Amino acid stimulation of ATP cleavage by two Ehrlich cell membrane preparations in the presence of ouabain

Two membrane fractions prepared from the Ehrlich ascites-tumor cell show non-identical stimulatory responses to certain amino acids in their Mg²⁺-dependent activity to cleave ATP, despite the presence of ouabain and the absence of Na⁺ or K⁺. The first of these, previously described, shows little ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase activity, and is characteristically stimulated by the presence of certain diamino acids with low $\text{pK}{}_2$, and at pH values suggesting that the cationic forms of these amino acids are effective. The evidence indicates that these effects are not obtained through occupation of the kinetically discernible receptor site serving characteristically for the uphill transport of these amino acids into the Ehrlich cell. The second membrane preparation was purified with the goal of concentrating the ($\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}$)-ATPase activity. It also is stimulated by the model diamino acid, 4-amino-1-methylpiperidine-4-carboxylic acid, and several ordinary amino acids. The diamino acids were most effective at pH values where the neutral zwitterionic forms might be responsible. Among the optically active amino acids tested, the effects of ornithine and leucine were substantially stronger for the l than for d isomers. The list of stimulatory amino acids again corresponds poorly to any single transport system, although the possibility was not excluded that stimulation might occur for both preparations by occupation of a membrane site which ordinarily is kinetically silent in the transport sequence. The high sensitivity to deoxycholate and to dicyclohexylcarbodiimide of the hydrolytic activity produced by the presence of l-ornithine and 4-amino-1-methyl-piperidine-4-carboxylic acid suggests that the stimulatory effect is not merely a general intensification of the background Mg⁺-dependent hydrolytic activity.     

Cation/proton antiport systems in Escherichia coli.

Three distinct systems which function as proton/cation antiports have been identified in $\text{E.}\text{coli}$ by the ability of the ions to dissipate the ΔpH component of the protonmotive force in everted vesicles. System I exchanges H⁺ for K⁺, Rb⁺ or Na⁺; System II has Na⁺ and Li⁺ as substrates; and System III catalyzes proton exchange for Ca²⁺, Mn²⁺ or Sr²⁺.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$ and $\text{K}{}_{\mathrm{m}}$ of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the $\text{K}{}_{\mathrm{m}}$ for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the $\text{K}{}_{\mathrm{m}}$ for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high $\text{K}{}_{\mathrm{m}}$ and is not additive with the Na⁺-dependent flux.     

Crystallographic study of plastocyanins

Crystals of plastocyanins from pea and corn leaves have been obtained. Both are suitable for X-ray structure analysis with a resolution up to 1.8 Å. The crystal form of plastocyanin from pea leaves belongs to the space group P2₁2₁2₁ with unit cell dimensions: $\text{a = 49.0}\text{A}\text{?}\text{, b = 53.3}\text{A}\text{?}\text{, c = 82.6}\text{A}\text{?}$. The assumed number of protein molecules per asymmetric unit of the unit cell is two. Crystals of the oxidized (Cu²⁺) and reduced (Cu⁺) forms are isomorphic. No essential differences in spot intensities for the main zone with a resolution of 3 Å were revealed. The crystal form of plastocyanin from corn leaves belongs to the space group P1 with unit cell parameters: $\text{a = 24.8}\text{A}\text{?}\text{, b = 30.0}\text{A}\text{?}\text{, c = 58.5}\text{A}\text{?}$ and α = 96° 10′, β = 87°08′, γ = 78°40′. The assumed number of protein molecules per asymmetric unit is two.     

Temperature-dependent relationship between K+ influx,Mg2+-ATPase activity,transmembrane potential and membrane lipid composition in mycoplasma

