Membrane potentials in mitochondrial preparations as measured by means of a cyanine dye

Changes in the fluorescent intensity of the dye 3,3′-dipropylthiodicarbocyanine iodide were measured in suspensions of hamster liver mitochondria upon the development of a K⁺ diffusion potential by the addition of valinomycin and upon the development of the energized state by the addition of succinate or ATP. The changes (large decreases) seen with the addition of succinate or ATP (inhibitable by NaCN and oligomycin respectively) were comparable to those recorded upon the addition of valinomycin to mitochondria suspended in media containing low concentrations of K⁺. The change observed with succinate was partially reversed by the addition of either 2,4-dinitrophenol or ADP. Oligomycin prevented the reversal seen with ADP. Decreases in fluorescent intensity were also recorded when succinate was added to suspensions of inner membranes (prepared from rat liver mitochondria) containing the dye. With submitochondrial particles (also from rat liver mitochondria), however, increases in fluorescent intensity were seen upon the addition of succinate or ATP. These observations are consistent with the idea that a large negative (internal) potential develops across the inner membrane of the mitochondrion during energization and with other aspects of the chemiosmotic hypothesis.     

Inhibition of mitochondrial respiration by loss of intra-mitochondrial K

1. A system is described in which intra-mitochondrial K⁺ concentration can be manipulated by the use of dinitrophenol and valinomycin.

2. As mitochondria lose K⁺ a striking inhibition of O₂ consumption occurs with both succinate and DPN-linked substrates, but not if tetramethyl-p-phenylenediamine + ascorbate serves as substrate. Respiration can be re-activated by adding K⁺ to the medium.

3. Results are discussed in terms of sensitivity of electron transport or substrate dehydrogenation to intra-mitochondrial K⁺ content.     

The sodium cycle. II. Na-coupled oxidative phosphorylation in Vibrio alginolyticus cells

The role of Na⁺ in Vibrio alginolyticus oxidative phosphorylation has been studied. It has been found that the addition of a respiratory substrate, lactate, to bacterial cells exhausted in endogenous pools of substrates and ATP has a strong stimulating effect on oxygen consumption and ATP synthesis. Phosphorylation is found to be sensitive to anaerobiosis as well as to HQNO, an agent inhibiting the Na⁺-motive respiratory chain of V. alginolyticus. Na⁺ loaded cells incubated in a K⁺ or Li⁺ medium fail to synthesize ATP in response to lactate addition. The addition of Na⁺ at a concentration comparable to that inside the cell is shown to abolish the inhibiting effect of the high intracellular Na⁺ level. Neither lactate oxidation nor Δω generation coupled with this oxidation is increased by external Na⁺ in the Na⁺-loaded cells. It is concluded that oxidative ATP synthesis in V. alginolyticus cells is inhibited by the artificially imposed reverse ΔPNa, i.e., [Na⁺]_in > [Na⁺]_out. Oxidative phosphorylation is resistant to a protonophorous uncoupler (0.1 mM CCCP) in the K⁺-loaded cells incubated in a high Na⁺ medium, i.e., when ΔpNa of the proper direction ([Na⁺]_in < [Na⁺]_out) is present. The addition of monensin in the presence of CCCP completely arrests the ATP synthesis. Monensin without CCCP is ineffective. Oxidative phosphorylation in the same cells incubated in a high K⁺ medium (ΔpNa is low) is decreased by CCCP even without monensin. Artificial formation of ΔpNa by adding 0.25 M NaCl to the K⁺-loaded cells (Na⁺ pulse) results in a temporary increase in the ATP level which spontaneously decreases again within a few minutes. Na⁺ pulse-induced ATP synthesis is completely abolished by monensin and is resistant to CCCP, valinomycin and HQNO. 0.05 M NaCl increases the ATP level only slightly. Thus, V. alginolyticus cells at alkaline pH represent the first example of an oxidative phosphorylation system which uses Na⁺ instead of H⁺ as the coupling ion.     

