Neuromuscular modulation in Limulus by both octopamine and proctolin

Both octopamine and proctolin potentiate nerve-evoked skeletal muscle contractions in the horseshoe crab, Limulus. The threshold concentration for octopamine was 10^?9 to 10^?8M, while for proctolin it was 3 × 10^?9M. Norepinephrine and dopamine produced effects similar to octopamine but at higher thresholds; tyramine and serotonin were ineffective. Octopamine caused significant increases in amplitudes of excitatory postsynaptic potentials (epsps) of muscle fibers, but had little effect on muscle fiber input resistance or membrane potential. Also, octopamine did not affect depolarization of muscle fibers and subsequent contraction due to the direct action of exogenously applied glutamate. These results suggest that octopamine potentiates nerve-evoked contractions primarily by facilitating release of neuromuscular transmitter. At concentrations above 10^?7M, however, octopamine sometimes caused muscle spikes in response to motoneuron stimulation, a finding that suggests that octopamine may also have some postsynaptic action. Proctolin potentiated the muscle contractions evoked by glutamate but had little effect on glutamate-evoked muscle fiber depolarization, muscle fiber input resistance, or membrane potential. Thus, proctolin appears to act directly on skeletal muscle to enhance contractility. The proctolin-induced potentiations of contraction were sometimes accompanied by modest increases in epsp amplitude, so that unlike lobster skeletal and Limulus cardiac neuromuscular preparations, proctolin may have a secondary direct synaptic effect. Both octopamine and proctolin have been found in Limulus cardiac ganglion. This potential access to the hemolymph and the relatively low threshold concentrations needed for physiological action suggest that octopamine and proctolin could function as hormonal modulators of neuromuscular function in Limulus.     

Mivazerol, a Novel α2-Agonist and Potential Anti-Ischemic Drug, Inhibits KCl-Stimulated Neurotransmitter Release in Rat Nervous Tissue Preparations

Abstract: In this study, we have investigated the effect of mivazerol, [3-(1H-imidazol-4-yl)methyl-1]-2-hydroxy-benzamide hydrochloride, a new α₂-agonist lacking hypotensive properties and a potential anti-ischemic drug, on the evoked release of norepinephrine, aspartate, and glutamate in tissue preparations from hippocampus, spinal cord T1–T5 section, rostrolateral ventricular medulla, and nucleus tractus solitarii of the brainstem of rat. A simple and efficient in vitro procedure to study pharmacologically the release of norepinephrine and glutamate is described. Tissues were chopped into (0.3 × 0.2 × 0.2 mm³) sections and the resulting minces were used for this study. Exposure to KCl (10–75 mM) for 5 min served as a stimulus for the release response. One, S (for aspartate and for glutamate release), or two such stimuli, S₁ and S₂ (for norepinephrine release) were conducted. The release of norepinephrine (+150% above baseline) was inhibited in a dose-dependent manner by mivazerol in hippocampus (IC₅₀ = 1.5 × 10^?8M), spinal cord (IC₅₀ = 5 × 10^?8M), rostrolateral ventricular medulla (IC₅₀ = 10^?7M), and nucleus tractus solitarii (IC₅₀ = 7.5 × 10^?8M), and by clonidine in hippocampus (IC₅₀ = 5 × 10^?8M), spinal cord (IC₅₀ = 4.5 × 10^?8M), rostrolateral ventricular medulla (IC₅₀ = 2.5 × 10^?7M), and nucleus tractus solitarii (IC₅₀ = 10^?7M). This effect was counteracted by the selective α₂-antagonists yohimbine and rauwolscine. A significant glutamate and aspartate release response was also induced by KCl (35 mmol/L) in hippocampus (+250 and +135%, respectively) and spinal cord (+120 and +55%, respectively), in vitro. However, neither mivazerol nor clonidine, at doses up to 10 µM, had any significant effect on KCl-induced glutamate release in spinal cord, whereas mivazerol blocked completely the release of both amino acids in hippocampus and only the release of aspartate in spinal cord. On the other hand, clonidine (1 µM) was only effective in reducing by 40% the release of aspartate in hippocampus. These data indicate that (1) inhibition of KCl-induced norepinephrine release by mivazerol is mediated by its action on α₂-adrenergic receptors; (2) at concentrations selective for α₂-adrenergic receptors, only mivazerol was effective in blocking the KCl-induced glutamate release in hippocampal tissue; and (3) at the same concentrations, both mivazerol and clonidine were unable to inhibit glutamate release in the spinal cord. These data suggest that prevention of hyperadrenergic activity by mivazerol in perioperative patients may be mediated through its effect on the release of norepinephrine and/or the release of glutamate and aspartate in regions of the CNS that are involved in the control of cardiovascular homeostasis.     

