Mg-ATP binding: its modification by spermine, the relevance to cytosolic Mg2+ buffering, changes in the intracellular ionized Mg2+ concentration and the estimation of Mg2+ by 31P-NMR (original) (raw)
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Regulation of cellular magnesium
Frontiers in Bioscience, 2000
TABLE OF CONTENTS 1. Abstract 2. Mg 2+ as an intracellular messenger 3. Changes in Serum Mg 2+ Level 4. Changes in Cellular Mg 2+ Content 4.1. Mg 2+ extrusion following beta-adrenergic receptor stimulation, and role of cAMP 4.2 Is there a role for alpha-adrenergic receptors in Mg 2+ extrusion? 4.3. Other agents that induce Mg 2+ extrusion 4.4. Hormones and stimulatory agents involved in Mg 2+ accumulation 5. Mg 2+ transport across the cell plasma membrane 6. Physiological significance of Mg 2+ changes within mitochondria 7. Conclusions 8. Acknowledgment 8. References
Analytical Biochemistry, 1997
of the b-ATP peak relative to the a-ATP is now widely Using Mg 2/ macroelectrodes based on the sensor ETH used (1). To convert this measured chemical shift into 7025 and accurate Mg 2/ -EDTA buffer solutions, the apthe [Mg 2/ ] i , a knowledge of the dissociation constant parent Mg 2/ -ATP dissociation constant (K app ) was meafor Mg 2/ -ATP in solutions similar to that of the intrasured at 25 and 37ЊC in background solutions mimicking cellular milieu is required, the apparent dissociation the cationic intracellular milieu of muscle cells. The constant (K app ). 5 Despite the importance of the K app only mean { SD (in mM) at 25ЊC was 157.0 { 13 (n Å 4), 127.5 a limited number of measurements have been carried { 12.0 (n Å 11), 101.0 { 9.0 (n Å 4) and at 37ЊC was 106.6 out in solutions containing physiological concentra-{ 9.6 (n Å 4), 87.4 { 4.9 (n Å 4), 78.1 { 2.0 (n Å 4) at pH tions of K / and Na / at the physiological pH value of values of 6.7, 7.2, and 7.7, respectively. The dependence 7.2 and these values are summarized in . (A of K app at 25ЊC on the ionic strength was also measured, detailed tabulation of values obtained under varying the mean { SD (mM) being 61.9 { 2.2 (n Å 3), 127.5 { 12 conditions can be found in ). The values of K app in (n Å 11), and 243.0 { 11.8 (n Å 3) at ionic strengths of at both 25 and 37ЊC differ by roughly a factor 0.087, 0.156 (normal background), and 0.3 M, respectively. of 2. Since the estimated [Mg 2/ ] i is directly proportional These values are larger than the K app values most com-
Na+/K+-ATPase: modes of inhibition by Mg2+
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1991
Adding 15 mM free Mg z+ decreased Vmx of the Na+/K+-ATPase reaction. Mg z+ also decreased the Ko~ for K ÷ activation, as a mixed inhibitor, but the increased inhibition at higher K + concentrations diminished as the Na + concentration was raised. Inhibition was greater with Rb + but less with Li + when these cations substituted for K + at pH 7.S, while at pH 8.5 inhibition was generally less and essenti~ly the same with all three cations: implying an association between inhibition and ion occlusion. On the other hand, Mg z+ increased the K0. s for Na+-activation of the Na+/K +-ATPase and Na+-ATPase reactions, as a mixed inhibitor. Changing incubation pH or temperature, or adding dimethylsulfoxide affected inhibition by Mg z+ and Ko. s for Na + diversely. Presteatly-state kinetic studies on enzyme phosphorylation, however, showed competition between Mg 2+ and Na +. In the K+-phospbatase reaction catalyzed by this enzyme Mg 2+ was a (near) competitor toward K +. Adding Na + with K + inhibited phosphatase activity, but under these conditions 15 mM Mg 2+ stimulated rather than inhibited; still higher Mg z÷ concentrations then inhibited with K + plus Na +. Similar stimulation and inhibition occurred when Mn 2+ was substituted for Mg z+, although the concentrations required were an order of magnitude less. In all these experiments no ionic substitutions were made to maintain ionic strength, since alternative cations, such as choline, produced various specific effects them~vcs. Kinetic analyses, in terms of product inhibition by Mg z+, require Mg 2+ release at multiple steps. The data are acconunodated by a scheme for the Na+/K+-ATPase with three alternative points for release: before MgATP binding, before K + release and before Na + binding. The latter alternatives necessitate two Mg ~-+ ions bound simultaneo~y to the enzyme, presumably to divalent cation-sites associated with the phosphate and the nucleotide domains of the active site.
