Role Of Mg2 Research Articles

The role of Mg2+ in the hydrolytic activity of the isolated chloroplast ATPase: Study by high-performance liquid chromatography

The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F1 ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima, Km values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg2+. Although the equations giving the reaction rate are similar in the two cases, the calculated values of Km are widely different. The value obtained from the classical mechanism does not agree with KD, the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na+ is more easily explained as a competition between this ion and the activating Mg2+, than by the classical mechanism.

Interaction of mitochondrial F1-ATPase with trinitrophenyl derivatives of ATP and ADP. Participation of third catalytic site and role of Mg2+ in enzyme inactivation.

Relatively high ATP concentrations show an unexpected lack of inhibition of the hydrolysis of low concentrations of trinitrophenyl ATP (TNP-ATP) by mitochondrial F1-ATPase. In striking contrast low TNP-ATP concentrations markedly inhibit the hydrolysis of much higher ATP concentrations. The three catalytic sites undergoing sequential conformational changes have different conformations at any instant of catalysis, and only two need to be filled for rapid, steady-state ATP hydrolysis. The remaining site has low affinity for ATP (Kd 2 mM) but about 10(4) greater affinity for TNP-ATP (Km and Kd about 0.2 microM). Thus 500 microM ATP does not prevent binding of less than 1 microM TNP-ATP. As the site binding the TNP-ATP undergoes sequential conformational changes the TNP-ATP undergoes sequential conformational changes the TNP-ATP is hydrolyzed and products are released. The results give strong support to the view that all three catalytic sites proceed equivalently in ATP as well as TNP-ATP hydrolysis. The conformation that has the lowest affinity for ATP has over a 10-fold greater affinity for ADP (Kd 150 microM) and may be akin to the conformation to which ADP binds during net ATP synthesis by the ATP synthase. The recognition of these features was made possible by new information obtained from detailed studies of the interactions of Mg2+, TNP-ADP, TNP-ATP, ATP, and noncatalytic sites on initial and steady-state hydrolysis rates.

The dephosphorylation reaction of the Ca(2+)-ATPase from plasma membranes.

The breakdown of phosphoenzyme (EP) of the Ca(2+)-ATPase from pig red blood cell membranes was studied at 37 degrees C by means of a rapid chemical quenching technique. When the enzyme was phosphorylated with [gamma-32P]ATP in media without added MgCl2, all the EP formed disappeared along two single exponential curves, a rapid one with k(app) = 90 +/- 10 s-1 and a slow one with k(app) = 0.7 +/- 0.3 s-1. The amount of EP involved in each reaction was close to 50% of the EP present at the beginning. Only EP of rapid breakdown could account for the steady-state hydrolysis of ATP observed under the same experimental conditions. ADP accelerated the slow reaction 45-fold (k(app) = 31 +/- 9 s-1) with K0.5 = 740 +/- 120 microM as if this reaction represented the decay of CaE1P, which donated its phosphate to water slowly in the forward direction and rapidly to ADP in the reverse direction of the cycle. Combination of Mg2+ with K0.5 = 26.3 +/- 5.0 microM at a single class of site in E1 before phosphorylation increased EP of rapid breakdown at the expense of ADP-sensitive EP so that, at nonlimiting concentrations of Mg2+ in the phosphorylation media, all EP decomposed at high rate. Rapid decomposition was observed even with enough CDTA to chelate most of the Mg2+ remaining from phosphorylation, suggesting that the role of Mg2+ during dephosphorylation was to accelerate the transition CaE1P-->CaE2P, preparing EP for hydrolysis. The combination of ATP at a single class of site with Km = 845 +/- 231 microM accelerated the hydrolysis of CaE2P. Calmodulin alone had no effects on dephosphorylation but enhanced acceleration of hydrolysis of CaE2P by ATP making the decay of EP under these conditions the fastest among those measured. Comparison of the rates of dephosphorylation of EP made in the presence of Mg2+ with those of steady-state Ca(2+)-ATPase activity with and without calmodulin showed that the transition CaE1P-->CaE2P and decomposition of CaE2P by hydrolysis are compatible with their role as obligatory intermediate reactions in the cycle of hydrolysis of ATP by the Ca(2+)-ATPase.