Electrophysiological studies of fetal mouse olfactory bulb explants during development of synaptic functions in culture

Explants of 18- or 19-day fetal mouse olfactory bulb have been maintained in culture for periods up to 5 weeks. Compound action potentials can be evoked in the bulb explants by 1 day in vitro, and by 3–4 days, synaptically mediated slow wave discharges can be demonstrated in bicuculline (10^?5 M). The capability of the bulb explants to generate these slow-wave discharges has also been revealed by the introduction of picrotoxin (10^?5 M), d-tubocurarine (10^?4 M) and chloride-free medium, but not of strychnine (up to 3 × 10^?5 M). The data indicate early functional development of inhibitory, as well as excitatory, synaptic systems. In addition, the selective and reversible depression of these slow wave potentials by GABA (1–5 × 10^?4 M), but not by glycine (up to 3 × 10^?3 M), indicates a GABA-ergic component in the inhibitory network. Single unit extracellular recordings have been obtained from the presumptive mitral cells which, in culture, are spontaneously active even as early as 1–2 days after explantation. Correlative Bodian silver-impregnations demonstrate the presence of neurons in these explants which resemble typical mitral cells. Studies of mitral cells using paired stimuli suggest the development in vitro of an inhibitory system analogous to that known to suppress the excitability of their in situ counterparts following orthodromic or antidromic activation. These data, as well as the pharmacological sensitivities of the mitral cells in culture to GABA (5 × 10^?4 M) and bicuculline (10^?5 M), indicate that granule-to-mitral synapses may develop characteristic functions in olfactory bulb explants.     

Interleukin-2 Modulates Evoked Release of [3H]Dopamine in Rat Cultured Mesencephalic Cells

Abstract: Mesencephalic cell cultures were used as a model to investigate the effects of interleukin-2 (IL-2) on evoked release of [³H]dopamine ([³H]DA) and γ-[³H]-aminobutyric acid ([³H]GABA). At low concentrations (10^?13-10^?12M), IL-2 potentiated [³H]DA release evoked by the excitatory amino acids N-methyl-D-aspartate (NMDA) and kainate, whereas higher IL-2 concentrations (10^?9-10^?8M) had no effect. IL-2 (10^?14-10^?8M) modulated K⁺-evoked [³H]DA release in a biphasic manner, with low concentrations (10^?12-10^?11M) of IL-2 potentiating and higher concentrations (10^?9-10^?8M) inhibiting K⁺-induced [³H]DA release. IL-2 (10^?14-10^?8M) by itself failed to alter spontaneous [³H]DA release. The inhibition by IL-2 of K⁺-evoked [³H]DA release was reversible and not due to neurotoxicity, as preexposure to IL-2 (10^?8M) had no significant effect on the subsequent ability of dopaminergic cells to take up and to release [³H]DA. Under our experimental conditions, IL-2 (10^?8 M) did not alter Ca²⁺-independent [³H]GABA release evoked by either K⁺ or NMDA. The results of this study indicate that IL-2 is able to potentiate [³H]DA release evoked by a number of different stimuli, including K⁺ depolarization and activation of both NMDA and non-NMDA receptor subtypes in mesencephalic cell cultures. IL-2 is active at very low concentrations, a finding that indicates a potent effect of IL-2 on dopaminergic neurons and implicates a physiological role for this cytokine in the modulation of DA release.     

A peptide action in a lobster neuromuscular preparation

The neuropeptide proctolin causes a sustained contraction of the opener muscle of the dactyl of the lobster walking leg. This substance acts directly on the muscle at concentrations as low as 10^?10M. The contraction is dependent on extracellular calcium. Neither a significant depolarization nor a detectable change in the input resistance accompanies the response. No presynaptic action of proctolin is indicated; excitatory and inhibitory junctional potential sizes and the frequency of spontaneous miniature excitatory junctional potentials are unaffected.     

