler and Duncan, 1968b). Arrhenius i plots showed that the two components had markedly differing temperature sensitivities and, further, that the enzyme activity of the Na+K+-Mg2+ ATPase was completely suppressed at temperatures below 5 C. However, in the poikilothermic frog, which will live and function normally at such temperatures, the Arrhenius t plot of the Na+-K+-Mg2+ ATPase of a brain microsomal preparation shows that enzyme activity continues at 1 C (Bowler and Duncan, 1968a). These results raise the question of the temperature sensitivity of the Na+K+-Mg2+ ATPase of the plasma membranes of hibernating mammals whose body temperature falls below 5 C (Kayser, 1961). If active cation transport is to continue during hibernation, enzyme activity of the Na+-K+-Mg2+ ATPase must not be supressed at these low temperatures. There appear to be two possible alternatives. Either hibernating mammals have a Na+-K+-Mg2+ ATPase whose temperature sensitivity resembles that of the poikilothermic frog, or, during preparation for hibernation, this enzyme is converted from the typical mammalian pattern into one in which enzyme activity continues below 5 C. In the present experiments the temperature sensitivity of the ATPases of a microsomal preparation from the brains of hedgehogs, using

In order to determine the capacity of renal cells to transport actively electrolytes at 0 C, separated rabbit renal tubules were incubated at this temperature and the effects of various inhibitors on Na, K, Cl, and water content and the fluxes of Na and K were measured. In contrast to previous studies with kidney slices, these observations indicate that cell K is completely exchangeable at 0 C and significant active transport persists. Thus, at 0 C as at higher temperatures, anoxia, strophanthidin, 2,4-DNP, and cyanide reduce the transcellular concentration gradients for Na, K, and Cl and cause the tubules to swell. In the presence of strophanthidin, both influx and efflux of K are slowed. It is concluded that at 0 C, as at higher temperatures, active cation transport by renal tubule cells accounts for the maintenance of transcellular electrolyte concentration gradients and limits colloid osmotic swelling.

Summary 1. There are 7 well-established red-cell antigen (blood group) loci. The R-O system has 3 phenotypes, R, O and i, identified by the ‘naturally occurring’ antibodies, anti-R and anti-O. The R and O substances are also present in soluble form in some body secretions. The expression of R and O is controlled by a dominant gene I, epistatic in effect, at an independent locus from that of R. The systems, A, C, M-L, B, D and X-2 are identified by means of ‘immune-type’ antibodies, and several of the loci have multiple alleles. An isoenzymic form of serum alkaline phosphatase is associated with the R-O system. The frequency for the genes at the various loci has been determined in a limited number of breeds. 2. Some sheep red cells have high K+ and low Na+ concentrations (HK type, or Key), others have low K+ and high Na+ concentrations (LK type or Kea). Two other rare forms exist; Key type which is HK but with lower than normal K+ values, and Kep type which has approximately equal Na+ and K+ concentrations. The red cells of foetuses and newborn lambs have high K+ levels irrespective of their potassium genotype. HK cells have 3–4 times greater (Na+-K+)-activated ATPase activity, a 3–4 times increased rate of active K+ transport and a larger number of ouabain-binding sites than LK cells. Antigen M is present on homozygous HK and heterozygous LK red cells, and antigen L is present on homozygous and heterozygous LK red cells. Sensitization of LK cells with anti-L stimulates active K+ transport and ATPase activity and exposes a larger number of ouabain-binding sites in these cells. Anti-M has no effect. The red cells of newborn lambs only show weak L and M antigen activity. It is postulated that L antigen inhibits cation transport in LK cells by masking the pump sites on the membrane. Immature red cells in LK-type sheep have a high rate of active K+ transport and yet have L antigen present. No satisfactory explanation for this has yet been advanced. There is no conclusive evidence that the potassium types have any significance from the point of view of adaptation or sheep breeding. The potassium-gene frequencies are known for a large number of breeds. 3. Two allelic genes, Hb4 and HbB control 3 haemoglobin phenotypes, A, AB, and B. Foetal haemoglobin (HbF) is present in foetuses and newborn lambs. Sheep with HbA also synthesize small amounts of another haemoglobin (HbC) and under conditions of severe anaemia, synthesis of HbC takes over from that of HbA. No change in HbB is observed in anaemia. A rare haemoglobin (HbD) has been found in 3 Yugoslavian sheep. Hbs A, B, C and F differ in their physicochemical properties; they share the same alpha chains but their non-alpha chains differ in a number of amino acids. HbD differs from HbA in one amino acid in the alpha chain. Certain genetic aspects are discussed. There is some evidence that sheep with HbA are less fertile than those with HbB. The gene frequencies for Hb are known for a large number of breeds. 4. Two isoenzymic forms of carbonic anhydrase are found in red-cell lysates and these are controlled by a pair of allelic autosomal genes, producing 3 phenotypes, CAF, CAFS and CAS. Only a few breeds have been studied but CAF is apparently quite rare. 5. An unidentified protein, designated ‘X’ is present in electrophoretic separations of haemolysates from some sheep. Its presence is dominant to its absence. Polymorphism at this locus is present in all breeds so far studied. 6. A deficiency of reduced glutathione (GSH) in red cells is found in some sheep and is inherited as an autosomal recessive disorder. Sheep with this deficiency have lower red-cell K+ and Naf concentrations than normal and it is suggested that the HK GSH-deficient sheep may be Ked type sheep. This deficiency has so far only been found with certainty in one breed of sheep. 7. In sheep twin chimeras, admixture of red-cell antigens, haemoglobin and ‘X’ protein types has been found. Various aspects of chimerism, which occurs only rarely in sheep, are discussed. 8. The significance of the genetic variation is discussed in the light of the physiology and immunology of the red cell and of the sheep itself.

