Summary A strain of Saccharomyces cerevisiae, known to produce multiple isoaccepting forms of several tRNA's which differ from a standard wild type strain, has been studied genetically. The multiple isoaccepting tRNA phenotype behaves as if it is caused by a single recessive mutation. Five tetrads were analyzed and all showed a 2:2 segregation of mutant to wild type profiles for Phe-tRNAPhe. Furthermore, the multiple isoacceptors for the other tRNA's in the mutant strain are probably caused by the same mutation, since Tyr-tRNATyr and Val-tRNAVal also exhibit 2:2 segregation for mutant versus wild type tRNA profiles and the segregation pattern is the same as that for Phe-tRNAPhe. 相似文献
Six purified amino acid acceptor tRNA species were examined by polyacrylamide gel electrophoresis. Small differences in migration were observed under conditions that preserve the conformation of tRNA. When tRNA was heated in the presence of either 10 mM acetate or EDTA at 60° a change in migration was observed for tRNAGlu. No difference in migration was seen between Val-tRNAVal and tRNAVal. When tRNA was denatured by heating in 4M urea and applied to a gel containing the same amount of urea, all tRNA species migrated approximately the same distance with the exception of tRNALeu V, which showed an appreciable slower migration. From the difference in migration of tRNALeu V as compared to tRNAVal and 5 S RNA, the difference in chain length between tRNAVal and tRNALeu V was estimated to be approximately 9 nucleotides. 相似文献
Magnesium-dependent adenosine triphosphatase has been purified from sheep kidney medulla plasma membranes. The purification, which is based on treatment of a kidney plasma membrane fraction with 0.5% digitonin in 3 mm MgCl2, effectively separates the Mg2+-ATPase from (Na+ + K+)-ATPase present in the same tissue and yields the Mg2+-ATPase in soluble form. The purified enzyme is activated by a variety of divalent cations and trivalent cations, including Mg2+, Mn2+, Ca2+, Co2+, Fe2+, Zn2+, Eu3+, Gd3+, and VO2+. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme shows two bands with Rf values corresponding to molecular weights of 150,000 and 77,000. The larger peptide is phosphorylated by [γ-32P]ATP, suggesting that this peptide may contain the active site of the Mg2+-ATPase. The Mg2+-ATPase activity is unaffected by the specific (Na+ + K+)-ATPase inhibitor ouabain. 相似文献
The effects of regional and global ischemia on cellular electrical activity and on arrhythmias induced by reperfusion were studied at different Mg2+ concentrations (Mg2+o, 0, 1.2, and 4.8 mM) in perfused rat hearts. Surface electrograms and transmembrane potentials were recorded during control, 10 min of ischemia (perfusion arrest or coronary ligation), and reperfusion. Increasing Mg2+o from 0-4.8 mM decreased heart rate, did not alter action potential morphology, and had a strong antiarrhythmic action on reperfusion following coronary ligation. At low and normal Mg2+o, the incidence of tachyarrhythmias was between 70 and 80%. Global ischemia led to progressive atrioventricular block and the final ventricular beating rate was similar at all Mg2+o despite unequal initial values. The severity of arrhythmias was similar to that found after regional ischemia in Mg2+o = 0, but much lower at normal and high Mg2+o. The resting depolarization induced by coronary ligation decreased as Mg2+o was raised, but such a relation was not seen during global ischemia where the depolarization was less marked. The action potential duration did not vary with the ventricular rate between 160 and 380 beats per min but increased considerably when sinus rate was markedly slowed (40 to 80 bpm) by raising Mg2+o to 9.6 mM. Our data show that a high Mg2+o exerts a strong protection against reperfusion arrhythmias regardless of the type of ischemia. Modulation of the sinus rhythm by Mg2+ may contribute to its protective effect by decreasing K+o accumulation and Na+i loading during ischemia. 相似文献