The temperature-dependent relationship between K⁺ active influx, Mg²⁺-ATPase activity, transmembrane potential (ΔΨ) and the membrane lipid composition has been investigated in mycoplasma PG3. Native organisms were grown in a medium containing 10 μg/ml cholesterol and either oleic plus palmitic (chol (+), O + P) or elaidic (chol (+), E) acids. Adapted cells were grown in a medium free of exogenous cholesterol and supplemented with elaidic acid (chol (?), E).Arrhenius plots of ⁴²K⁺ active influx gave a linear relationship for (chol (+), O + P) cells ($\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?9}\text{kcal}$). On the other hand, when oleic plus palmitic acids are replaced by elaidic acid, an upward discontinuity appears between 28 and 30°C, which is associated with a large increase in the apparent activation energy of the process ($\text{t > 30°}\text{C}\text{, E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?24}\text{kcal}\text{; t < 30°}\text{C}\text{, E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?40}\text{kcal}$).Finally, a biphasic response with a break at approx. 23°C ($\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?7}\text{kcal}\text{, t > 23°}\text{C}$; $\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?44}\text{kcal}\text{, t < 23°}\text{C}$) is observed for (chol (?), E) organisms. From the lack of correspondence between these effects on the K⁺ influx and the temperature dependence of both the Mg²⁺-ATPase activity and ΔΨ, it is suggested that changes in the membrane lipid composition affect the K⁺ transport at the level of the K⁺ carrier itself.Differential scanning calorimetry, steady-state fluorescence polarization of diphenylhexatriene and freeze-fracture electron microscopy experiments further suggest that the effect is largely due to modifications of the membrane microviscosity and that the K⁺ carrier is associated with the most fluid lipid species present in the membrane.     

Basal and potassium stimulated, calcium dependent, endorphins release from pituitary cells.

Mouse pituitary tumor cells grown in tissue culture release endorphins spontaneously to the culture medium. Depolarization of these cells by incubation with high K⁺ concentration (56 mM) increased 2–3 folds the release of endorphins. The K⁺ evoked release was Ca⁺⁺ dependent by that: $\text{a}$, removal of Ca⁺⁺ ions inhibited 90% of K⁺ stimulated release. $\text{b}$, ethyleneglycol-bis (β-aminoethyl ether) N,N′-tetraacetic acid (EGTA) inhibited release of endorphins in the presence of high K⁺ and Ca⁺⁺. It is suggested that dual regulatory system inhibit and/or stimulate $\text{in-vivo}$ release of endorphins from the pituitary glands.     

Electrically silent cotransport of Na+, K+ and Cl− in ehrlich cells

A cotransport system for Na⁺, K⁺ and Cl^? in Ehrlich cells is described. It is insensitive towards ouabain but specifically inhibited by furosemide and other ‘high ceiling’ diuretics at concentrations which do not affect other pathways of the ions concerned. As the furosemide-sensitive fluxes of these ions are not affected by changes in membrane potential, and as their complete inhibition by furosemide does not appreciably alter the membrane potential, they appear to be electrically silent. Application of the pulse-response methods in terms of irreversible thermodynamics reveals tight coupling between the furosemide-sensitive flows of Na⁺, K⁺ and Cl^? ($\text{q}$ close to unity for all three combinations) at a stoichiometry of 1 : 1 : 2. The site for each of the ions appears to be rather specific: K⁺ can be replaced by Rb⁺ but not by other cations tested whereas Cl^? can be poorly replaced by Br^? but not by NO₃^?, in contradistinction to the Cl^?-OH^? exchange system. The cotransport system appears to function in cell volume regulation as it tends to make the cell swell, thus counteracting the shrinking effect of the ouabain-sensitive (Na⁺, K⁺) pump.The experiments presented could not clarify whether the cotransport process is a primary or secondary active one; while incongruence between transport and conjugated driving force seems to indicate primary active transport, it is very unlikely that hydrolysis of ATP supplies energy for the transport process, since there is no stimulation of ATP turnover observable under operation of the cotransport system.     

Gibberellin biosynthesis in a cell-free system from immature seeds of Pisum sativum

A cell-free system from immature pea seeds converts ¹⁴C-labelled $\text{ent}$-kaurene to $\text{ent}$-kaurenol, $\text{ent}$-kaurenal, $\text{ent}$-kaurenoic acid, $\text{ent}$-7α-hydroxykaurenoic acid, and gibberellin A₁₂-aldehyde. The latter becomes converted further to 13-hydroxygibberellin A₁₂, gibberellin A44, gibberellin A₁₂-alcohol, and several unidentified products. Thus the biosynthesis of gibberellins $\text{via ent}$-kaurene is now established for a member of the $\text{Leguminosae}$. It is the first time that 13-hydroxylation of gibberellins has been observed in a cell-free system and that gibberellin A₁₂-alcohol has been obtained in any biological system.     