A wide study, extending even to analysis in homogenates and subcellular fractions, is now being made in order to elucidate the exact mechanism of linkage between active cation transport and metabolism. Many wonderful achievements and hypotheses seem to have been published; nevertheless, the exact mechanism is still obscure. The fact that separated mammalian cerebral tissues, suitably maintained in vitro, show remarkable metabolic responses to adequate electrical stimulation has been explored mainly by McIlwain and his colleagues. Their comprehensive studies concerning this phenomenon have led to many valuable findings, and that concerning ganglioside may be especially excellent (1-3). However, at the present stage, it cannot be said that the biochemical origin of these metabolic responses and its physiological roles are clear. In addition, except for their achievement, other achievements along this line are still very few and some differences in the results also can be observed. From the point of view of the present studies concerning active transport and metabolism, there is even more of a necessity for studies along this line. Therefore, we have taken a somewhat different experimental procedure to manifest the role of external sodium in metabolism of separated mammalian cerebral cortical tissue in both the presence and absence of electrical stimulation in correlation to the effects of potassium and calcium.

N,N′-Dicyclohexylcarbodiimide (DCCD) was found to be a potent inhibitor of the membrane-bound ATPase of Streptococcus faecalis but did not inhibit the solubilized form of the enzyme. Inhibited membrane-bound ATPase was reactivated by releasing the enzyme from the membrane. Conversely, sensitivity to DCCD was restored by reconstituting the ATPase-membrane complex from solubilized ATPase and depleted membranes. The results indicate that DCCD reacts covalently with a membrane component and inhibits the enzyme indirectly, perhaps by means of a transmitted effect on its conformation. When added to intact cells, DCCD reduced the rate of glycolysis yet the ATP content of the cells was as high in the presence of the inhibitor as in its absence. DCCD strongly inhibited the degradation of ATP which ensued when glycolysis was stopped, either by removal of glucose or by addition of iodoacetate. The results suggest that DCCD inhibits ATPase in vivo, just as it does in isolated membrane preparations. The inhibition of over-all glycolysis may be secondary to the inhibition of ATP degradation. DCCD also inhibited a number of energy-dependent transport processes, including the accumulation of K+ by exchange for H+ and Na+ and the uptake of phosphate and alanine. Inhibition was observed whether glucose or arginine served as energy source. The findings are consistent with the hypothesis that the membrane-bound ATPase of S. faecalis mediates energy transductions involved in membrane transport; inhibition of transport by DCCD would be a consequence of inhibition of the ATPase. Alternatively, DCCD may bind to sites on the membrane to inhibit indirectly both transport and ATP hydrolysis.