Enhancement by tetraphenylboron of inhibition of mitochondrial respiration induced by 1-methyl-4-phenylpyridinium ion (MPP)

The effect of tetraphenylboron (TPB⁻), an activator of a membrane transport of lipophilic cations, on the inhibition of mouse liver mitochondrial respiration induced by a neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP⁺), and by some structurally related compounds was studied. Of the compounds tested, MPP⁺ and 4-phenylpyridine (4-PP) significantly inhibited the respiration in an ADP-activated oxidation of substrates (state 3). TPB⁻, dose-dependently, shortened the lag time of MPP⁺-induced inhibition and thus lowered the concentrations of MPP⁺ for the inhibition. However, TPB⁻, even at the high concentration (10 μM), did not significantly affect 4-PP-induced inhibition. Carbonyl-cyanide-m-chlorophenylhydrazone (CCCP) blocked the respiratory inhibition by MPP⁺, independent of K⁺ concentration in the medium, and valinomycin blocked the inhibition only in the medium containing high K⁺ concentration. Determination of the intramitochondrial MPP⁺ concentration revealed about 1000-fold concentrated MPP⁺ from that in the medium during the incubation with TPB⁻, indicative of potentiation of MPP⁺ transport into mitochondria by TPB⁻. This might account for the enhancement of respiratory inhibition by MPP⁺. In the case of 4-PP, it will penetrate the mitochondrial membrane and intrinsically inhibit the respiration, but cannot accumulate in mitochondria. The present results indicate that, although the inhibitory potency of MPP⁺ per se is similar to 4-PP, MPP⁺ will be highly concentrated within mitochondria by the membrane potential, as the drive force for its transport.     

Transient hyperpolarization of yeast by glucose and ethanol

At pH 7, addition of glucose under anaerobic conditions to a suspension of the yeast Saccharomyces cerevisiae causes both a transient hyperpolarization and a transient net efflux of K⁺ from the cells. Hyperpolarization shows a peak at about 3 min and a net K⁺ efflux at 4–5 min. An additional transient hyperpolarization and net K⁺ efflux are found after 60–80 and 100 min, respectively. Addition of 2-deoxyglucose instead of glucose does not lead to hyperpolarization of the cells or K⁺ efflux. At low pH, neither transient hyperpolarization nor a transient K⁺ efflux are found. With ethanol as substrate and applying aerobic conditions, both a transient hyperpolarization and a transient K⁺ efflux are found at pH 7. The fluorescent probe 2-(dimethylaminostyryl)-1-ethylpyridinium appears to be useful for probing changes in the membrane potential of S. cerevisiae. It is hypothesized that the hyperpolarization of the cells is due to opening of K⁺ channels in the plasma membrane. Accordingly, the hyperpolarization of the cells at pH 7 is almost completely abolished by 1.25 mM K⁺, whereas the same amount of Na⁺ does not reduce the hyperpolarization     

Inhibition of K+ efflux prevents mitochondrial dysfunction, and suppresses caspase-3-, apoptosis-inducing factor-, and endonuclease G-mediated constitutive apoptosis in human neutrophils

Neutrophils die rapidly via apoptosis and their survival is contingent upon rescue from constitutive programmed cell death by signals from the microenvironment. In these experiments, we investigated whether prevention of K⁺ efflux could affect the apoptotic machinery in human neutrophils. Disruption of the natural K⁺ electrochemical gradient suppressed neutrophil apoptosis (assessed by annexin V binding, nuclear DNA content and nucleosomal DNA fragmentation) and prolonged cell survival within 24–48 h of culture. High extracellular K⁺ (10–100 mM) did not activate extracellular signal-regulated kinase (ERK) and Akt, nor affected phosphorylation of p38 MAPK associated with constitutive apoptosis. Consistently, pharmacological blockade of ERK kinase or phosphatidylinositol 3-kinase (PI 3-kinase) did not affect the anti-apoptotic action of KCl. Inhibition of K⁺ efflux effectively reduced, though never completely inhibited, decreases in mitochondrial transmembrane potential (ΔΨ_m) that preceded development of apoptotic morphology. Changes in ΔΨ_m resulted in attenuation of cytochrome c release from mitochondria into the cytosol and decreases in caspase-3 activity. Culture of neutrophils in medium containing 80 mM KCl with the pan-caspase inhibitor Z-VAD-FMK resulted in slightly greater suppression of apoptosis than KCl alone. High extracellular KCl also attenuated translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) from mitochondria to nuclei. The DNase inhibitor, aurintricarboxylic acid (ATA) partially inhibited nucleosomal DNA fragmentation, and the effects of ATA and 80 mM KCl were not additive. These results show that prevention of K⁺ efflux promotes neutrophil survival by suppressing apoptosis through preventing mitochondrial dysfunction and release of the pro-apoptotic proteins cytochrome c, AIF and EndoG independent of ERK, PI 3-kinase and p38 MAPK. Thus, K⁺ released locally from damaged cells may function as a survival signal for neutrophils.     