Analysis of two chloride requirements for sodium-dependent amino acid and glucose transport by intestinal brush-border membrane vesicles of fish

Intestinal brush border vesicles of a Mediterranean sea fish (Dicentrarchus labrax) were prepared using the Ca²⁺-sedimentation method. The transport of glucose, glycine and 2-aminoisobutyric acid is energized by an Na⁺ gradient ($\text{out > in}$). In addition, amino acid uptake requires Cl^? in the extravesicular medium (2-aminoisobutyric acid more than glycine). This Na⁺- and Cl^?-dependent uptake is electrogenic, since it can be stimulated by negative charges inside the vesicles. The specific Cl^? requirement of glycine and 2-aminoisobutyric acid transport is markedly influenced by pH, a change from 6.5 to 8.4 reducing the role played by Cl^?. In the presence of Cl^?, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 2-aminoisobutyric acid uptake is reduced and its $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ is enhanced. Cl^? affects also a non-saturable Na⁺-dependent component of this amino acid uptake. Amino acid transport is also increased by intravesicular Cl^? (2-aminoisobutyric acid less than glycine). This effect is more concerned with glucose uptake, which can be then multiplied by 2.3. A concentration gradient ($\text{in > out}$) as well as the presence of Na⁺ in the incubation medium seems to enter into this requirement. This intravesicular Cl^? effect is not influenced by pH between 6.5 and 8.4.     

Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell

The possible presence and properties of the Ca²⁺-dependent K⁺ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187+Ca²⁺, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K⁺ (balanced by the uptake of Na⁺) and a stimulation of both the influx and the efflux of ⁸⁶Rb. These effects were antagonized by quinine, a known inhibitor of the Ca²⁺-dependent K⁺ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca²⁺-dependent K⁺ channel whose characteristics are similar to those described in other cell systems.     

The Relation between Membrane Potential and the Transport Activity of Systems a and L in Plasma Membrane Vesicles of the Ehrlich Cell

Plasma membrane vesicles isolated from Ehrlich ascites tumor cells have been used to investigate the role of the transmembrane potential in the energetics of Systems A and L. As expected, Na⁺-dependent System A was responsive to changes in membrane potential. System L activity, as measured by transport of 2-aminonorbornane-2-carboxylic acid (BCH), was shown to be Na⁺-independent and was not altered by changes in the membrane potential. The combination of valinomycin and nigericin decreased accumulation of MeAIB but not that of BCH. The presence of nigericin alone caused a significant decrease in uptake by System A and a decrease in uptake by System L to a lesser degree. The inhibitory action of nigericin might reflect its ability to dissipate the Na⁺ gradient rather than an effect on K⁺ or H⁺ flows. The results indicate that modes of energization not produced through the transmembrane potential must account for any uphill operation of System L.     

Factors affecting the relative magnitudes of the ouabain-sensitive and the ouabian-insensitive fluxes of thallium ion in erythrocytes

A maximal rate of the ouabain-sensitive ²⁰⁴Tl influx in human erythrocytes can be attained at trace concentrations of Tl⁺ in Mg²⁺ isotonic media free of K⁺ and Na⁺. The maximal influx of Tl⁺ from isotonic Mg(NO₃)₂ at 20°C and pH 7.4 was $\text{0.45}\text{mM}\text{· 1}{}^{\mathrm{?1}}\text{· h}{}^{\mathrm{?1}}$ with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.025 mM. In contrast to the active influx of Tl⁺, the passive Tl⁺ fluxes were neither saturated nor influenced by external cations in the range of concentrations of Tl⁺ and K⁺ studied. The rate constants of Tl⁺ passive fluxes in human and cat erythrocytes can be related to pH by the equation $\text{log}\text{k}{}_{\mathrm{<mtext>in(out)</mtext>}}\text{= –A + B ·}\text{pH}$, where $\text{A}$ and $\text{B}$ are empirical constants for particular conditions. The apparent activation energy was 16 and 11 kcal/mol in sulphate and nitrate media, respectively. Tl⁺ and the alkali metal cations seem to overcome a common barrier in the erythrocyte membrane. Nevertheless, the rate of the passive penetration of Tl⁺ is about two orders of magnitude faster than those of K⁺ or Rb⁺. An extra non-Coulombic interaction between Tl⁺ and membrane ligands appears to be involved providing an accumulation of Tl⁺ somewhere in the vicinity of the membrane barrier and increasing the diffusion fluxes of Tl⁺ in both directions.     