Measurement of cellular K and Na concentrations in growing Escherichia coli indicates that the osmololity of the medium is a major determinant of the cell K concentration. In contrast, the cell Na concentration is independent of the medium osmolality and is largely dependent on the Na concentration of the medium. Sudden changes in the osmolality of the medium lead to rapid changes in K content. Washing the cells with solutions of lower osmolality results in a very rapid loss of K, which is greater in more dilute and in cold solutions. A sudden increase in the osmolality of the growth medium produces a rapid uptake of K by a mechanism whose rate is a saturable function of the K concentration of the medium and which appears to involve an exchange of K for cellular H.

Methyl phenyldiazenecarboxylate (azoester) has been widely used for the intracellular oxidation of glutathione in the red blood cell. However, the reactions of azoester are complex, and azoester is unstable in aqueous solution. Using 86 Rb we have studied the effects of azoester on the cation transport and permeability of rat lenses. Lenses were exposed to both freshly prepared solutions of azoester (rapid delivery) and solutions which were allowed to stand for 35 min before lens exposure (delayed delivery). Rapid delivery of azoester effects a rapid oxidation of lens glutathione. In the presence of glucose this effect is diminished, presumably because the glutathione level is maintained by the NADPH generated by the pentose shunt mechanism. In contrast, delayed delivery of azoester does not affect lens glutathione content. Lenses pretreated with azoester (rapid delivery) without glucose for 15 min manifest a 68–73% decrease in 86 Rb uptake. Delayed delivery of azoester without glucose causes a similar decrease in 86 Rb uptake. Glucose medium improves the 86 Rb uptake of similarly treated lenses (both rapid and delayed delivery), but does not restore the uptake to normal values. In addition, azoester-treated lenses (rapid delivery) incubated without glucose manifest a 29% increase in 86 Rb run-out. In contrast, delayed delivery of azoester without glucose causes a 32% decrease in 86 Rb run-out. In the presence of glucose both rapid and delayed delivery of azoester effect a normal rate of 86 Rb run-out. Both rapid and delayed delivery of azoester result in a marked decrease in lens ATP content.

Previous studies have suggested that malaria induces changes in erythrocytic membrane permeability and susceptibility to osmotic lysis. The present study investigated erythrocytic transport of sodium with cells from Rhesus monkeys infected with Plasmodium knowlesi. Red blood cell sodium concentration was significantly elevated in 37 parasitized animals (21.8+/-1.2 mM; mean +/-SEM), as compared to 23 control animals (10.0+/-0.38 mM). The cellular sodium increased with the density of parasitemia and the cellular potassium decreased in proportion to the elevation of sodium. Nonparasitized as well as parasitized erythrocytes possessed this abnormality of cation metabolism. Effective chloroquine therapy reversed the changes over a period of 4 days. Active sodium outflux rate constants were depressed in animals with malaria (0.202+/-0.012), as compared to controls (0.325+/-0.027). Passive sodium influx rate constants were higher in infected monkeys (0.028+/-0.002) than in control animals (0.019+/-0.002). The cross incubation of malarial plasma with normal red blood cells induced a 22% diminution in active sodium outflux but no changes were observed in sodium influx. It is concluded that malaria alters erythrocytic sodium transport in all erythrocytes. The elevated intracellular sodium concentration is the net result of decreased sodium outflux and increased sodium influx. The plasmodium organism or the affected host may produce a circulating substance that is deleterious to erythrocytic membrane cation transport.