Excitatory neuromuscular transmission in crayfish: Calcium dependence is unaffected by picrotoxin

Picrotoxin, 1 × 10^?5M to 1.6 × 10^?3M, had little or no effect on the amplitude of intracellularly recorded excitatory junctional potentials (EJPs) at extracellular calcium concentrations [Ca²⁺]₀ ranging from 0.5 to 15 mM. The slope of the log EJP vs. log[Ca²⁺]₀ relationship was approximately 1 with or without picrotoxin. The reduction of EJP amplitude resulting from the addition of 5 × 10^?5M GABA was largely reversed by 10^?5M picrotoxin.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage—current relationship of the membrane. By including γ-aminobutyrate (GABA) in the bathing medium, the voltage—current relationship was made linear in the depolarizing direction over a range of 15–20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and asparate functioning synergistically.     

Studies on the viscosity of solutions of bovine liver glutamate dehydrogenase and on related hydrodynamic models; effect of toluene on enzyme association

The viscosity of bovine liver glutamate dehydrogenase solutions was studied at 10 and 20° C in 0.2.M sodium phosphate buffer at pH 7, in the concentration range 0.1–8 mg/ml. A method for the study of the viscosity of very dilute solutions of associating enzymes is described. It was found that the reduced specific viscosity η_sp/c of glutamate dehydrogenase continuously increases with increasing enzyme concentration, from about 4 ml/g at the lowest concentrations to about 16 ml/g at 8 mg/ml. In the presence of 10^?3M GTP and 10^?3M NADH the viscosity increase is much smaller and the results can be extrapolated to zero enzyme concentration to yield an intrinsic viscosity [η] = 3.2. The values of η_sp/c in phosphate buffer alone apparently extrapolate to the same value of [η], or to a value close to it. We also observe that, in the presence of toluene η_sp/c increases very much more with enzyme concentration: η_sp/c already equals 16 ml/g at a concentration of 0.75 mg/ml. These observations are in good agreement with the hypothesis that the active oligomer of glutamate dehydrogenase (MW = 312,000) associates with increasing enzyme concentration to form linear rodlike polymers of indefinite length. This association is strongly diminished by the addition of 10^?3M GTP, 10^?3M NADH. Toluene, on the other hand, promotes reversible association to linear polymers of very high molecular weight. The transverse and axial rotary frictional coefficients of macroscopic bodies, similar to a physical model for the structure of glutamate dehydrogenase recently advanced, were determined. Assuming that the viscosity of the model is equal to that of an ellipsoid of rotation with identical frictional coefficients, we calculate [η] = 3.26 ml/g according to Kuhn and 3.20 ml/g according to Simha, for the glutamate dehydrogenase oligomer, in good agreement with the result derived from the study of enzyme solutions.     

Experimental studies upon a bundle of tonic fibres in the locust extensor tibialis muscle

The bundle of tonic fibres situated at the proximal end of the locust metathoracic extensor tibialis muscle is innervated by the dorsal unpaired median neurone (DUMETi) as well as by the slow excitatory (SETi)) and common inhibitor (CI) neurones. It is not innervated by the fast excitatory neurone (FETi).These fibres contract spontaneously and rhythmically. The myogenic rhythm can be modified by neural stimulation.Spontaneous slow depolarizing potentials resembling the pacemaker potentials of insect cardiac muscle were demonstrated in these fibres.The actions of glutamate on the tonic muscle fibres are not compatible with its being a specific excitatory transmitter. Glutamate can stimulate weak contractions of the muscle, but this action is inhibited when chloride ions are removed from the saline.10^?6 M Octapamine hyperpolarizes the tonic fibre membrane. Octopamine, GABA and glutamate all inhibit the myogenic contractions and reduce the force of the neurally evoked contractions.The tonic muscle is very responsive to proctolin. At 5 × 10^?11 M proctolin enhances the force and increases the frequency of myogenic contractions. At 10^?9 M it depolarizes the muscle membrane potential, and at that and higher concentrations it causes the muscle to contract. At 2 × 10^?7 M proctolin induces contractures which resemble those evoked by sustained high-frequency neural stimulation. Iontophoretic experiments show that proctolin receptors occur at localized sites on the tonic fibre membrane.     