Mg2+ in various concentrations was added to purified Rubisco in vitro to gain insight into the mechanism of molecular interactions
between Mg2+ and Rubisco. The enzyme activity assays showed that the reaction between Rubisco and Mg2+ was two order, which means that the enhancement of Rubisco activity was accelerated by low concentration of Mg2+ and slowed by high concentration of Mg2+. The kinetics constant (Km) and Vmax was 1.91 μM and 1.13 μmol CO2 mg−1 protein∙min−1, respectively, at a low concentration of Mg2+, and 3.45 μM and 0.32 μmol CO2∙mg−1 protein∙min−1, respectively, at a high concentration of Mg2+. By UV absorption and fluorescence spectroscopy assays, the Mg2+ was determined to be directly bound to Rubisco; the binding site of Mg2+ to Rubisco was 0.275, the binding constants (KA) of the binding site were 6.33 × 104 and 5.5 × 104 l·mol−1. Based on the analysis of the circular dichroism (CD) spectra, it was concluded that the binding of Mg2+ did not alter the secondary structure of Rubisco, suggesting that the observed enhancement of Rubisco carboxylase activity
was caused by a subtle structural change in the active site through the formation of the complex with Mg2+. 相似文献
The accessibility of the s4U base in native tRNAVal from E.coli was monitored by studying the binding of various mercurials. The relative binding order HgBr2[unk]HgCl2CH3HgOAc[unk]CH3HgCl[unk]PCMB parallels approximately the steric requirements of linear HgX2 or RHgX compounds for SN2 displacement by sulfur, although other factors are operative. Para-chloromercuri-benzoate (PCMB) does not bind the thiolated nucleotide unless the tertiary structure of the tRNA is opened up by removal of Mg2+ ions and heating to 40°. Under these conditions, equilibrium dialysis measurements using 14C-labeled PCMB showed one binding site (n = 0.93) with an association constant, K1, of 9 × 104M−1. 相似文献
Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg2+ plus K+. Mn2+ was less effective and Ca2+ could not substitute for Mg2+. However, adding Ca2+ with Mg2+ or substituting Mn2+ for Mg2+ activated it appreciably in the absence of added K+, and all three divalent cations decreased apparent affinity for K+. Inhibition by Na+ decreased with higher Mg2+ concentrations, when Ca2+ was added, and when Mn2+ was substituted for Mg2+. Dimethyl sulfoxide, which favorsE2 conformations of the enzyme, increased apparent affinity for K+, whereas oligomycin, which favorsE1 conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg2+ and Mn2+, favoring (under these conditions)E2 conformations, are effective, whereas Ca2+, favoringE1, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K+, and although Ca2+ and Mn2+ are fairly effective, Mg2+ is a poor substitute for K+, while Na+ at these sites favorsE1 conformations. K+ increases theKm for substrate, but both Ca2+ and Mn2+ decrease it, perhaps by competing with K+. On the other hand, phosphatase activity in the presence of Na+ plus K+ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg2+ and Mn2+, but not by adding Ca2+; this is consistent with stimulation occurring through facilitation of an E1 to E2 transition, perhaps an E1-P to E2-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg2+ plus Na+ alone or Mg2+ plus Na+ plus low K+: this effect of oligomycin may reflect acceleration, in the absence of adequate K+, of an alternative E2-P to E1 pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence. 相似文献
Incubation of F1 in the presence of Mg2+ results in a pronounced lag in its ATPase activity measured with the ATP-regenerating system. A decrease of the initial rate of ATPase induced by Mg2+ is also observed when free nucleotides were separated from the enzyme by Sephadex gel filtration. No inhibition is observed when F1 treated to remove tightly bound nucleotides was preincubated in the presence of Mg2+. Mg2+-induced inhibition of ATPase activity of nucleotide-depleted F1 can be restored by an addition of low concentrations of ADP. In all cases the inhibited ATPase can be activated by the ADP-removing system /phosphoenol pyruvate + pyruvate kinase/. It is concluded that i/ Mg2+-induced inhibition of the ATPase activity of F1 is due to the formation of an inactive F1. ADP complex; and ii/ unusual inhibition of oligomycin-sensitive ATPase by ADP /Fitin et al., Biochem. Biophys. Res. Communs. 1979, , 434/ is directed to F1 component of the complete mitochondrial ATPase system. 相似文献