The use of the fluorescent chelate probe, chlorotetracycline, in mitochondria is described. The probe shows a high fluorescence in the presence of mitochondria which may be ascribed to binding of the probe to membrane-associated Ca(++) and Mg(++). The fluorescence excitation and emission spectra are diagnostic of binding of the probe to Ca(++) in coupled mitochondria and Mg(++) in uncoupled mitochondria. The fluorescence polarization spectra are diagnostic of the cations having a moderately high mobility in the membrane environment. The effects of exogenous EDTA and of endogenous Mn(++) indicate that the probe is primarily visualizing actively accumulated Ca(++) on the inner surface of the inner membrane. By employing the Ca(++) transport inhibitor, Tb(+++), the fluorescence changes associated with metabolic alterations are shown to arise partly from cation transport and partly through alterations in the binding properties of the inner surface of the membrane. Chlorotetracycline is a probe for divalent cations associated with the membrane and is of general utility in the study of cation migrations in cellular and subcellular systems.

Cation transport in steam-grown SiO2 has been investigated. Variations in the migration kinetics and mobility indicate that trapping in the SiO2 surface region dominates ionic motion towards the Si–SiO2 interface. A simple model has been proposed to explain the observed differences in migration kinetics. Es wird der Kationentransport in aus der Dampfphase gezogenem SiO2 untersucht. Anderungen in der Wanderungskinetik und der Beweglichkeit zeigen, das das Anhaften an der SiO2-Oberflache uber die Ionenbewegung zur Si–SiO2-Zwischenflache dominiert. Es wird ein einfaches Modell zur Erklarung der beobachteten Unterschiede der Wanderungskinetik vorgeschlagen.

Mechanisms which determine sodium and potassium content and volume of rat thymic and human chronic lymphocytic leukemia (CLL) lymphocytes have been studied. The deleterious effect of cell isolation on monovalent cation content was proven by comparing thymus sodium and potassium concentration to that of thymocytes prepared from autologous hemithymus. In vivo distribution ratios of sodium-24 and potassium-42 between thymus water and plasma water were very similar to the distribution ratios of non-radioactive isotopes (sodium-23 and potassium-39). The similar lymphocyte: thymocyte ratio of (a) cell volume (1.48), (b) cell sodium plus potassium (1.47) and (c) cell water (1.50) demonstrated the close correlation of lymphocyte volume with monovalent cation content and water content. Steady-state CLL lymphocyte sodium (32 ± 1.9 mM) and potassium (131 ± 5.1 mM) and thymocyte sodium (31 ± 1.2 mM) and potassium (136 ± 3.9 mM) were similar; however, these steady-state levels were maintained by quantitatively different membrane functions. Radiopotassium and radiosodium uptake by thymocytes was more rapid than by CLL lymphocytes. Ouabain-sensitive potassium influx was 2.4 times greater in thymic (8.70 ± 2.28 mmoles/cm2/min × 10−8) than in CLL (3.24 ± 0.45 mmoles/cm2/min × 10−8) lymphocytes. Potassium exodus was also slower in CLL lymphocytes as compared to thymocytes. Ouabain-sensitive sodium accumulation and ouabain-insensitive sodium accumulation were also slower in CLL lymphocytes than in rat thymocytes. Half-maximal ouabain inhibition of sodium entry was 7.5 × 10−3M in thymic and CLL lymphocytes. The inhibitory effect of ouabain on sodium and potassium transport was easily reversible. Oligomycin inhibited ouabain-sensitive potassium accumulation in both lymphocyte types. Four lines of evidence indicate the presence in the lymphocyte of a system of leaks and pumps, the latter subserved by a ouabain and oligomycin-sensitive (sodium-potassium) ATPase: (a) steady-state monovalent cation gradient (K ∼ 20:1, Na ∼ 5:1), (b) the inability to maintain normal sodium and potassium gradients at cold temperature and in the presence of ouabain, (c) the effect of ouabain and oligomycin on active potassium influx and (d) the restitution of steady-state sodium and potassium concentration after cell isolation, ouabain treatment and cold exposure. CLL lymphocytes as compared to rat thymocytes have a decreased rate of ouabain-insensitive sodium uptake and potassium exodus requiring a reduced rate of active sodium extrusion and potassium accumulation to maintain steady-state cation content. Ouabain-sensitive ATPase is difficult to locate in lymphocytes in vitro possibly because it comprises a very small proportion of membrane ATPase since magnesium activated ecto-ATPase in intact lymphocytes is 1500 to 2500 times that of the intact erythrocyte. The inhibition by ouabain of blast transformation, mitosis, amino acid accumulation and nucleic acid synthesis in vitro, may reflect the importance of ouabain-sensitive ATPase and monovalent cation transport in the function of lymphoid cells.