Effect of glutamate on extracellular potassium activity in isolated retina of cyprinid fish

The effect of ( -)glutamate on extracellular K⁺ activity of the isolated retina of the cyprinid fish, roach, was investigated using double-barrelled K⁺-sensitive micro-electrodes. Application of μM-mM glutamate to the retina as a “puff” from an atomizer induced a transient rise in extracellular K⁺ activity, which was maximal 50–100 μm below the photoreceptor surface. The effect was concentration-dependent, but not related to the state of light adaptation of the retina. In the presence of dinitrophenol or ouabain, the glutamate-induced increase in extracellular K⁺ activity was maintained.

The following conclusions are made. (1) The most likely cellular origin(s) of the glutamate-induced rise in extracellular K⁺ activity are the photoreceptors and/or the Muller (glial) cells. (2) The mechanism responsible for returning the extracellular K⁺ activity to normal depends strongly on metabolic, Na⁺, K⁺ pump activity. (3) The effect parallels the action of endogenous glutamate, and may be important for modulation of neurotransmission in the intact retina.     

ADP binding and ATP synthesis by reconstituted H-ATPase from chloroplasts

The H⁺-ATPase from chloroplasts, CF₀F₁, was isolated, purified and reconstituted into asolectin liposomes. The enzyme was brought either into the oxidized state or into the reduced state, and the rate of ATP synthesis was measured after energisation of the proteoliposomes with an acid—base transition ΔpH (pH_in = 5.0, pH_out = 8.5) and a K⁺/valinomycin diffusion potential, Δφ (K⁺_in = 0.6 mM, K⁺_out = 60 mM). A rate of 250 s⁻¹ was observed with the reduced enzyme (85 s⁻¹ in the absence of Δφ). A rate of 50 s⁻¹ was observed with the oxidized enzyme under the same conditions (15 s⁻¹ in the absence of Δφ). The reconstituted enzyme contained 2 ATP_bound per CF₀F₁ and 1 ADP_bound per CF₀F₁. Upon energisation the enzyme was activated and 0.9 ADP per CF₀F₁, was released. Binding of ADP to the active reduced enzyme was observed under different conditions. In the absence of phosphate the rate constant for ADP binding was 10⁵ M⁻¹·s⁻¹ under energized and de-energized conditions. In the presence of phosphate the rate of ADP binding drastically increased under energized conditions, and strongly decreased under de-energized conditions.     

Ion dependent efflux from neuronal and glial cell cultures

A spontaneous efflux of choline originating from the cytoplasmic free choline compartment and, partly, from metabolized form was measured from neurons and glial cells in culture. The efflux was stimulated by an excess of K⁺ and by the absence of Ca²⁺ ions from the incubation medium in both types of culture. The two effects did not appear to be synergistic.