At least ten distinct ATP-hydrolyzing activities are associated with mitochondria, endoplasmic reticulum-, Golgi-, and plasma membrane-enriched fractions from the marine diatom, Nitzschia alba. These activities are divided into four groups: Ca2+-dependent, Mg2+-dependent monovalent cation-stimulated, Mg2+-anion-stimulated ATPases, and Mg2+-dependent nucleotidases.The Mg2+-dependent activities hydrolyze nucleoside triphosphates and, in some membranes, nucleoside diphosphates. Molar ratios of 1:2 are preferred. However, their divalent cation requirements are not specific, and they can effectively utilize Ca2+, Mn2+, Mg2+, or Zn2+. The most effective inhibitors of the Mg2+-dependent activities are oligomycin, NaN3, and NaF.Optimal activity of the Mg2+-dependent monovalent cation-stimulated ATPase is obtained at Na+, or Na+ plus K+ concentrations of 100–300 mm. Under these high salt conditions, ATP is hydrolyzed almost exclusively, and Mg2+ is specifically required for activation. Preference is for a molar ratio of , and the sulfhydryl-blocking agents, p-chloromecuribenzoate, N-ethylmaleimide, and iodoacetamide strongly or completely inhibit ATP hyrolysis. 相似文献
An ATPase activity stimulated by divalent ions (Mg2+, Ca2+, Mn2+, Zn2+) has been observed in intact hamster fibroblasts cultured in vitro (BHK line). Such activity has been determined by the incubation (30 min at 37°C) of washed cell suspensions (about 1 mg of proteins) in a medium containing 100 mM NaCl, 20 mM KCl, 15 mM Tris—HCl (pH 7.4), 10 mM NaHCO3, 5 mM glucose and equimolar concentrations of ATP and divalent cation. Mg2+-ATPase activity is insensitive to ouabain and lacks specificity towards nucleoside triphosphate substrates. AMP and ADP are not hydrolyzed under these conditions. Apparent Km of 0.76 mM and Vmax of 1.46 μmol Pi · mg proteins?1 · h?1 have been calculated for Mg-ATP complex. This ATPase is an ectoenzyme, therefore its activity could be used as a suitable index of the action of chemicals like chromium compounds known for their cytotoxic effects on membrane functions.Salts of trivalent (CrCl3) and hexavalent (K2Cr2O7) chromium at concentrations ranging from 1 mM to 5 mM inhibit Mg2+-ATPase. The inhibition by K2Cr2O7 is observed after pretreatment of the cells with this compound followed by its absence from the assay medium “per se” for Mg2+-ATPase, and it is referred to the alterations of membrane bound enzyme structures by the oxidizing hexavalent chromium. The inhibition by CrCl3 is mainly evident when this compound is present in the incubation medium, and is referred to the interaction of trivalent chromium with Mg2+-ATP as it is partially reversed by increasing Mg2+-ATP concentration. 相似文献
Certain biochemical characteristics of an adenylate cyclase that is activated by low concentrations of histamine (Ka, 8 μm) and that is present in cell-free preparations from the dorsal hippocampus of guinea pig brain have been studied. Histamine increased the maximal reaction velocity of adenylate cyclase without altering the Km (0.18 mm) for its substrate, MgATP. Increasing concentrations of free Mg2+ stimulated enzymatic activity; the kinetic properties of this activation by Mg2+ suggest the existence of a Mg2+ allosteric site on the enzyme. Histamine increased the affinity of this apparent site for free Mg2+. Free ATP was a competitive inhibitor with respect to the MgATP substrate. The apparent potency of free ATP as an inhibitor increased in the presence of histamine. In the presence of Mg2+, low concentrations of Ca2+ markedly inhibited adenylate cyclase activity; half-maximal inhibition of both