Probing the hammerhead ribozyme structure with ribonucleases.

Susceptibility to RNase digestion has been used to probe the conformation of the hammerhead ribozyme structure prepared from chemically synthesised RNAs. Less than about 1.5% of the total sample was digested to obtain a profile of RNase digestion sites. The observed digestion profiles confirmed the predicted base-paired secondary structure for the hammerhead. Digestion profiles of both cis and trans hammerhead structures were nearly identical which indicated that the structural interactions leading to self-cleavage were similar for both systems. Furthermore, the presence or absence of Mg2+ did not affect the RNase digestion profiles, thus indicating that Mg2+ did not modify the hammerhead structure significantly to induce self-cleavage. The base-paired stems I and II in the hammerhead structure were stable whereas stem III, which was susceptible to digestion, appeared to be an unstable region. The single strand domains separating the stems were susceptible to digestion with the exception of sites adjacent to guanosines; GL2.1 in the stem II loop and G12 in the conserved GAAAC sequence, which separates stems II and III. The absence of digestion at GL2.1 in the stem II hairpin loop of the hammerhead complex was maintained in uncomplexed ribozyme and in short oligonucleotides containing only the stem II hairpin region. In contrast, the G12 site became susceptible when the ribozyme was not complexed with its substrate. Overall the results are consistent with the role of Mg2+ in the hammerhead self-cleavage reaction being catalytic and not structural.

Molecular Recognition and Mechanism of Action of Hammerhead Ribozyme-Metalloenzyme

The structure of a hammerhead ribozyme does not change significantly in the presence or absence of Mg2+ ions, an indication that the role of Mg2+ ions is catalytic rather than structural. Accordingly, kinetics and molecular orbital calculations indicate that ribozymes are metalloenzymes. Kinetic analysis supports the idea that an important part of the binding energy (reflected in Kd) is utilized to provide the driving force for catalysis, so that the observed binding energy (Km reflected in) represents only what is left over after this utilization. It is also possible to produce nuclease-resistant ribozymes.

Crystal structures of ribonuclease HI active site mutants from Escherichia coli.

In order to investigate the relationships between the three-dimensional structure and the enzymic activity of E. coli RNase HI, three mutant proteins, which were completely inactivated by the replacements of three functional residues, Asp10 by Asn (D10N), Glu48 by Gln (E48Q), and Asp70 by Asn (D70N), were crystallized. Their three-dimensional structures were determined by x-ray crystallography. Although the entire backbone structures of these mutants were not affected by the replacements, very localized conformational changes were observed around the Mg(2+)-binding site. The substitution of an amide group for a negatively charged carboxyl group in common induces the formation of new hydrogen bond networks, presumably due to the cancellation of repulsive forces between carboxyl side chains with negative charges. These conformational changes can account for the loss of the enzymic activity in the mutants, and suggest a possible role for Mg2+ in the hydrolysis. Since the 3 replaced acidic residues are completely conserved in sequences of reverse transcriptases from retroviruses, including human immunodeficiency virus, the concepts of the catalytic mechanism deduced from this structural analysis can also be applied to RNase H activity in reverse transcriptases.

Mg2+ affects the binding of ADP but not ATP to 3-phosphoglycerate kinase. Correlation between equilibrium dialysis binding and enzyme kinetic data.