The stimulation produced by an excess of K⁺ (100 mM) was blocked in neurons by 0.5 μM BaCl₂ and in glia cells by 0.1 μM BaCl₂ (in the presence of 30 mM K⁺). The stimulation produced by the absence of Ca²⁺ instead was not blocked by Ba²⁺ ions in either of the two types of culture. The results suggest that the stimulation induced by K⁺ (high concentration and long time of incubation) might be of biochemical rather than physiological nature and that choline may be driven out of the cells in correlation with the K⁺ gradient. The greater sensitivity of glial cells to K⁺ ions may also suggest a supportive role of these cells with respect to neurons, as they seem capable of furnishing choline for neuronal needs during depolarization.     

Expression of an inwardly rectifying K+ channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

Mg2+ ion effect on conformational equilibrium of poly A . 2 poly U and poly A poly U in aqueous solutions

Differential UV spectroscopy and thermal denaturation were used to study the Mg²⁺ ion effect on the conformational equilibrium in poly A · 2 poly U (A2U) and poly A · poly U (AU) solutions at low (0.01 M Na⁺) and high (0.1 M Na⁺) ionic strengths. Four complete phase diagrams were obtained for Mg²⁺–polynucleotide complexes in ranges of temperatures 20–96 °C and concentrations (10⁻⁵–10⁻²) M Mg²⁺. Three of them have a ‘critical’ point at which the type of the conformational transition changes. The value of the ‘critical’ concentration ([Mg_t²⁺]_cr=(4.5±1.0)×10⁻⁵ M) is nearly independent of the initial conformation of polynucleotides (AU, A2U) and of Na⁺ contents in the solution. Such a value is observed for Ni²⁺ ions too. The phase diagram of the (A2U+Mg²⁺) complex with 0.01 M Na⁺ has no ‘critical’ point: temperatures of (3→2) and (2→1) transitions increase in the whole Mg²⁺ range. In (AU+Mg²⁺) phase diagram at 0.01 M Na⁺ the temperature interval in which triple helices are formed and destroyed is several times larger than at 0.1 M Na⁺. Using the ligand theory, a qualitative thermodynamic analysis of the phase diagrams was performed.     

A negative resistance region underlies the triggering property of membrane potential in human T-lymphocytes

Steady-state current-voltage relationships (SSCVRs) of the plasma membrane of human T-lymphocytes were studied at the physiological temperature of 37°C by using the whole-cell patch-clamp technique. SSCVRs displayed a characteristic N-like shape with a negative resistance region (NRR) in a voltage range of −45 to −35 mV. The majority of cells assayed revealed SSCVR patterns crossing the V-axis at three points (in mV): V₁ = −55 to −45, V₂ = −40 to −35, V₃ = −30 to −10. SSCVRs of T-cells activated by phytohaemagglutinin (48–96 h) also displayed NRR, but crossed the V-axis at one point only (V₁ = −55 to −60 mV). It implies the possibility of two stable levels of membrane potential (V₁ and V₃) for the resting T-cells, but only one (V₁) for activated T-cells. These data thus account for the triggering property of T-cell membrane potential previously reported. The NRR can be explained on the basis of the Hodgkin-Huxley type n⁴j model of K⁺ channel kinetics. According to the model the possibility for a membrane to have on or two stable levels of membrane potential depends on the ratio of selective K⁺ conductance to non-selective leaky conductance (G_k/G_leak). The steady-state level of K⁺ conductance in resting T-lymphocytes proved to be sensitive to Ca²⁺. Buffering Ca²⁺ ions from either external or internal solution resulted in an appreciable increase in K⁺ conductance. The possibility for membrane potential have two stable levels of membrane potential in connection with the Ca²⁺ dependence of K⁺ conductance was supposed to be important for Ca²⁺-signalling during T-cell activation.     