basal and histamine-stimulated enzyme activity occurred at 40 μm Ca2+. Other divalent cations, including Zn2+, Cu2+, and Cd2+, were also inhibitory. Of the divalent cations tested, only Co2+ and Mn2+ could replace Mg2+ in supporting histamine-stimulated adenylate cyclase activity. The nucleoside triphosphates GTP and ITP increased basal adenylate cyclase activity and markedly potentiated the stimulation by histamine. Preincubation of adenylate cyclase with 5′-guanylylimidodiphosphate dramatically increased enzyme activity; in this activated state, the adenylate cyclase was relatively refractory to further stimulation by histamine or F?. The subcellular distribution of histamine-sensitive adenylate cyclase activity was studied in subfractions from guinea pig cerebral cortex. The highest total and specific activities were observed in those fractions enriched in nerve endings, while adenylate cyclase activity was not detectable in the brain cytosol fraction. A possible physiological role for this histamine-sensitive adenylate cyclase in neuronal function is discussed. 相似文献
Several aspects of Mg2+ homeostasis were investigated in cultured chicken heart cells using the fluorescent Mg2+ indicator, FURAPTRA. The concentration of cytosolic Mg2+ ([Mg2+]i) is 0.48 ± 0.03 mM (n = 31). To test whether a putative Na/Mg exchange mechanism controls [Mg2+]i below electrochemical equilibrium, we manipulated the Na+ gradient and assessed the effects on [Mg2+]i. When extracellular Na+ was removed, [Mg2+]i increased; this increase was not altered in Mg-free solutions, but was attenuated in Ca-free solutions. A similar increase in [Mg2+]i, which was dependent upon extracellular Ca2+, was observed when intracellular Na+ was raised by inhibiting the Na/K pump with ouabain. These results do not provide evidence for Na/Mg exchange in heart cells, but they suggest that Ca2+ can modulate [Mg2+]i. In addition, removing extracellular Na+ caused a decrease in intracellular pH (pHi), as measured by pH-sensitive microelectrodes, and this acidification was attenuated when Cat+ was also removed from the solution. These results suggest that Ca2+ and H+ interact intracellularly. Since changes in the Na+ gradient can also alter pHi, we questioned whether pH can modulate [Mg2+]i. pHi was manipulated by the NH4Cl prepulse method. NH4+-evoked changes in pHi, as measured by the fluorescent indicator BCECF, were accompanied by opposite changes in [Mg2+]i; [Mg2+]i changed by –0.16 mM/unit pH. These NH4+-evoked changes in [Mg2+]i were not caused by movements of Mg2+ or Ca2+ across the sarcolemma or by changes in cytosolic Ca2+. Additionally, pHi was manipulated by changing extracellular pH (pHo). When pHo was decreased from 7.4 to 6.3, pHi decreased by 0.64 units and [Mg2+]i increased by 0.12 mM; in contrast, when pHo was raised from 7.4 to 8.3, pHi increased by 0.6 units and [Mg2+]i did not change significantly. The results of our investigations suggest that Ca 2+ and H+ can modulate [Mg2+]i, probably by affecting cytosolic Mg2+ binding and/or subcellular Mg2+ transport and that such redistribution of intracellular Mg2+ may play an important role in Mg2+ homeostasis in cardiac cells. 相似文献
Aurovertin forms a complex with soluble beef heart mitochondrial ATPase (F1) while exhibiting a biphasic fluorence enhancement. The effect of substrate, activators and inhibitors of F11 of the fluorescence of the aurovertin-F1 complex is reported. The aurovertin-F1 complex can exist in two different states, one showing low fluorescence and the other with high fluorescence. Transition into the low fluorescence state is induced by various nucleoside triphosphates (ATP ± Mg2+, ITP ± Mg2+, GIP + Gg2+, and AMP-P(NH)P ± Mg2+). The rate and extent of fluorescence decrease caused by nucleotide addition (except that caused by ATP) is dependent on the presence of added Mg2+. The inhibitors of ATPase activity (AMP-P(NH)P, GMP-P(NH)P and EDTA) at concentrations that inhibit hydrolysis of ATP did not prevent the ATP induced decrease