The role of Mg2+ in the binding of ADP and ATP to pig muscle and yeast 3-phosphoglycerate kinases has been studied by equilibrium dialysis. Whereas the Kd of ATP binding varies between 0.17 and 0.23 mM (S.E.M. 0.03 mM) for both enzymes, independently of the presence of Mg2+, the Kd values for ADP and MgADP binding are in the range 0.18-0.27 mM (S.E.M. 0.04 mM) and 0.05-0.06 mM (S.E.M. 0.01 mM) respectively. Thus Mg2+ exclusively tightens the interaction of ADP, but not of ATP, with the protein molecule. Although the equilibrium dialysis data are consistent with a model possessing a single site for nucleotides, the existence of a much weaker secondary site (with a Kd value at least two orders of magnitude larger) cannot be excluded. The binding of AMP and adenosine to pig muscle 3-phosphoglycerate kinase is weaker than binding of MgATP; the respective Kd values are 0.36 +/- 0.05 mM and 0.65 +/- 0.05 mM. Thus, in addition to the interaction of the alpha-phosphate that is detectable by crystallography [Banks, Blake, Evans, Haser, Rice, Hardy, Merrett and Phillips (1979) Nature (London) 279, 773-777], the beta- and/or gamma-phosphate(s) of MgATP may also interact with the enzyme molecule. The fact that MgADP binds more tightly than ADP is consistent with its stronger inhibition of the reaction catalysed by the enzyme between 3-phosphoglycerate and MgATP. MgADP is a product of this reaction, and inhibits it competitively with both substrates; as an inhibitor its KI is comparable with the Kd found in binding studies. At the same time, the Km value for MgADP in the reverse reaction (0.18 +/- 0.05 mM; mean +/- S.E.M.) is higher than these constants; this may be due either to a different kinetic mechanism in this direction of the enzymic reaction, or to different binding modes of MgADP as inhibitor and as substrate. The reason why inhibition by MgADP is competitive with 3-phosphoglycerate may be that its binding prevents the specific change in conformation that the enzyme undergoes [Harlos, Vas and Blake (1992) Proteins 12, 133-144] when it binds 3-phosphoglycerate.

Ribonuclease H. A magnesium-dependent endonuclease. Role of Mg2+ in substrate binding and evidence for a distinct polynucleotide binding regulatory domain.

Ribonuclease H. A magnesium-dependent endonuclease. Role of Mg2+ in substrate binding and evidence for a distinct polynucleotide binding regulatory domain.

Role of Mg2+ ions in the conformational change reported by fluorescein 5'-isothiocyanate modification of Na+,K(+)-ATPase.

The role of Mg2+ in the conformational change reported by fluorescein 5'-isothiocyanate modification of Na,K-ATPase has been studied by stopped-flow fluorometry. K+ causes a fluorescence quench that is reversed by Na+. The principal experimental observations are as follows: (1) Mg2+ decreases the apparent affinity of the enzyme for K+ but does not affect the maximum rate of the K+ quench. (2) The amplitude of the K+ quench depends hyperbolically on the K+ concentration, and the maximum amplitude is unaffected by the Mg2+ concentration. (3) The rate at which Na+ reverses the K+ quench depends inversely on the Mg2+ concentration. (4) The amplitude of the Na+ reversal also decreases with increasing Mg2+ concentration. The data are quantitatively explained by a model that assumes only two enzyme conformations, detectable by their fluorescence emission. Mg2+ increases Kd for K+ from 14 to 223 mM. At 22 degrees C, Kd = 0.16 mM for Mg2+ dissociation from E1, and the heat of Mg2+ binding, delta H degrees, is 11.4 kcal mol-1. Kd is more than an order of magnitude larger for Mg2+ dissociation from E2K. Mg2+ binding does not affect the forward (E1K-->E2K) rate constant (kf), but decreases the reverse rate constant (kr) thus increasing the equilibrium constant for the reaction (Kc = kf/kr) 6-fold. Therefore, Mg2+ is not directly involved in the conformational transition, but the study supports proposals that Mg2+ binding and release may help to regulate the transport cycle by shifting the distribution of enzyme between E1 and E2 conformers.

Modeling Study on the Cleavage Step of the Self-Splicing Reaction in Group I Introns