— Adrenalectomy in the rat was shown to lower seizure threshold measured with the volatile convulsant hexafluorodiethyl ether. Although the expected hyponatraemia and hyperkalaemia were demonstrated in the adrenalectomized rats, there was no associated alteration in Na-K stimulated Mg-ATPase activity either in the whole rat brain or in a microsomal fraction. These results in brain tissue are contrasted with the marked decrease in this enzyme that occurs in kidney tissue of the rat following adrenalectomy. It was suggested that the activity of the enzyme system responsible for active cation transport responds directly in proportion to the work of active transport required by the individual tissue.

Cultured chick embryo fibroblasts accumulate Ca2+ in the presence of Mg2+ and ATP. The uptake is highly specific; Mn2+ inhibits it, and other nucleotide triphosphates are without effect. The presence of oxalate, Na+ and/or K+ increases the amount of accumulated Ca2+. The cation transport is inhibited by mersalate, oligomycin, and hydroxylamine. The capacity of fibroblasts to energetically transport and/or bind Ca2+ resides in components of the plasma membrane. The function of this Ca2+ uptake may be to control motility in ameboid-like cells.

Nitrate salts resulted in a large stimulation of exudation from detopped tobacco and also increased cation transport to the exudate, but only if nutrient solutions applied previously to detopping had become depleted in salts. Such plants were not necessarily nitrogen deficient. Pregrowth in ammonium nitrogen resulted in the same effect as pregrowing with low amount of salts. Pregrowth with high levels of salt other than nitrate, however had the same effect as pregrowing with a high level of nitrate. There was no evidence that synthesis of nitrate reductase was necessary for the stimulation of exudation by nitrate. The nitrate response was usually apparent within 1 h. Glucose added with nitrate at 10°C resulted in prolonged and increased exudation for 32 days following detopping. Mg (NO3)2 as a single salt was toxic in terms of exudation production. Only one cycle of response to nitrate on exudation could be obtained from detopped plants. A cycle could be obtained when nitrate was added many days after detopping if nitrate had not been added previously since detopping.

Studies were performed on Na and K transport by red blood cells of the freshwater turtle under anaerobic and aerobic conditions. Although it had previously been assumed that cation transport in turtle red blood cells was dependent on respiration, the present data show greater Na efflux rates in N2 than in O2. However, ouabain inhibited Na transport by the same amount quantitatively in O2 and N2 gas phases. Thus there was no difference in ouabain-sensitive or "pump" Na transport rates. Na influx rates were higher in nitrogen than in air and potassium influx rates were not significantly different under aerobic and anaerobic conditions. Moreover in the absence of sodium in the bathing medium no difference between air and nitrogen could be discovered. Finally with ethacrynic acid plus ouabain there was an additional decrease in Na efflux but there was a persisting difference between air and nitrogen. These studies do not rule out the existence of a ouabain-insensitive ethacrynic acid-inhibitable flux; however, they suggest that at least part of the activation of Na efflux observed in N2 was due to increased exchange diffusion.