Effect of N-ethylmaleimide on leucine transport in chang liver cells. I. Stimulatory effect on Na+-independent transport at low concentrations of leucine

Pretreatment of Chang liver cells with $\text{N-}\text{ethylmaleimide}$ (0.5 or 1 mM) stimulated Na⁺-independent uptake of leucine at low concentrations (?1 mM). The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the uptake of leucine measured in Na⁺-replete medium was completely blocked by the addition of $\text{b-2-}\text{aminobicyclo}\text{[2,2,1]}\text{heptane}\text{-2-}\text{carboxylate}$ (5 mM), which shows that the L system participates in the stimulation. The Na⁺-dependent uptake of glycine was depressed by $\text{N-}\text{ethylmaleimide}$ pretreatment. The stimulation of the Na⁺-independent component of leucine uptake continued for at least 30 min after $\text{N-}\text{ethylmaleimide}$ treatment, while the inhibition of glycine uptake was progressive with time and the Na⁺-dependent uptake of leucine became depressed later, after the treatment. It has been demonstrated that treatment of cells with $\text{N-}\text{ethylmaleimide}$ is capable of increasing the Na⁺-independent influx of leucine and at the same time slightly decreasing the efflux of it. These results suggest that $\text{N-}\text{ethylmaleimide}$ attacks the Na⁺-independent system of amino acid transport at the reactive SH groups(s) of relevant protein(s) in favor of specific activation of that system in this cell.     

Letter: Studies on histidinol dehydrogenase preliminary crystallographic data

Crystals of histidinol dehydrogenase (EC 1.1.1.23) from Salmonella typhimurium have been obtained in large size suitable for single crystal X-ray studies. The following crystal data were obtained on examination of a number of X-ray precession photographs; crystal system monoclinic, $\text{a = 149.6}\text{A}\text{?}$, $\text{b = 88.9}\text{A}\text{?}$, $\text{c = 105.6}\text{A}\text{?}$, β = 124.5 °, space group C2. The density of the crystal measured by flotation in bromobenzene/xylene mixture is 1.186 g/cm³. There is one dimer molecule of molecular weight 80,000 in one crystallographically asymmetric unit.     

Saturation behavior of ascites tumor cell chloride exchange in the presence of gluconate

Steady state Cl^? flux across the Ehrlich mouse ascites cell membrane was studied when gluconate replaced Cl^? in the external medium. Saturation behavior was observed; $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was 23.9 mM Cl^? and V was 758 μmol · g^?1 dry weight · h^?1. The cells lost K⁺, Cl^? and H₂O, consistent with relative impermeability to gluconate, and the Cl^? efflux rate coefficient was elevated. The results indicate that a major portion of Cl^? exchange occurs as a membrane transport process and suggest that the process is sensitive to intracellular Cl^? levels.     

Properties of carnitine transport in rat kidney cortex slices

The properties of carnitine transport were studied in rat kidney cortex slices. Tissue: medium concentration gradients of 7.9 for L-[methyl-¹⁴C]carnitine were attained after 60-min incubation at 37°C in 40 μM substrate. L- and D-carnitine uptake showed saturability. The concentration curves appeared to consist of (1) a high-affinity component, and (2) a lower affinity site. When corrected for the latter components, the estimated K_m for L-carnitine was 90 μM and $\text{V = 22}\text{nmol/min}$ per ml intracellular fluid; for D-carnitine, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 166}\text{μM}$ and $\text{V = 15}\text{nmol/min}$ per ml intracellular fluid. The system was stereospecific for L-carnitine. The uptake of L-carnitine was inhibited by (1) D-carnitine, γ-butyrobetaine, and (2) acetyl-L-carnitine. γ-Butyrobetaine and acetyl-L-carnitine were competitive inhibitors of L-carnitine uptake. Carnitine transport was not significantly reduced by choline, betaine, lysine or γ-aminobutyric acid. Carnitine uptake was inhibited by 2,4-dinitrophenol, carbonyl cyanide $\text{m-}\text{chlorophenylhydrazone}$, N₂ atmosphere, KCN, $\text{N-}\text{ethylmaleimide}$, low temperature (4°C) and ouabain. Complete replacement of Na⁺ in the medium by Li⁺ reduced L- and D-carnitine uptake by 75 and 60%, respectively. Complete replacement of K⁺ or Ca²⁺ in the medium also significantly reduces carnitine uptake. Two roles for the carnitine transport system in kidney are proposed: (1) a renal tubule reabsorption system for the steady-state maintenance of plasma carnitine; and (2) maintenance of normal carnitine levels in kidney cells, which is required for fatty acid oxidation.     