A three-dimensional model of the Tetrahymena thermophila group I intron is used to further explore the catalytic mechanism of the transphosphorylation reaction of the cleavage step. Based on the coordinates of the catalytic core model proposed by Michel and Westhof (Michel, F., Westhof, E. J. Mol. Biol. 216, 585-610 (1990)), we first converted their ligation step model into a model of the cleavage step by the substitution of several bases and the removal of helix P9. Next, an attempt to place a trigonal bipyramidal transition state model in the active site revealed that this modified model for the cleavage step could not accommodate the transition state due to insufficient space. A lowering of P1 helix relative to surrounding helices provided the additional space required. Simultaneously, it provided a better starting geometry to model the molecular contacts proposed by Pyle et al. (Pyle, A. M., Murphy, F. L., Cech, T. R. Nature 358, 123-128. (1992)), based on mutational studies involving the J8/7 segment. Two hydrated Mg2+ complexes were placed in the active site of the ribozyme model, using the crystal structure of the functionally similar Klenow fragment (Beese, L.S., Steitz, T.A. EMBO J. 10, 25-33 (1991)) as a guide. The presence of two metal ions in the active site of the intron differs from previous models, which incorporate one metal ion in the catalytic site to fulfill the postulated roles of Mg2+ in catalysis. The reaction profile is simulated based on a trigonal bipyramidal transition state, and the role of the hydrated Mg2+ complexes in catalysis is further explored using molecular orbital calculations.

Identification of a Mg(2+)- and guanyl nucleotide-dependent glucagon receptor cycle by use of permeabilized canine hepatocytes.

We have investigated (by use of intact and saponinpermeabilized canine hepatocytes) the roles of Mg2+ and guanyl nucleotides in regulating glucagon-receptor interactions. In contrast to intact cells, saponinpermeabilized hepatocytes bind [[125I]iodo-Tyr10]glucagon according to a single first-order process and exhibit a single apparent dissociation constant for glucagon binding during steady-state incubations. Further analysis of the permeabilized cell system demonstrated (a) the temperature-sensitive action of Mg2+ to enhance the extent and affinity of glucagon-receptor interactions at steady-state, (b) the conversion of Mg(2+)-independent hormone-receptor complexes to Mg(2+)-dependent complexes, (c) the effect of guanyl nucleotides to inhibit specifically the Mg(2+)-dependent component of glucagon-receptor interactions, (d) the more rapid association of glucagon with receptor during cell incubations occurring in the presence of guanyl nucleotides or in the absence of Mg2+, and (e) the ability of guanyl nucleotides to induce both high and low affinity states of glucagon-receptor interactions. Additional experiments identified an effect of cell incubations in the presence of glucagon to limit the subsequent binding of hormone, the ability of GDP, GTP, or guanosine-5'-3-O-(thio)triphosphate (GTP gamma S) to dissociate previously bound glucagon, and a specific requirement for GDP to re-activate the glucagon receptor for additional cycles of hormone binding. A model is presented in which (a) glucagon binds to receptor in a Mg(2+)-independent fashion, (b) glucagon-receptor complexes are converted to a Mg(2+)-dependent state, (c) guanyl nucleotide exchange initiates both an alteration in glucagon-receptor affinity and the subsequent dissociation of hormone, and (d) in the context of the intact cell, G protein-mediated hydrolysis of GTP to GDP is required to reinitialize the system.

Role of magnesium ion in the interaction between chromomycin A3 and DNA: binding of chromomycin A3-Mg2+ complexes with DNA.

Chromomycin A3 is an antitumor antibiotic which blocks macromolecular synthesis via reversible interaction with DNA template only in the presence of divalent metal ions such as Mg2+. The role of Mg2+ in this antibiotic-DNA interaction is not well understood. We approached the problem in two steps via studies on the interaction of (i) chromomycin A3 and Mg2+ and (ii) chromomycin A3-Mg2+ complex(es) and DNA. Spectroscopic techniques such as absorption, fluorescence, and CD were employed for this purpose. The results could be summed up in two parts. Absorption, fluorescence, and CD spectra of the antibiotic change upon addition of Mg2+ due to complex formation between them. Analysis of the quantitative dependence of change in absorbance of chromomycin A3 (at 440 nm) upon input concentration of Mg2+ indicates formation of two types of complexes with different stoichiometries and formation constants. Trends in change of fluorescence and CD spectroscopic features of the antibiotic in the presence of Mg2+ at different concentrations further corroborate this result. The two complexes are referred to as complex I (with 1:1 stoichiometry in terms of chromomycin A3:Mg2+) and complex II (with 2:1 stoichiometry in terms of chromomycin A3:Mg2+), respectively, in future discussions. The interactions of these complexes with calf thymus DNA were examined to check whether they bind differently to the same DNA. Evaluation of binding parameters, intrinsic binding constants, and binding stoichiometry, by means of spectrophotometric and fluorescence titrations, shows that they are different. Distinctive spectroscopic features of complexes I and II, when they are bound to DNA, also support that they bind differently to the above DNA. Measurement of thermodynamic parameters characterizing their interactions with calf thymus DNA shows that complex I-DNA interaction is exothermic, in contrast to complex II-DNA interaction, which is endothermic. This feature implies a difference in the molecular nature of the interactions between the complexes and calf thymus DNA. These observations are novel and significant to understand the antitumor property of the antibiotic. They are also discussed to provide explanations for the earlier reports that in some cases appeared to be contradictory.