Activity of Various Growth Substances in the Avena First Internode Test

The elongation growth of the Avena first internode segments was studied in the presence of one or several of the following growth substances: indoleacetic acid (IAA), 6-fur-furylamino purine (FAP, kinetin), 6-benzylamino purine (BAP), gibberellin A₃ (GA₃) and A₄₊₇ (GA₄₊₇), and abscisic acid (ABA). The cytokinins at concentrations of 10^?7 to 10^?6M stimulated growth with 4 to 6 per cent but this effect was not statistically significant. Concentrations higher than 5 × 10^?6M inhibited growth. FAP and BAP (from 10^?8M to 10^?6M) had no significant interaction with any other growth substance used. The two-factor interactions of IAA × ABA, IAA × GA₃, and GA₃× ABA, as well as the three-factor interaction IAA × ABA × GA₃ were significant. However, the IAA × ABA interaction was significant only when high concentration (10^?6M) of ABA was used. The growth inhibition produced by 10^?7 and 10^?6M ABA was overcome by about equimolar concentrations of IAA. The stimulation of growth by GA₃ and GA₄₊₇ (10^?9 to 10^?7M) was prevented by simultaneous application of ABA, and it was reduced significantly by application of IAA (10^?7 to 10^?8M). GA3 at 10^?8M combined with different concentrations of IAA gave slightly higher elongation than IAA alone but the observed values were significantly lower than expected assuming independent additive action.     

Stimulation of Growth of Peppermint (Mentha piperita) by Phosfon, a Growth Retardant

The shoot growth and fresh weight of Mentha piperita grown in soil were stimulated at concentrations of 1.26 × 10^?5M to 7.77 × 10^?4M phosfon (2,4-dichlorobenzyl tributyl phosphonium chloride) while higher concentrations resulted in retardation of growth. Concentrations of 6.30 × 10^?7M to 3.78 × 10^?5M caused retardation of growth in mineral nutrient solution, and even death at the highest concentrations. However, when the M. piperita plants were grown in mineral nutrient solutions at concentrations of phosfon which had been sequentially lowered from 2.52 × 10^?8M to 2.52 × 10^?12M, the shoot growth and fresh weight were stimulated as in the case of plants grown in phosfon treated soil.     

Chemical sensitivity of the hyperneural nerve-muscle preparation of the American cockroach

The hyperneural muscle of Periplaneta americana responded with sustained contracture to applications of l-glutamic acid at near 10^?4 M. d-glutamic acid was much less active. The responses of a particular preparation to glutamate were usually extremely consistent and highly reproducible; however, some preparations showed no response to l-glutamic acid even at 10^?2 M whereas neurally evoked responses were normal. High magnesium, low calcium perfused onto the preparations blocked neurally evoked contractions. The glutamate response was blocked reversibly in low calcium solutions. suggesting that the glutamate effect, when present, was presynaptic.Dopamine, acetylcholine, 5-hydroxytryptamine, synephrine, Rogitine^®, strychnine, strychnine, pentobarbital, and picrotoxin, all suspected to varying degrees of some action on insect central or peripheral synaptic transmission, had no effect on the patterns of neurally evoked contracture of the hyperneural muscle. A new transducer is described for use with low force insect muscle contractions.     

Blockade of central beta-adrenergic receptors by tazolol (1-isopropylamino-3-(2-thiazoloxy)-2-propanol).

Tazolol, a β₁-adrenergic agonist in heart, had no intrinsic β-adrenergic agonist activity with respect to cyclic AMP-generating systems in rat cerebral cortical slices or with respect to firing of rat cerebellar Purkinje cells. Instead, tazolol proved to be a relatively potent and specific β-adrenergic antagonist. The IC₅₀ for (±) tazolol in antagonizing (-) isoproterenol-elicited accumulation of cyclic AMP in rat cortical slices was 7 × 10^?7M. The IC₅₀ in antagonizing [³H] dihydroalprenolol-binding in rat cortical homogenates was 2.9 × 10^?7 M. Tazolol was about 10 fold more potent in both cases than the β-antagonist, (±) sotalol. Tazolol antagonized the inhibitory, β-adrenergically mediated effects of iontophoretically applied norepinephrine on firing of cerebellar Purkinje cells. The inhibitory effects of γ-aminobutyric acid on firing of Purkinje cells were not altered by tazolol. Tazolol appeared to lack significant local anesthetic activity as evidenced by its lack of effect on spike height in spontaneous firing Purkinje cells.     