The effects of ion concentration, pH, and presence of competing ions on the sodium and potassium binding properties of rat liver cell microsomes were studied. Typical adsorption isotherms were obtained in the concentration dependence studies, with saturation being reached when 1.2 to 1.4 m.eq. cations were retained per gm. of microsome Kjeldahl nitrogen. The retention was shown to be due to a binding to specific sites rather than to a trapping of the cations. The binding showed a sharp pH dependence in the range 6.0 to 7.5. The presence of one cation depressed the binding of the other, indicating that Na+ and K+ as well as H+ ions compete for the same sites. Potassium was bound slightly more strongly than sodium, while hydrogen was bound about 105 times more strongly than either. Calculations show that the binding follows the simple mass law. Similarities between adsorption by microsomes and adsorption by synthetic cation exchange resins are discussed and compared to some of the characteristics of electrolyte behavior in living systems. A possible ion exchange elution, active cation transport mechanism is suggested, involving the preferential elution of Na+ out of the cell by H+ ions produced by metabolism.

The enzyme Na-K-activated ATPase, which is closely related to the active transport system of sodium and potassium in human erythrocytes, was found to occur in the epithelium of the lens of the cat, calf, and rabbit in relatively high activities. No significant activity could be detected in anterior and posterior capsule, cortex, and nucleus. Various properties of the enzyme system were determined and compared with corresponding properties of the transport system, reported by other investigators. Both the enzyme and the transport system were found to be located in the lens epithelium. They were both inhibited by ouabain, and there was good agreement between the half-maximal inhibition concentrations for the two systems in both calf and rabbit lens, when determined in media with the same K level. In the enzyme system as well as the transport system, Rb could replace K. In the rabbit lens the pH optimum of the enzyme was 7.3 as compared to 7.5 for the Rb uptake. The temperature coefficients of the two systems were 2.4 and 2, respectively. The ratios of equivalents of cation transported to moles of ATP hydrolyzed by the epithelial Na-K ATPase ranged from 2.51 to 4.15 (av. 3.07) for the three cations actively transported in calf and rabbit lens. It was concluded that the epithelial Na-K ATPase and the active cation-transport system of the lens are identical or very closely related.

The rates of active transport of calcium in vitro by everted gut-sacs prepared from the proximal small intestine of the rat have been quantified and expressed in absolute units. A maximal rate of transport has been measured. The bulk of the calcium transferred to the serosal surface of the gut-sac is ionized calcium, suggesting that the process is an active cation transport mechanism. The active transfer is relatively specific for Ca++, and no significant accumulation of Mg++, Sr++, Ba++ or K+ in the fluid bathing the serosal surface could be demonstrated. The active transport of calcium in vitro is greater with gut-sacs from growing than from older rats, and it is greater with gut-sacs from pregnant than from nonpregnant rats. The results suggest that the active transport mechanism can increase the intestinal absorption of calcium facultatively to meet the needs of the organism.

Freshly prepared duck erythrocytes, incubated either in plasma or an isotonic synthetic medium containing norepinephrine ([K] of both media approximately 2.5 mM), maintain water and electrolyte composition in the steady state (upper steady state) for at least 90 min. If incubated in the synthetic medium without norepinephrine or in plasma to which a beta-adrenergic blocking agent (propranolol) is added, the cells lose both water and electrolyte (predominantly KCl) until a new steady state is reached (lower steady state). Reaccumulation of water and electrolyte from isotonic solutions toward the upper steady-state levels requires the addition of norepinephrine and KCl. Reaccumulation is maximal when the concentration of K and norepinephrine in the medium is 15 mM and 10(-7)M, respectively. Dibutyryl cyclic-AMP (10(-2)M) mimics norepinephrine in lower steady-state cells. Although an analogous effect in upper steady-state cells was not established with certainty, it is proposed that the catecholamine-induced net changes in water and electrolyte movement in duck erythrocytes are a consequence of stimulation of the activity of a membrane-bound adenyl cyclase system.

1. 1. Intracellular concentrations of sodium and potassium were measured in the excised gill of Carcinus maenas in media of different ionic compositions as a function of salinity. 2. 2. The increase in respiration observed with decrease in salinity was independent of the activity of the conventional Na+/K+ linked pump. 3. 3. No evidence was obtained to suggest that the energy for the Na+/K+ pump was supplied by glycolysis. 4. 4. Evidence is presented which shows that the ionic composition of the medium has a pronounced effect on respiration, lactic acid and carbon dioxide production and the formation of glycogen and glucose.