The role of Mg2+ and K+ in the phosphorylation of Na+,K+-ATPase by ATP in the presence of dimethylsulfoxide but in the absence of Na+

We have previously demonstrated that Na+,K+-ATPase can be phosphorylated by 100 μM ATP and 5 mM Mg2+ and in the absence of Na+, provided that 40% dimethylsulfoxide (Me2SO) is present. Phosphorylation was stimulated by K+ up to a steady-state level of about 50% of Etot (Barrabin et al. (1990) Biochim. Biophys. Acta 1023, 266–273). Here we describe the time-course of phosphointermediate (EP) formation and of dephosphorylation of EP at concentrations of Mg2+ from 0.1 to 5000 μM and of K+ from 0.01 to 100 mM. The results were simulated by a simplified version of the commonly accepted Albers-Post model, i.e. a 3-step reaction scheme with a phosphorylation, a dephosphorylation and an isomerization/deocclusion step. Furthermore it was necessary to include an a priori, Mg2+- and K+-independent, equilibration between two enzyme forms, only one of which (constituting 14% of Etot) reacted directly with ATP. The role of Mg2+ was two-fold: At low Mg2+, phosphorylation was stimulated by Mg2+ due to formation of the substrate MgATP, whereas at higher concentrations it acted as an inhibitor at all three steps. The affinity for the inhibitory Mg2+-binding was increased several-fold, relative to that in aqueous media, by dimethylsulfoxide. K+ stimulated dephosphorylation at all Mg2+-concentrations, but at high, inhibitory [Mg2+], K+ also stimulated the phosphorylation reaction, increasing both the rate coefficient and the steady-state level of EP. Generally, the presence of Me2SO seems to inhibit the dephosphorylation step, the isomerization/deocclusion step, and to a lesser extent (if at all) the phosphorylation reaction, and we discuss whether this reflects that Me2SO stabilizes occluded conformations of the enzyme even in the absence of monovalent cations. The results confirm and elucidate the stimulating effect of K+ on EP formation from ATP in the absence of Na+, but they leave open the question of the molecular mechanism by which Me2SO, inhibitory Mg2+ and stimulating K+ interact with the Na+,K+-ATPase.

Endothelial-dependent sexual dimorphism in vascular smooth muscle: role of Mg2+ and Na+.

