Effect of magnesium ion (Mg2+) and magnesium adenosinetriphosphate ion (MgATP2-) on pigeon kidney pyruvate carboxylase

Kinetic methods have been used to determine whether Mg²⁺ and MgATP^2? play an important role in regulating pigeon kidney pyruvate carboxylase (pyruvate: CO₂ ligase (ADP), EC 6.4.1.1.). Mg²⁺ not only forms a complex with ATP^4? (MgATP^2?) but is also required for the enzyme activation (and probably for the binding of MgATP^2? to this enzyme). Contrary to the results of other investigators, the MgATP^2? complex was not found to activate pigeon kidney pyruvate carboxylase. We could not demonstrate homotropic cooperativity with MgATP^2? complex. Excess Mg²⁺ induces allosteric stimulation of the enzymatic activity at different concentrations of MgATP^2?. With different Mg²⁺ concentrations, changes also occured in the apparent Km^? and Vmax-values. Without excess of Mg²⁺ only about 2 % of the total enzymic activity available could be demonstrated in the presence of MgATP^2?. It is concluded that Mg²⁺ exhibits a homotropic cooperative effect and is required for the activation of this enzyme. Mg²⁺ may bind either to a specific effector site, at the active site, or at the binding site for MgATP^2? which is capable of functioning as an effector site and in this way facilitates the carboxylation of pyruvate.     

Identification of a second GTP-bound magnesium ion in archaeal initiation factor 2

Eukaryotic and archaeal translation initiation processes involve a heterotrimeric GTPase e/aIF2 crucial for accuracy of start codon selection. In eukaryotes, the GTPase activity of eIF2 is assisted by a GTPase-activating protein (GAP), eIF5. In archaea, orthologs of eIF5 are not found and aIF2 GTPase activity is thought to be non-assisted. However, no in vitro GTPase activity of the archaeal factor has been reported to date. Here, we show that aIF2 significantly hydrolyses GTP in vitro. Within aIF2γ, H97, corresponding to the catalytic histidine found in other translational GTPases, and D19, from the GKT loop, both participate in this activity. Several high-resolution crystal structures were determined to get insight into GTP hydrolysis by aIF2γ. In particular, a crystal structure of the H97A mutant was obtained in the presence of non-hydrolyzed GTP. This structure reveals the presence of a second magnesium ion bound to GTP and D19. Quantum chemical/molecular mechanical simulations support the idea that the second magnesium ion may assist GTP hydrolysis by helping to neutralize the developing negative charge in the transition state. These results are discussed in light of the absence of an identified GAP in archaea to assist GTP hydrolysis on aIF2.     

Interaction of thiamine diphosphate with magnesium ion

The action of magnesium ion on the exchange rate of the proton in C2 of thiamine and thiamine diphosphate is studied at different values of pD. Above pD 5 the ion Mg2+ increases this exchange rate. The phenomenon is markedly enhanced for TDP rather than thiamine and increases with pD. Below pD 5 magnesium decreases the exchange rate. This decrease is greater for TDP than for thiamine. The maximum effect is reached at a magnesium concentration of 0.5/1 for thiamine and of 1/1 for TDP. T1 measurements are made for different pH values with and without magnesium ion. Results seem to prove that an increase in pD values from 3.9 to 5.9 leads to an accentuation of the molecules "folded" form. Nevertheless for a given pD value the TDP-Mg complex seems to have a more "folded" form than TDP.     

Reversal of the bactericidal reaction of serum by magnesium ion

Muschel, Louis H. (University of Minnesota, Minneapolis), and Jean E. Jackson. Reversal of the bactericidal reaction of serum by magnesium ion. J. Bacteriol. 91:1399-1402. 1966.-Magnesium ion was found to reverse the bactericidal action of the antibody-complement system. Concentrations of 0.03 to 0.11 m MgSO(4) or MgCl(2) were effective, provided that the reaction of the antibody-complement system did not proceed longer than 50 min. Salts of other monovalent and divalent cations, the polyamine, spermine, and several anticomplementary substances were inactive in reversing the serum bactericidal reaction. Thus, the bactericidal reaction of serum may be bacteriostatic under certain conditions, and this finding is compatible with the persistence of infection in certain diseases despite high levels of antibody.     