Glutamate transporter GLAST/EAAT1 directs cell surface expression of FXYD2/gamma subunit of Na, K-ATPase in human fetal astrocytes

Na⁺-dependent uptake of excitatory neurotransmitter glutamate in astrocytes increases cell energy demands primarily due to the elevated ATP consumption by glutamine synthetase and Na⁺, K⁺-ATPase. The major pool of GLAST/EAAT1, the only glutamate transporter subtype expressed by human fetal astrocytes in undifferentiated cultures, was restricted to the cytoplasmic compartment. Elevated glutamate concentrations (up to 50 μM) stimulated both glutamate uptake and Na⁺, K⁺-ATPase activity and concomitantly increased cell surface expression of GLAST and FXYD2/γ subunit of Na⁺, K⁺-ATPase. Intracellular accumulation of glutamate or its metabolites per se was not responsible for these changes since metabolically inert transport substrate, d-aspartate, exerted the same effect. Nanomolar concentrations of TFB-TBOA, a novel nontransportable inhibitor of glutamate carriers, almost completely reversed the action of glutamate or d-aspartate. In the same conditions (i.e. block of glutamate transport) monensin, a potent Na⁺ ionophore, had no significant effect neither on the activation of Na⁺, K⁺-ATPase nor on the cell surface expression of γ subunit or GLAST. In order to elucidate the roles of γ subunit in the glutamate uptake-dependent trafficking events or the activation of the astroglial sodium pump, in some cultures γ subunit/FXYD2 was effectively knocked down using siRNA silencing. Unlike the blocking effect of TFB-TBOA, the down-regulation of γ subunit had no effect neither on the trafficking nor activity of GLAST. However, the loss of γ subunit effectively abolished the glutamate uptake-dependent activation of Na⁺, K⁺-ATPase. Following withdrawal of siRNA from cultures, the expression levels of γ subunit and the sensitivity of Na⁺, K⁺-ATPase to glutamate/aspartate uptake have been concurrently restored. Thus, the activity of GLAST directs FXYD2 protein/γ subunit to the cell surface, that, in turn, leads to the activation of the astroglial sodium pump, presumably due to the modulatory effect of γ subunit on the kinetic parameters of catalytic subunit(s) of Na⁺, K⁺-ATPase.     

Ca entry through the store-mediated pathway directly activates only the K current but the subsequent Ca release from the store activates both K and Cl currents in submandibular gland acinar cells of the rat

The store-mediated Ca²⁺ entry was detected in single and cluster of rat submandibular acinar cells by measuring the Ca²⁺ activated ionic membrane currents. In the cells where intracellular Ca²⁺ was partly depleted by stimulation with submaximal concentration of acetylcholine (ACh) under a Ca²⁺-free extracellular condition, an employment of external Ca²⁺ in the absence of ACh caused a sustained increase of the K⁺ current without affecting the Cl⁻ current. A renewed ACh challenge without external Ca²⁺ caused repetitive spikes of both K⁺ and Cl⁻ currents due to the Ca²⁺ release. SK & F 96365 inhibited the generation of the sustained K⁺ current and refilling of the Ca²⁺ store following the Ca²⁺ readmission. It is suggested that the Ca²⁺ enters the cell through the store-mediated pathway near the K⁺ channels and is taken up by the store. Thus, only Ca²⁺ released from the store can activate both the K⁺ and Cl⁻ currents.     

Spinach-thylakoid phosphorylation: Studies on the kinetics of changes in photosystem antenna size, spill-over and phosphorylation of light-harvesting chlorophyll a/b protein

The kinetics of LHCP phosphorylation and associated changes in photosystem cross-section and energy ‘spill-over’ from PS II to PS I have been examined in isolated spinach chloroplasts. During an initial phosphorylation period of 3–6 min, in the presence of saturating concentrations of Mg²⁺, the increase in PS I and decrease in PS II cross-section are largely completed, as judged by both measurements of the steady-state redox state of Q and fluorescence yield changes. This corresponds to a period of rapid ³²P incorporation into the low-molecular weight LHCP polypeptide. Subsequent to this initial 3–6-min period there is substantial further phosphorylation of both LHCP polypeptides, which is not accompanied by significant changes in photosystem cross-section, even after the chloroplasts had been unstacked with extensive mixing of PS I and PS II by Mg-removal. It is suggested that there exists a specific ‘mobile’ population of LHCP molecules which is rapidly phosphorylated and which may be enriched in the low-molecular-weight polypeptide. In addition, measurements of the kinetics of the ‘spill-over’ changes upon either Mg²⁺ addition or removal indicate that the continued phosphorylation of LHCP is able to increase the ‘spill-over’ process under favourable ionic conditions.     