1. In isolated aortae of the male rat [Mg2+]o withdrawal and concomitant reduction in [Na+]o (to 84 mM) induced significant increases of basal tone, but, surprisingly, this did not occur in intact aortae removed from female rats. Such tension development, however, was observed in endothelium-denuded aortic preparations from both sexes. These observed gender-related differences were not dependent on animal strain or types of tissue preparations. 2. No tension development was observed in aortae obtained from castrated males treated with oestradiol. Aortic tissues of sexually-immature male and female rats exhibited marked tension development when exposed to 0 mM [Mg2+]o and low [Na+]o. 3. Tension development in Mg(2+)-free, low-Na+ media was not tachyphylactic and completely dependent on extracellular Ca2+; addition of 1.2 mM Mg2+ to the Mg2+ and Na(+)-deficient incubation media relaxed the increase in tension to a normal basal level. 4. Two known endothelial-derived relaxant factor (EDRF) inhibitors, methylene blue and haemoglobin, induced tension development in female aortae with intact endothelium exposed to Mg(2+)-Na+ deficient media, while use of a specific inhibitor of EDRF-derived nitric oxide, viz., NG-monomethyl-L-arginine (L-NMMA), resulted in potentiation of tension development in male, but not in female, aortae. This effect of L-NMMA was antagonized by L-arginine. 5. The Ca ionophore, A23187, partially relaxed contractile responses in male aortae (with intact endothelium) which were followed by potentiated contractions. Endothelium-dependent vasodilator responses to A23187 (10(-10)-10(-6) M) of aortic rings from male or female rats in normal Krebs-Ringer bicarbonate solution were not different.6. These results suggest that: (a) as in vascular smooth muscle cells, Mg2+ plays an important role in Ca2 + homeostasis in endothelial cells, probably via Na+-Ca2+ exchange; and (b) sex steroid hormones, probably the female sex hormone, 17-beta-oestradiol, may regulate contractile responses of intact vascular smooth muscle by modifying endothelium functions through such Mg2 '-regulated internal Natdependent Ca2+ entry. These data may help to explain why female subjects, despite Mg deficiency, unlike male subjects, are protected against ischaemic heart disease and cerebrovascular disease until menopause.

Effect of Mg2+ on Na(+)-dependent inositol transport. Role for Mg2+ in etiology of diabetic complications

Effect of Mg2+ on Na(+)-dependent inositol transport. Role for Mg2+ in etiology of diabetic complications

Isocitrate lyase from Phycomyces blakesleeanus. The role of Mg2+ ions, kinetics and evidence for two classes of modifiable thiol groups

Isocitrate lyase was purified from Phycomyces blakesleeanus N.R.R.L. 1555(-). The native enzyme has an Mr of 240,000. The enzyme appeared to be a tetramer with apparently identical subunits of Mr 62,000. The enzyme requires Mg2+ for activity, and the data suggest that the Mg2(+)-isocitrate complex is the true substrate and that Mg2+ ions act as a non-essential activator. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated an ordered Uni Bi mechanism and the kinetic constants of the model were calculated. The spectrophotometric titration of thiol groups in Phycomyces isocitrate lyase with 5.5'-dithiobis-(2-nitrobenzoic acid) gave two free thiol groups per subunit of enzyme in the native state and three in the denatured state. The isocitrate lyase was completely inactivated by iodoacetate, with non-linear kinetics. The inactivation data suggest that the enzyme has two classes of modifiable thiol groups. The results are also in accord with the formation of a non-covalent enzyme-inhibitor complex before irreversible modification of the enzyme. Both the equilibrium constants for formation of the complex and the first-order rate constants for the irreversible modification step were determined. The partial protective effect of isocitrate and Mg2+ against iodoacetate inactivation was investigated in a preliminary form.

Supra-additive stimulation of adenylate cyclase activity by prostaglandin E2 and D-Ala2-met-enkephalinamide in the guinea-pig superior cervical ganglion: role of Mg2+ions.

Agonists modulation of Mg2(+)-dependent adenylate cyclase activity has been studied in guinea-pig superior cervical ganglion crude membrane preparations. In the absence of receptors ligands, Mg2+ stimulates the enzyme in a concentration-dependent manner. The dose-activation curve shows heterogeneity and two components with "higher" and "lower" apparent affinity states, are extrapolated. In the presence of D-Ala2-met-enkephalinamide only one component is present and the apparent affinity of the ganglionic adenylate cyclase system for the divalent cation as well as Vmax are inhibited. On the contrary, prostaglandin E2 increases affinity and Vmax values of the lower and, to a lesser extent, of the higher Km component. When the two drugs are tested in combination, not only the inhibitory effect of the opiate is overcome, but a large increase of the apparent affinities and Vmax values for both components is obtained, suggesting the involvement of the Mg2(+)-regulated subunits of the adenylate cyclase system in the supra-additive stimulation mechanism of the enzyme.