Matrix magnesium and the permeability of heart mitochondria to potassium ion

Isolated beef heart mitochondria were treated with A23187 in the presence of different concentrations of Mg2+ or EDTA to establish varying levels of total mitochondrial Mg2+. The Mg2+ content was related to the rate of passive swelling of the mitochondria in potassium acetate and other potassium salts in which swelling is presumed to depend on K+ entry via an endogenous K+/H+ antiport. Swelling in these salts does not commence until Mg2+ has been depleted from an initial value of 36 nmol X mg-1 of protein to 8 nmol/mg-1, or less. Below this level, swelling increases linearly with decreasing Mg2+ to a maximum rate at 2 nmol of Mg2+ X mg-1. Rotenone-treated heart mitochondria suspended in 75 mM potassium acetate at pH 7.80 show no delta pH by 5,5-dimethyl-2,4-oxazolidinedione distribution. Distribution of methylamine also shows essentially no delta pH under these conditions when allowance is made for binding of [14C]methylamine by mitochondrial membranes under these conditions. Addition of A23187 results in a small and transient delta pH (delta pH less than 0.14, acid interior) as measured by methylamine distribution. Estimation of the maximum matrix free Mg2+ concentration from the maximum delta pH observed and the external free Mg2+ concentration at equilibrium with A23187 shows that swelling is not initiated until matrix free Mg2+ is decreased to below 150 microM. An independent estimate of free Mg2+ using a null-point procedure gives a lower, but quite similar value (50 microM) for maximum matrix free Mg2+ when swelling commences. The large depletion of total and free Mg2+ that is required to activate swelling in potassium acetate (and presumably K+/H+ antiport activity) does not appear to be compatible with previous indications that free Mg2+ acts as a "carrier brake" to regulate K+ extrusion from the mitochondrion on such an antiport (Garlid, K. D. (1980) J. Biol. Chem. 255, 11273-11279). The removal of a tightly bound component of mitochondrial Mg2+ is closely related to increased K+ permeability and increased passive swelling in potassium salts. This Mg2+ appears to play a role in the maintenance of mitochondrial membrane structure and integrity.     

The structure and regulation of magnesium selective ion channels

The magnesium ion (Mg^2 +) is the most abundant divalent cation within cells. In man, Mg^2 +-deficiency is associated with diseases affecting the heart, muscle, bone, immune, and nervous systems. Despite its impact on human health, little is known about the molecular mechanisms that regulate magnesium transport and storage. Complete structural information on eukaryotic Mg^2 +-transport proteins is currently lacking due to associated technical challenges. The prokaryotic MgtE and CorA magnesium transport systems have recently succumbed to structure determination by X-ray crystallography, providing first views of these ubiquitous and essential Mg^2 +-channels. MgtE and CorA are unique among known membrane protein structures, each revealing a novel protein fold containing distinct arrangements of ten transmembrane-spanning α-helices. Structural and functional analyses have established that Mg^2 +-selectivity in MgtE and CorA occurs through distinct mechanisms. Conserved acidic side-chains appear to form the selectivity filter in MgtE, whereas conserved asparagines coordinate hydrated Mg^2 +-ions within the selectivity filter of CorA. Common structural themes have also emerged whereby MgtE and CorA sense and respond to physiologically relevant, intracellular Mg^2 +-levels through dedicated regulatory domains. Within these domains, multiple primary and secondary Mg^2 +-binding sites serve to staple these ion channels into their respective closed conformations, implying that Mg^2 +-transport is well guarded and very tightly regulated. The MgtE and CorA proteins represent valuable structural templates to better understand the related eukaryotic SLC41 and Mrs2–Alr1 magnesium channels. Herein, we review the structure, function and regulation of MgtE and CorA and consider these unique proteins within the expanding universe of ion channel and transporter structural biology.     

Ca(2+)-regulated ion channels

Due to its high external and low internal concentration the Ca(2+) ion is used ubiquitously as an intracellular signaling molecule, and a great many Ca(2+)-sensing proteins have evolved to receive and propagate Ca(2+) signals. Among them are ion channel proteins, whose Ca(2+) sensitivity allows internal Ca(2+) to influence the electrical activity of cell membranes and to feedback-inhibit further Ca(2+) entry into the cytoplasm. In this review I will describe what is understood about the Ca(2+) sensing mechanisms of the three best studied classes of Ca(2+)-sensitive ion channels: Large-conductance Ca(2+)-activated K(+) channels, small-conductance Ca(2+)-activated K(+) channels, and voltage- gated Ca(2+) channels. Great strides in mechanistic understanding have be made for each of these channel types in just the past few years.     

Kinetics of complexing activation by the magnesium ion on green crab (Scylla serrata) alkaline phosphatase.