of aurovertin fluorescence. EDTA at high concentration (>0.4 mM) enhanced the effect of ADP.The complex of aurovertin with F1 that had previously been treated with butanedione loses sensitivity to ATP. Addition of ADP to the system containing butanedione-treated enzyme caused a 2-fold greater enhancement of fluorescence than the addition of ADP to the control system. In contrast to the butanedione-treated enzyme, the complex of aurovertin with F1 previously treated at pH 5.6 loses sensitivity to ADP. Addition of ATP to this system lowered the fluorescence as in the system containing native enzyme.On the basis of the analyses of the aurovertin fluorescence changes and hydrolytic activity of F1, the existence of several types of ligand binding sties with varying degrees of specificity are proposed. It is further proposed that these sites are important in control of the conformation and the catalytic properties of the ATPase molecule. 相似文献
AimsLoss of magnesium (Mg2+) inhibits cell proliferation and augments nephrotoxicant-induced renal injury, but the role of Mg2+ has not been clarified in detail. We examined the effect of extracellular Mg2+ deprivation on a MEK–ERK cascade and cell proliferation using a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells.Main methodsMDCK cells were cultured in Mg2+-containing or Mg2+-free media. A HA-tagged constitutively active (CA)-MEK1 and a dominant negative (DN)-MEK1 were transfected into MDCK cells. The level of protein was examined by Western blotting. The intracellular free Mg2+ concentration ([Mg2+]i) was measured using a fluorescent dye, mag-fura 2. Cell proliferation was determined by WST-1 assay. Dead cells were identified by staining with annexin V-FITC and propidium iodide.Key findingsIn the presence of fetal calf serum (FCS), Mg2+ deprivation decreased phosphorylated-ERK1/2 (p-ERK1/2) levels and [Mg2+]i. Re-addition of Mg2+ increased p-ERK1/2 levels, which were inhibited by U0126, a specific inhibitor of a MEK–ERK cascade. Glutathione-S-transferase pull-down and coimmunoprecipitation assays showed that CA-MEK1 and DN-MEK1 binds with ERK1/2 in the presence of Mg2+. In contrast, neither CA-MEK1 nor DN-MEK1 bound to ERK1/2 in the absence of Mg2+. These results indicate that the MEK–ERK cascade is regulated by [Mg2+]i. Cell proliferation was increased by the treatment with FCS or the expression of CA-MEK1 in the presence of Mg2+, but was inhibited by Mg2+ deprivation. Mg2+ deprivation did not increase the number of dead cells.SignificanceMg2+ is involved in the regulation of the MEK–ERK cascade and cell proliferation in MDCK cells. 相似文献
Although different routes for the S-nitrosation of cysteinyl residues have been proposed, the main in vivo pathway is unknown. We recently demonstrated that direct (as opposed to autoxidation-mediated) aerobic nitrosation of glutathione is surprisingly efficient, especially in the presence of Mg2+. In the present study we investigated this reaction in greater detail. From the rates of NO decay and the yields of nitrosoglutathione (GSNO) we estimated values for the apparent rate constants of 8.9±0.4 and 0.55±0.06 M−1 s−1 in the presence and absence of Mg2+. The maximum yield of GSNO was close to 100% in the presence of Mg2+ but only about half as high in its absence. From this observation we conclude that, in the absence of Mg2+, nitrosation starts by formation of a complex between NO and O2, which then reacts with the thiol. Omission of superoxide dismutase (SOD) reduced by half the GSNO yield in the absence of Mg2+, demonstrating O2− formation. The reaction in the presence of Mg2+ seems to involve formation of a Mg2+•glutathione (GSH) complex. SOD did not affect Mg2+-stimulated nitrosation, suggesting that no O2− is formed in that reaction. Replacing GSH with other thiols revealed that reaction rates increased with the pKa of the thiol, suggesting that the nucleophilicity of the thiol is crucial for the reaction, but that the thiol need not be deprotonated. We propose that in cells Mg2+-stimulated NO/O2-induced nitrosothiol formation may be a physiologically relevant reaction. 相似文献