Effect of magnesium on depression of the monosynaptic reflex induced by 2-chloroadenosine or hypoxia in the isolated spinal cord of neonatal rats

Superfusion of the isolated spinal cord of neonatal rats (4-9 days postpartum) with physiological medium containing 2-chloroadenosine (2-CA) or anoxic medium (equilibrated with 95% N2-5% CO2) depressed the evoked monosynaptic reflex (MSR) recorded extracellularly from a ventral spinal root. The effectiveness of 2-CA or anoxic medium in depressing the MSR was significantly reduced when the concentration of Mg2+ in the physiological medium was lowered from 1.25 X 10(-3) M to zero. The absence of Mg2+ resulted in a 7-fold shift to the right of the concentration-response curve to 2-CA and a reduction in the maximal depression of the MSR from 100% to 65 +/- 4% (mean +/- S.E.M.) of control. A 10 min exposure to anoxic medium containing 1.25 X 10(-3) M Mg2+ decreased the amplitude of the MSR to 23 +/- 6% of control, whilst in zero Mg2+ a decrease to only 50 +/- 5% of control was observed. These data provide further evidence that the response to adenosine, at the A1-receptor, is sensitive to Mg2+ ion concentration and suggest that there is an absolute requirement for Mg2+ in order to obtain full expression of the adenosine effect. Furthermore, the data are consistent with the hypothesis that adenosine is an important mediator of hypoxia-induced depression of the evoked MSR in the spinal cord, and suggest a potential role for Mg2+ during or after exposure to hypoxia in altering the actions of adenosine on neuronal activity or synaptic events.

Essential role of magnesium in oxytocin-receptor affinity and ligand specificity

We have analysed, with the aid of a novel radioiodinated oxytocin (OT)-receptor antagonist, the role of Mg2+ in uterine OT-receptor function. The antagonist-receptor interaction was characterized by high affinity, reversibility and stereospecificity in Tris/HCl buffer containing 3 mmol of Mg2+/litre as well as buffer free of Mg2+. By contrast, omission of Mg2+ decreased the affinity of the receptor for OT by about 1500-fold; moreover, the stereospecificity of agonist, but not antagonist, binding was lost. Since guanine nucleotides had relatively minor effects in this system (less than or equal to 2-fold decrease in OT affinity), we suggest that the agonist-binding site of OT receptors is directly modulated by Mg2+, unlike other receptors, where the effects of bivalent cations are exerted via guanine-nucleotide-binding (G-) proteins. Thus the ligand recognition mechanism of OT receptors may be novel in this respect.

Adenylate cyclase stimulation and luteinizing hormone-receptor interaction in plasma membranes from rat testicular interstitial cells in relation to the chemical structure of the hormone. Role of Mg2+.

To understand more closely the structural requirements of the LH molecule necessary to stimulate adenylate cyclase, we studied the modulation of this enzyme in partially purified plasma membranes prepared from isolated interstitial cells of rat testis submitted to oLH and to some oLH derivatives and natural analogues. The role of Mg2+ was also investigated in relation to the structural modifications of oLH. Some new facts appeared in this study: 1. Methyl oLH, which exhibited the same ability as native oLH to stimulate cAMP accumulation and steroidogenesis in isolated cells, cannot induce the same level of maximal stimulation of adenylate cyclase as native oLH in plasma membranes. This phenomenon is related to the Mg2+ concentration, and the differences between maximal activation induced by methyl oLH and oLH were more apparent at a free Mg2+ concentration of 3.3 mmol/l than at lower concentrations. The maximal activity (in terms of native oLH) of other alkyl derivatives, such as ethyl or isopropyl oLH, on the contrary, was similar in isolated plasma membranes and in intact cells suggesting that the differential behaviour of the membranes specifically concerns the methyl derivative. 2. Guanidyl oLH and guanidyl porcine LH, which were able to induce cAMP accumulation in intact cells, did not exhibit any stimulating activity in plasma membranes. 3. Among the natural analogues, hCG and pLH are distinguished by a lower maximal activity (by comparison with oLH) particularly at high Mg2+ concentration. This work shows that changes in the LH structure have an impact not only on the parameters of the adenylate cyclase complex but also on the transduction of the hormone signal and its modulation by Mg2+.