Some pharmacological properties of neuromuscular transmission in the oviduct of the locust,Locusta migratoria

The effects of various pharmacological agents on neurally evoked contractions of the visceral muscles of the oviduct of Locusta migratoria have been examined. The pentapeptide, proctolin, at low concentrations (10^?11 M?10^?10 M), induced an increase in the amplitude of neurally evoked contractions and basal tonus, and induced the appearance and increased the frequency of myogenic contractions. Glutamate, at 10^?4 M, produced a small transient contraction which in some preparations was accompanied by a reduction in amplitude of neurally evoked contractions. Octopamine, at 10^?6 M, reduced the amplitude of neurally evoked contractions and also resulted in a relaxation of the muscles. The octopaminergic effects were inhibited by the α-aminergic antagonist phentolamine. Neurally evoked contractions were unaffected by dopamine, 5-HT or the acetylcholine receptor antagonists atropine and hexamethonium. Acetylcholine increased the amplitude of neurally evoked contractions, but only at the high concentration of 10^?3 M. The possible role of proctolin and glutamate as excitatory neuro-transmitters and the inhibitory action of octopamine is discussed.     

The excitatory glutamate-activated channel recorded in cell-attached and excised patches from the membranes of tail,leg and stomach muscles of crayfish

Summary Glutamate activated, excitatory single channel currents were recorded from 5 different muscles of crayfish (Austropotamobius torrentium) from abdomen, legs and stomach. Cell-attached and outside-out excised membrane patches with G-seals were studied. At –70 mV membrane potential and 19 °C, single channel currents activated by 0.5 mM glutamate had an amplitude of –7.6 pA, a mean open time of 0.22 ms and a mean burst length of 0.58 ms. These values did not show significant differences in all muscles investigated. The distributions of open times and of burst durations were described by single exponentials. The distributions of closed times could be fitted only by at least two exponentials. The short component of on average 0.1 ms represented closings within bursts, a longer component of on average 0.9 ms grouping of bursts. Burst durations (but not individual open times) increased with rising glutamate concentration: the relative open time of the channel was approximately proportional to glutamate concentration between 0.1 and 5 mM. The channels described above could not be activated by the glutamate analogues kainate and NMDA, but were about 10 times more sensitive to quisqualate than to glutamate. Quisqualate elicited single channel currents of the same amplitude as those triggered by glutamate. Compared at the same concentrations, channel open times and burst durations were about 4 times longer in quisqualate than in glutamate. A model describing the kinetics of the glutamate-activated excitatory channels is discussed. In addition, a type of Ca-independent, depolarization-activated K⁺-channel is reported.     

The mode of action of phenylhydrazine hydrochloride on the locust neuromuscular system

The action of the carbonyl reagent phenylhydrazine hydrochloride (Phen. HCl) on locust excitatory neuromuscular systems was studied by examining the effects of this compound on the mechanical and electrical properties of the retractor unguis and extensor tibiae muscles of the locust Schistocerca gregaria.Low concentrations of Phen. HCl (10^?9 w/v to 2·5 × 10^?5 w/v) potentiated the muscle contractions and the excitatory post-synaptic potential (EPSP), the optimum concentration being about 10^?5 w/v. 10^?8 w/v Phen. HCl increased miniature EPSP frequency, but this increase became less pronounced as the concentration was raised, and no increase at all was observed at 10^?5 w/v. There was no change in miniature EPSP amplitude at any concentration. Higher concentrations of Phen. HCl (> 2·5 × 10^?6 w/v) depressed the neurally evoked contraction, the EPSP, and the response of the muscle to iontophoretically applied l-glutamate. A gradual increase in muscle input conductance was observed on perfusion with these high concentrations of Phen. HCl. The presence of magnesium in the bathing fluid (15 m-moles/l.) reduced the effectiveness of Phen. HCl in potentiating the EPSP and delayed or reduced the increase in input conductance observed on perfusion with high concentrations of Phen. HCl.The results indicate that low concentrations of Phen. HCl act presynaptically, possibly by depolarizing the excitatory nerve terminals. Higher concentrations may act directly on the post-synaptic glutamate receptors.     