Effects of ATP and monovalent cations on Mg2+ inhibition of (Na,K)-ATPase
Archives of Biochemistry and Biophysics, 1986
The hydrolysis of ATP catalyzed by purified (Na,K)-ATPase from pig kidney was more sensitive to Me inhibition when measured in the presence of saturating Na+ and K+ concentrations [(Na,K)-ATPase] than in the presence of Na+ alone, either at saturating [(Na,Na)-ATPase] or limiting [(Na,O)-ATPase] Naf concentrations. This was observed at two extreme concentrations of ATP (3 mM where the low-affinity site is involved and 3 I.LM where only the catalytic site is relevant), although Mgz+ inhibition was higher at low ATP concentration. In the case of (Na,Na)-ATPase activity, inhibition was barely observed even at 10 mM free Mgz+ when ATP was 3 mM. When (Na,K)-ATPase activity was measured at different fixed Kf concentrations the apparent Ki for Me inhibition was lower at higher monovalent cation concentration. When K+ was replaced by its congeners (Rb+, NH:, Li'), Mgz+ inhibition was more pronounced in those cases in which the dephosphorylating cation forms a tighter enzyme-cation complex after dephosphorylation. This effect was independent of the ATP concentration, although inhibition was more marked at lower ATP for all the dephosphorylating cations. The Ko.5 for ATP activation at its low-affinity site, when measured in the presence of different dephosphorylating cations, increased following the sequence Rb+ > K+ > NH: > Li+ > none. The Ko.5 values were lower with 0.05 mM than with 10 mM free Me but the order was not modified. The trypsin inactivation pattern of (Na,K)-ATPase indicated that Mg2+ kept the enzyme in an El state. Addition of K+ changed the inactivation into that observed with the E2 enzyme form. On the other hand, K+ kept the enzyme in an Ez state and addition of M$+ changed it to an El form. The Ko.5 for KCl-induced El-to-E2 transformation (observed by trypsin inactivation profile) in the presence of 3 mM MgClz was about 0.9 mM. These results concur with two mechanisms for free Mgzf inhibition of (Na,K)-ATPase: "product" and dead-end. The first would result from Mgz+ interaction with the enzyme in the E,(K) occluded state whereas the second would be brought about by a MS+-enzyme complex with the enzyme in an El state. o 1666 Academic PRSS, IN Magnesium can act in two opposite ways on (Na,K)-ATPase3: (i) as an essential ac
The reaction of Mg2+ with the Ca2+-ATPase from human red cell membranes and its modification by Ca2+
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1986
(1) Media prepared with CDTA and low concentrations of Ca 2+, as judged by the lack of Na+-dependent phosphorylation and ATPase activity of (Na++ K +)-ATPase preparations are free of contaminant Mg 2 +. (2) In these media, the Ca2+-ATPase from human red cell membranes is phosphorylated by ATP, and a low Ca2+-ATPase activity is present. (3) In the absence of Mg 2+ the rate of phosphorylation in the presence of 1/~M Ca 2+ is very low but it approaches the rate measured in Mg2+-containing media if the concentration of Ca 2+ is increased to 5 mM. (4) The Kca for phosphorylation is 2/~M in the presence and 60/~M in the absence of Mg 2+. (5) Results are consistent with the idea that for catalysis of phosphorylation the Ca2+-ATPase needs Ca 2+ at the transport site and Mg 2+ at an activating site and that Ca 2+ replaces Mg 2+ at this site. (6) Under conditions in which it increases the rate of phosphorylation, Ca 2 + is without effect on the Ca2+-ATPase activity in the absence of Mg 2+ suggesting that to stimulate ATP hydrolysis Mg 2+ accelerates a reaction other than phosphorylation. (7) Activation of the EIP)E2P reaction by Mg 2+ is prevented by Ca 2+ after but not before the synthesis of EIP from E l and ATP, suggesting that Mg 2+ stabilizes E 1 in a state from which Mg 2+ cannot be removed by Ca 2+ and that Ca 2+ stabilizes EIP in a state insensitive to Mg 2+. (8) The response of the Ca2+-ATPase activity to Mg 2+ concentration is biphasic, activation with a KMg = 88 #M is followed by inhibition with a K i = 9.2 mM. Ca 2+ at concentration up to 1 mM acts as a dead-end inhibitor of the activation by Mg 2+, and Mg 2+ at concentrations up to 0.5 mM acts as a dead-end inhibitor of the effects of Ca 2 + at the transport site of the Ca 2 +-ATPase.