As with mammalian enzymes, green crab (Scylla serrata) alkaline phosphatase can be activated by Mg2+ through a time-dependent course. The activation is mainly a Vmax effect. Tsou's method was used to study the kinetic course of activation. The results show that the enzyme was activated by a complexing scheme that had not been previously identified: the enzyme first reversibly and quickly binds Mg2+ and then undergoes a slow reversible course to activation, with a relatively high activation energy (78 +/- 4 kJ/mol) and a slow conformational change. The activation reaction is a single molecule reaction, and the apparent activation rate constant is independent of Mg2+ concentration if the concentration is sufficiently high. The microscopic rate constants of activation and the association constant were determined from the measurements. The proposed scheme may also be applied to the Mg2+ activation mechanism for mammalian enzyme, to explain why the activation rate is time-dependent and not diffusion controlled. Substrate binding was also shown to affect the activation rate constant.     

Differentiation of the slow-binding mechanism for magnesium ion activation and zinc ion inhibition of human placental alkaline phosphatase

The binding mechanism of Mg(2+) at the M3 site of human placental alkaline phosphatase was found to be a slow-binding process with a low binding affinity (K(Mg(app.)) = 3.32 mM). Quenching of the intrinsic fluorescence of the Mg(2+)-free and Mg(2+)-containing enzymes by acrylamide showed almost identical dynamic quenching constant (K(sv) = 4.44 +/- 0.09 M(-1)), indicating that there is no gross conformational difference between the M3-free and the M3-Mg(2+) enzymes. However, Zn(2+) was found to have a high affinity with the M3 site (K(Zn(app.)) = 0.11 mM) and was observed as a time-dependent inhibitor of the enzyme. The dependence of the observed transition rate from higher activity to lower activity (k(obs)) at different zinc concentrations resulted in a hyperbolic curve suggesting that zinc ion induces a slow conformational change of the enzyme, which locks the enzyme in a conformation (M3'-Zn) having an extremely high affinity for the Zn(2+) (K*(Zn(app.)) = 0.33 microM). The conformation of the M3'-Zn enzyme, however, is unfavorable for the catalysis by the enzyme. Both Mg(2+) activation and Zn(2+) inhibition of the enzyme are reversible processes. Structural information indicates that the M3 site, which is octahedrally coordinated to Mg(2+), has been converted to a distorted tetrahedral coordination when zinc ion substitutes for magnesium ion at the M3 site. This conformation of the enzyme has a small dynamic quenching constant for acrylamide (K(sv) = 3.86 +/- 0.04 M(-1)), suggesting a conformational change. Both Mg(2+) and phosphate prevent the enzyme from reaching this inactive structure. GTP plays an important role in reactivating the Zn-inhibited enzyme activity. We propose that, under physiological conditions, magnesium ion may play an important modulatory role in the cell for protecting the enzyme by retaining a favorable geometry of the active site needed for catalysis.     

Calcium,magnesium and zinc ion coordination equilibria of vincristine

The zinc ion coordination of vincristine was studied by polarography; the analogous calcium ion coordination process was studied potentiometrically by a calcium ion selective electrode. In both cases, complexes of 1:1 composition were formed. The formation constant of the calcium complex was found to be 1g K = 3.27 ± 0.1. On the basis of the substitution of zinc in its vincristine complex by calcium and magnesium ions respectively, the ratio of the corresponding stability constants could be estimated as K_Zn:K_Ca (and K_Zn:K_Mg) ∼ 10⁵−3 × 10⁴. The complex formation processes proved to be pH-independent in the pH range 3.4–5.5, indicating that the metal ions are coordinated by the unprotonated oxygen donor atoms of vincristine.     

Photoinitiated ion movements in bilayer membranes containing magnesium octaethylporphyrin.

A photocurrent produced by planar lipid bilayers containing Mg-octaethylporphyrin in the presence of oxygen has been investigated to determine if the current is due to movement of the MgOEP+ ion in the bilayer. Photoexcitation of the MgOEP is known to produce MgOEP+ in the bilayer when an electron acceptor is present. However, the aqueous electron acceptors ferricyanide and methyl viologen (MV+2) have opposite effects on the photocurrent. Ferricyanide decreases the photo current, even in the presence of oxygen, whereas methyl viologen increases the photocurrent, but only when oxygen is present. We attribute most of the photocurrent to the movement of superoxide anion. The difference in effect between ferricyanide and methyl viologen is attributed to the different rates of reduction of O2 by reduced MV+ (fast) vs. ferrocyanide (slow) and the known competition between ferricyanide and oxygen as the acceptor for the photoexcited porphyrin. It is inferred that most of the MgOEP is localized in the polar region of the lipid bilayer. Addition of ferrocyanide to the aqueous phase on one side of the bilayer, to trap MgOEP+ produced on the other side by MV+2, fails to increase the lifetime of the photovoltage. With a pH gradient across the bilayer, we observed only 5% of the photovoltage expected for the selective transport of H+ or OH- by MgOEP+. Thus, these measurements set the lower limit for the cross bilayer transit time of MgOEP+ or its charge in the range of 0.1-0.5 s.