Interaction of Kinetin and Indoleacetic Acid in the Avena Straight-Growth Test

Kinetin has a stimulating effect in the Avena straight-growth test. The action of different concentrations of kinetin, 2.5 × 10^?7, 2.5 × 10^?6 and 2.5 × 10^?5M, in combination with different concentrations of IAA was studied in this test. It was shown that the effect of low IAA concentrations, 0.25 × 10^?7 and 1 × 10^?7M, was strongly enhanced by the addition of all the kinetin concentrations investigated. The effect of the highest IAA concentrations, 25 × 10^?7 and 100 × 10^?7M, on the other hand, was inhibited relatively strongly by the highest employed concentration of kinetin. The results are explained as due to a kinetin-produced increase of auxin in the coleoptile segment, which in combination with low IAA concentrations can lead to a growth stimulation and with high IAA concentrations to a growth inhibition. Since kinetin in purification and chromatography of auxin can partly follow IAA, thereby affecting the quantitative yield, it is emphasized that, prior to the test, auxin extracts containing cytokinins should be freed from the latter by, for example, gel filtration or paper electrophoresis.     

Comparative Effects and Concentration of Picloram, 2,4,5-T and Dicamba in Tissue Culture

In nutrient agar comparative concentrations (10^?3 to 10^?5M) of (2,4,5-trichlorophenoxy)acetic acid (2,4,5-T) were generally more inhibitory to the growth of tissue cultures of soybean (Glycine max (L.) Merrill cv. Acme) and cottonwood (Populus deltoides Marsh.) than were either 4-amino-3,5,6-trichloropicolinic acid (picloram) or 3,6-dichloro-o-anisic acid (dicamba). Compared to untreated tissue dicamba or picloram at 10^?6M in the nutrient agar resulted in a 200 % increase in the growth of soybean tissue. At 10^?5 and 10^?6M dicamba also produced an increase in the growth of cottonwood tissue. Greatest absorption of picloram and dicamba by tissue cultures from agar occurred during the first 24 h after treatment. However, absorption remained nearly static thereafter for 14 days. More dicamba was absorbed by soybean and cottonwood tissue cultures than either picloram or 2,4,5-T.     

Adenosine Triphosphatases Bound to Plasmalemma, Mitochondria, and Microsomes or Present in the Cytoplasmic Phase from Potato Tubers

Between pH 4–10, basal ATPase activity, measured in the absence of mineral ions, was 10 to 100 times higher in the final cytoplasmic supernatant from potato tuber homogenates than in the membraneous fractions (purified plasmalemma, purified mitochondria and microsomes). The soluble ATPase was slightly inhibited, whereas the membrane-bound ATPases were all stimulated by Mg²⁺ ions. A further stimulation by Na⁺ or K⁺ ions was only observed in purified plasmalemma or mitochondria, at alkaline pH (7.5–9.5). At a fixed (Na⁺+ K⁺) concentrations (80 mM), this last stimulation was much greater in purified mitochondria (350%) than in plasmalemma (33%); it also increased with (Na⁺+ K⁺) concentrations up to 200 mM in mitochondria whereas, in plasmalemma, it was roughly constant for monovalent ion concentrations between 20 and 200 mM. General properties of the plasma membrane-bound ATPase have been determined, i.e. substrate specificity, activity variations with quantity of substrate, temperature, pH, etc. Divalent cations stimulated strongly the ATPase in the following order: Mn²⁺ > Mg²⁺ > Ca²⁺. The maximum ATP hydrolysis velocity for that part of ATPase activity which is strictly dependent on Mg²⁺ ions was 3.85 μmol × mg^?1 protein × h^?1. This plasma membrane ATPase was not sensitive to ouabaïn or to oligomycin.     

Magnesium ion effect on the helix-coil transition of DNA

The effect of magnesium ions on the parameters of the DNA helix-coil transition has been studied for the concentration range 10^?6–10^?1M at the ionic strengths of 10^?3M Na⁺. Special attention has been given to the region of low ion concentrations and to the effect of polyvalent metallic impurities present in DNA. It has been shown that binding with Mg⁺⁺ increases the DNA stability, the effect being observed mainly in the concentration range 10^?6–10^?4M. At[Mg⁺⁺]>10^?2M the thermal stability of DNA starts to decrease. The melting range extends to concentrations ～10^?5M and then decreases to 7–8°C at the ion content of 10^?3M. Asymmetry of the melting curves is observed at low ionic strengths ([Na⁺] = 10^?3M) and [Mg⁺⁺] ? 10^?5M. The results, analyzed in terms of the statistical thermodynamic theory of double-stranded homopolymers melting in the presence of ligands, suggest that the effects observed might be due to the ion redistribution from denatured to native DNA. An experimental DNA–Mg⁺⁺ phase diagram has been obtained which is in good agreement with the theory. It has been shown that thermal denaturation of the system may be an efficient method for determining the ion-binding constants for both native and denatured DNA.