Effects of moderate short-term potassium depletion in normal humans the role of prostaglandins

The role of prostaglandins (PG) in the effects of potassium (K⁺)depletion was studied in six normal women. A mean K⁺-deficit of 220 mEq was induced with and without concomitant treatment with indomethacin (150 mg/day). Mean serum K⁺ concentration decreased from 4.2 ± (S.E.) 0.1 to 3.2 ± 0.1 mEq/L without indomethacin and from 4.1 ± 0.1 to 3.2 ± 0.1 mEq/L with indomethacin. “Supine” and “upright” plasma renin activity (PRA) and plasma norepinephrine concentration (NE) were unaltered by K⁺ -depletion alone but decreased with indomethacin. Plasma aldosterone (PA) was suppressed during K⁺-depletion (control: 7.2 ± 2.6 ng/dl supine, 19.3 ± 8.1 ng/dl upright; K⁺-depletion: 2.6 ± 0.3 ng/dl supine, 5.5 ± 1.3 ng/dl upright) and was paralleled by a decrease in urinary aldosterone. K⁺-depletion decreased urinary PGE₂ from 667 ± 133 to 343 ± 60 ng/day (P < 0.025) without a change in PGF₂. The dose of exogenous angiotensin II (A II) which increased diastolic blood pressure by 20 mm Hg (pressor dose) was 7.1 ± 1.4 ng/kg/min during control and increased to 11.0 ± 0.7 ng/kg/min during K⁺-depletion (P < 0.05). Indomethacin increased the sensitivity to A II both during control (pressor dose: 4.9 ± 0.6 ng/kg/min) and K⁺- depletion (pressor dose: 6.0 ± 1.0 ng/kg/min). These results indicate that in healthy subjects, moderate short-term K⁺-depletion does not affect PRA or NE but decreases production of aldosterone and PGE₂ by the kidney. The changes in vascular sensitivity to exogenous A II during K⁺-depletion and indomethacin and the decreases in plasma NE and PRA during indomethacin may be explained by changes in vascular vasodilator PG.     

Influence of ATPase activity on PPi dependent H-transport in tonoplast vesicles of Acer pseudoplatanus

Tonoplast H⁺-ATPase and H⁺-pyrophosphatase (H⁺-PPase) were previously characterized in Acer pseudoplatanus cells (A. Pugin et al., Plant Sci., 73 (1991) 23–34; A. Fraichard et al., Plant Physiol. Biochem., 31 (1993) 349–359). The present study concerns the relationships between these two enzymes in vitro. ATP and PPi hydrolysis were additive and the inhibition of one did not affect the activity of the second one. ATP and PPi H⁺-transports were also additive. The H⁺ -PPase inhibition did not change ATP-dependent H⁺-transport but H⁺-ATPase inhibition inhibited the PPi dependent H⁺-transport. Because H⁺-PPase was reported to transport H⁺ and K⁺ into the vacuole (Davies et al., Proc. Natl. Acad. Sci. USA, 89 (1992) 11701–11705), these results led us to suggest that the inhibition of the H⁺-ATPase activity could modify the H⁺/K⁺ stoichiometry for the benefit of K⁺-transport.     

Screening for synergistic interactions in dilute polysaccharide solutions

A simple viscometric approach has been used to screen for binding interactions between different polysaccharides in very dilute solution where exclusion effects should be negligible. The method involves preparing stock solutions to approximately the same, low, viscosity (η_sp≈1), dialysing to identical ionic conditions, mixing in various proportions, and looking for departures from the initial common viscosity.

Mixtures of xanthan or de-acetylated xanthan with locust bean gum (LBG) or konjac glucomannan (KM) show massive enhancement of viscosity, as anticipated from the formation of synergistic gels at higher concentrations. However, no viscosity changes on mixing with LBG or KM were observed for other conformationally ordered bacterial polysaccharides (welan and rhamsan) or for alginate and pectin with sufficient Ca²⁺ to induce almost complete conversion to the dimeric ‘egg box’ form, demonstrating that conformational rigidity is not, in itself, sufficient for other polysaccharides to form heterotypic junctions with mannan or glucomannan chains.