Ribulose-1,5-bisphosphate oxygenase was activated by incubation with CO2 and Mg2+ and inactivated upon removal of CO2 and Mg2+ by gel filtration. The activity of the enzyme was dependent upon the preincubation concentrations of CO2 and Mg2+ and upon the preincubation pH. This indicated that activation involved the reversible formation of an equilibrium complex of enzyme-CO2-Mg. The kinetics of the activation process were the same as those described by G. H. Lorimer et al. ((1976) Biochemistry15, 529–536), for ribulose bisphosphate carboxylase and are consistent with the ordered reversible reaction sequence: The activity of the enzyme, after preincubation at constant concentrations of CO2 and Mg2+, increased as the pH was raised, suggesting that CO2 reacted with an enzyme group having an alkaline pK. Since CO2 and O2 interact competitively at the catalytic site, the activation of ribulose bisphosphate oxygenase by CO2 and Mg2+ indicates that the CO2 molecule which takes part in the activation process is not the same as that which becomes fixed during the carboxylase reaction. These results also indicate that the oxygenase and carboxylase functions of the catalytic site are tightly coupled rather than independent of one another. 相似文献
1-adrenaline, ACTH and glucagon activate the adenylate cyclase of rat adipocytes by decreasing its S0.5(Mg2+) (concentration yielding 0.5 Vmax) from its basal value of 11.5 to 1.2, 0.3 and 1.8 mM and by increasing its Ki(ATP4?) from 0.03 to 0.25; 0.62 and 0.16 mM respectively. The kinetic properties of the enzyme are regulated by its state of saturation with ATP4? or Mg2+; its saturation with ATP4? and citrate3? suppressed its basal and hormone-dependent activities. The hormone-dependent decrease in Km and increase in Vmax of the enzyme occur when shifting from suboptimal low concentrations of hormone and Mg2+ to optimal conditions, i.e., high concentration of hormone and low concentration of Mg2+. The increase in the state of saturation of the enzyme with Mg2+ decreases the hormone-dependent effects on Vmax and results in identical values of Km (0.14 mM) for its basal and 1-adrenaline dependent activities. CaCl2 saturation curves at 5 mM ATP with either 5, 10 or 20 mM MgCl2 show that the substitution of 5 mM MgCl2 by 10 mM and 20 mM MgCl2 increased the Ki(Ca2+) of the enzyme from 0.19 to 0.49 and 0.94 mM but decreased its Ki(CaATP) from 0.42 to 0.19 and 0.14 mM respectively. Only when the concentration of MgCl2 exceeded that of ATP did 1-adrenaline and ACTH activate the enzyme by increasing its Ki(Ca2+), although only ACTH increased its Ki(CaATP). An increase in energy charge would decrease the intracellular concentrations of Mg2+ and Ca2+ because ATP4? has stronger binding constants for Mg2+ and Ca2+ than ADP3? and AMP2?. Hence, the reported properties of the enzyme suggests that changes in energy charge may allow for metabolic feedback control of the hormonal responsiveness of the Mg2+, Ca2+, ATP4? -sensitive adenylate cyclase. 相似文献
The effects of extracellular Mg2+ on both dynamic changes of [Ca2+]i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca2+ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca2+ concentrations ([Ca2+]i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca2+]i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca2+]i (150 nM) for several hours. The initial [Ca2+]i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca2+]i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg2+ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg2+, caused an increase in basal [Mg2+]i from 0.8 ± 0.3 to 1.6 ± 0.5 mM. As compared to 1.4 mM extracellular Mg2+, 1 M thapsigargin induces, in 10 mM Mg2+, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca2+]i and a lower Ca2+ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca2+]i which was inhibited by high extracellular and intracellular Mg2+. 相似文献