Effects of magnesium and ATP on pre-steady-state phosphorylation kinetics of the Na+, K+ATPase
Biochimica Et Biophysica Acta-biomembranes, 1992
The aim of the present work s~as to elucidate the role pla~ed by ATP and Mg 2+ ions in the early steps .ff the Na+,K +-ATPase cycle. The approach was to follow pre-steady-state phosphorylation kinetics in Na +-containing K+-frce solutions under variable ATP and MgCI 2 concentrations. The experiments were performed with a rapid mixing apparatus at 20 5: 2°C. The concentrations of free and complexes species of Mg 2+ and ATP were calculated on the basis of a dissociation constant of 0.091 + 0.004 raM, estimated with Arsenazo lll under identical conditions. A simplified scheme were ATP binds to the ENa enzyme, which ix phosphorylated to MgEPNa and consequently dcl~hosphurylated returning to the ENa form, was used. In the absence of ADP and phosphate four rate constants are relevant: k! and k_ t, the on and off rate constants for ATP binding; k2, the transphosphorylation rate constant and k 3, the constant that governs the depbosphorylation rate. The values obtained were: kt ffi 0.025 + 0.003 ttM-i ms-1 for both free ATP and ATPMg; k_ t = 0.038 + 0.004 ms-~ for free ATP and 0.009 + 0.002 ms-t for ATPMg; k 2 ffi 0.199 4-0.005 ms-1; k3 ffi 0.0019 4-0.0002 ms-i. The model that seems best to explain the data is one where (i) the role of true substrate can be played equally well by free ATP or ATPMg, and (ii) free Mg 2+, an essential activator acts by binding to a specific Mg 2+ site on the enzyme mn.~eculc.
Magnesium homeostasis in mammalian cells
Frontiers in Bioscience, 2007
Mammalian cells tightly regulate cellular Mg 2+ content despite undergoing a variety of hormonal and metabolic stimulatory conditions. Evidence from several laboratories indicates that stimulatory conditions that increase cellular cAMP level result in a major mobilization of Mg 2+ from cells and tissues into the bloodstream. Conversely, hormones or agents that decrease cAMP level or activate protein kinase C signaling induce a major accumulation of Mg 2+ into the tissues. These Mg 2+ fluxes are quite large and fast suggesting the operation of powerful transport mechanisms. At front of the recent identification of several Mg 2+ entry mechanisms, the Mg 2+ extrusion pathway(s) still remain(s) poorly characterized. Similarly, it remains not completely elucidated the physiological significance of these Mg 2+ fluxes in the various tissues in which they occur. In the present review, we will attempt to provide a comprehensive framework of the modalities by which cellular Mg 2+ homeostasis and transport are regulated, as well as examples of cellular functions regulated by changes in cellular Mg 2+ level.
Archives of Biochemistry and Biophysics, 1992
Magnesium is the second most abundant cation within mammalian cells, trailing only potassium. It is also indispensable in the cell for a score of reactions and functions: it activates a large number of enzymes either directly or by forming complexes with and modifying substrates; it maintains proper conformations of nucleic acids and proteins; it regulates the operation of channels, receptors, and intracellular signalling molecules; it modulates photosynthesis, oxidative phosphorylation, muscle contraction, and nerve excitability;
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2005
To determine the nature of intracellular Mg 2+ stores and Mg 2+ release mechanisms in differentiated PC12 cells, Mg 2+ and Ca 2+ mobilizations were measured simultaneously in living cells with KMG-104, a fluorescent Mg 2+ indicator, and fura-2, respectively. Treatment with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), increased both the intracellular Mg 2+ concentration ([Mg 2+ ] i ) and the [Ca 2+ ] i in these cells. Possible candidates as intracellular Mg 2+ stores under these conditions include intracellular divalent cation binding sites, endoplasmic reticulum (ER), Mg-ATP and mitochondria. Given that no change in [Mg 2+ ] i was induced by caffeine application, intracellular IP 3 or Ca 2+ liberated by photolysis, it appears that no Mg 2+ release mechanism thus exists that is mediated via the action of Ca 2+ on membrane-bound receptors in the ER or via the offloading of Mg 2+ from binding sites as a result of the increased [Ca 2+ ] i . FCCP treatment for 2 min did not alter the intracellular ATP content, indicating that Mg 2+ was not released from Mg-ATP, at least in the first 2 min following exposure to FCCP. FCCP-induced [Mg 2+ ] i increase was observed at mitochondria localized area, and vice versa. These results suggest that the mitochondria serve as the intracellular Mg 2+ store in PC12 cell. Simultaneous measurements of [Ca 2+ ] i and mitochondrial membrane potential, and also of [Ca 2+ ] i and [Mg 2+ ] i , revealed that the initial rise in [Mg 2+ ] i followed that of mitochondrial depolarization for several seconds. These findings show that the source of Mg 2+ in the FCCP-induced [Mg 2+ ] i increase in PC12 cells is mitochondria, and that mitochondrial depolarization triggers the Mg 2+ release. D
3. Changes in Serum Mg 2+ Level 4. Changes in Cellular Mg 2+ Content
2011
2. Mg 2+ as an intracellular messenger 3. Changes in Serum Mg 2+ Level 4. Changes in Cellular Mg 2+ Content 4.1. Mg 2+ extrusion following beta-adrenergic receptor stimulation, and role of cAMP 4.2 Is there a role for alpha-adrenergic receptors in Mg 2+ extrusion? 4.3. Other agents that induce Mg 2+ extrusion 4.4. Hormones and stimulatory agents involved in Mg 2+ accumulation 5. Mg 2+ transport across the cell plasma membrane 6. Physiological significance of Mg 2+ changes within mitochondria 7. Conclusions 8. Acknowledgment 8. References