Interactions of carrageenans with LBG appear to depend on both conformation and the extent of aggregation. Mixtures of LBG with K⁺ kappa carrageenan in 100mM KCl (which is known to promote extensive aggregation of double helices) gave erratic values for rotational viscosity and showed typical gel-like mechanical spectra under low-amplitude oscillation. Disordered carrageenans (K⁺ kappa in water and lambda in 100mM KCl) showed no evidence of interaction with LBG. Negative results were also obtained for iota carrageenan under ionic conditions believed to promote ordering without significant aggregation (100mM KCl). However, under conditions where limited aggregation might be expected (iota carrageenan in 90 mM CaCl₂; Me₄N⁺ kappa carrageenan in 150 mM Me₄NI), significant reductions in viscosity were observed on mixing with LBG, which may indicate some intermolecular association but without the formation of an extended network structure.     

Effects of Tetraethylammoniumchloride (TEA), Vanadate, and Alkali Ions on the Lateral Leaflet Movement Rhythm of Desmodium motorium (Houtt.) Merr

The period (∼3-5 min) of the ultradian rhythm of the lateral leaflet movement of Desmodium motorium is strongly lengthened (≤30-40%) by the K⁺ channel blocker tetraethylammoniumchloride (20, 30, and 40 mM) and vanadate (0.5 and 1 mM), which is an effective inhibitor of the plasma membrane-bound H⁺ pump. The alkali ions K⁺, Na⁺, Rb⁺, and Cs⁺ (10-40 mM) shorten the period only slightly (≤ 10-15%). Li⁺ (5-30 mM), however, increases the period of the leaflet rhythm drastically (≤80%). We concluded that the plasmalemma-H⁺-ATP-ase-driven K⁺ transport through K⁺ channels is an essential component of the ultradian oscillator of Desmodium, as has been proposed for the circadian oscillator.     

Some properties of, and the effect of temperature on the (Mg + Ca) ATPase from immature and adult rat brain

1. 1. (Mg²⁺ + Ca²⁺) ATPases of microsomal and synaptic membrane preparations from immature and adult rat brain were activated by calcium (0.1–10 μM), maximal activation was found at 3 μM. The increase in (Mg²⁺ + Ca²⁺) ATPase seen during development was greatest in the synaptic membrane preparations.

2. 2. At 37°C both Na⁺ or K⁺ at concentrations higher than 30 mM inhibited the microsomal Mg²⁺ ATPase, but the (Mg²⁺ + Ca²⁺) ATPase was stimulated by both Na⁺ and K⁺. Synaptic membrane Mg²⁺ ATPase was inhibited by concentrations higher than 100 mM K⁺; Na⁺ however stimulated this enzyme at all concentrations. Much of this Na⁺ stimulated activity was ouabain sensitive. Synaptic membrane (Mg²⁺ + Ca²⁺) ATPase was stimulated by Na⁺ or K⁺, this stimulation follows approximate saturation kinetics with an apparent K_m of 18.8 mM Na⁺ or K⁺.

3. 3. Arrhenius plots of microsomal (Mg²⁺ + Ca²⁺) ATPase were curvilinear, but two intersecting lines with a break at 20°C could be fitted. The calculated energies of activation from these lines were very similar in immature and adult preparations. The synaptic membrane preparation (adult) also gave a curvilinear plot; but two intersecting lines with a break at 25°C could be fitted to the data. These lines had slopes of 21 and 28 Kcal mole⁻¹ above and below the break, respectively. The immature preparation when made using EDTA gave a Arrhenius plot of very similar form to the adult preparation. Without EDTA however the Arrhenius plot was complex with a plateau at 25–32°C. Pretreatment with EDTA activated the synaptic membrane (Mg²⁺ + Ca²⁺) ATPase from both immature and adult brain.