Multiple forms of cyclic nucleotide phosphodiesterases in normal and goitrous rat thyroid

The stoichiometry of H⁺ ejection coupled to electron flow from succinate to ferricyanide in the electron transport chain of mitochondria from Ehrlich ascites tumor and AS30-D hepatoma cells was determined. Values close to 4.0 for the $\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$ ejection ratio were found in both cell lines, with either Ca²⁺ or K⁺ (+ valinomycin) as charge-compensating permeant cation. The 4 H⁺ ejected were compensated by outward movement of two negative charges to reduce 2 Fe(CN)6^3? to 2 Fe(CN)6^4?, and the uptake of two positive charges in the form of the permeant cation. Experiments on (a) omission of rotenone (b) the effect of antimycin A and (c) depletion of endogenous NAD(P)-linked substrates showed that no significant endogenous electron flow or H⁺ ejection occurred, thus eliminating possible overestimation of the H⁺/2e^? ratio from endogenous substrates. These data on mitochondria from two tumor cell lines are fully consistent with earlier measurements of the H⁺/O stoichiometry for succinate and NADH oxidation in tumor mitochondria and with the $\text{H}{}^{\mathrm{+}}\text{2e}{}^{\mathrm{?}}$ stoichiometry for site 2 in normal rat liver mitochondria.     

Cellular uptake of lithium via amino acid transport system A

We now add to the agencies by which cells take up lithium the process of cotransport with neutral amino acids via System A. In the Ehrlich cell various natural and synthetic amino acids, depending on their structure, can cause substantial accelerations of Li⁺ uptake over a considerable range of levels of Na⁺, Li⁺ and H⁺. Half the maximal augmentation of uptake, namely 1.2 mequiv. Li/kg cell water per 15 min, was obtained for 5.4 mM alanine in a double-reciprocal plot. Alanine also stimulated the exodus of Li⁺ from the Ehrlich cell. The human red blood cell, lacking System A as it does, becomes an imperfect model for studying cellular uptake of Li⁺. Until the Li⁺ dependence of amino acid uptake in the reticulocyte is known, reticulocytosis can be suspected of contributing to the interpersonal variations seen in Li⁺-for-Na⁺ exchange.     

The basic asymmetry of Na+-dependent glycine transport in Ehrlich cells

Influx and efflux of glycine have been examined as a function of external and internal Na⁺ concentrations, respectively, when $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$. With $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$ it was found that at comparable external and cellular Na⁺ levels, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for efflux was larger by an order of magnitude than the value for influx and the $\text{V}$ for efflux was several times greater than the $\text{V}$ for influx. For both fluxes the major effect of Na⁺ was to decrease the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value. The observations are consistent with the conclusion that the Na⁺-dependent transport system is asymmetric per se. Influx and efflux of glycine were increased in a near linear manner by increasing the Na⁺ concentration from 13 to 100 mM, the half-time for glycine equilibration being a function of the Na⁺ concentration in absence of an electrochemical potential difference for Na⁺. In Na⁺-free media ($\text{[}\text{Na}{}^{\mathrm{+}}\text{] < 5}\text{mM}$) equilibration of glycine between cells and medium was not achieved after 60 min at 25°C. With $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$, efflux (or uptake) of glycine was not affected by internal (or external) K⁺ between 20 and 120 mM suggesting that K⁺ plays no direct role in Na⁺-dependent transport of glycine in Ehrlich cells.