Functional-structural analysis of threonine 25, a residue coordinating the nucleotide-bound magnesium in elongation factor Tu.
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Elongation factor (EF) Tu Thr-25 is a key residue binding the essential magnesium complexed to nucleotide. We have characterized mutations at this position to the related Ser and to Ala, which abolishes the bond to Mg2+, and a double mutation, H22Y/T25S. Nucleotide interaction was moderately destabilized in EF-Tu(T25S) but strongly in EF-Tu(T25A) and EF-Tu(H22Y/T25S). Binding Phe-tRNAPhe to poly(U).ribosome needed a higher magnesium concentration for the latter two mutants but was comparable at 10 mM MgCl2. Whereas EF-Tu(T25S) synthesized poly(Phe), as effectively as wild type, the rate was reduced to 50% for EF-Tu(H22Y/T25S) and was, surprisingly, still 10% for EF-Tu(T25A). In contrast, protection of Phe-tRNAPhe against spontaneous hydrolysis by the latter two mutants was very low. The intrinsic GTPase in EF-Tu(H22Y/T25S) and (T25A) was reduced, and the different responses to ribosomes and kirromycin suggest that stimulation by these two agents follows different mechanisms. Of the mutants, only EF-Tu(T25A) forms a more stable complex with EF-Ts than wild type. This implies that stabilization of the EF-Tu.EF-Ts complex is related to the inability to bind Mg2+, rather than to a decreased nucleotide affinity. These results are discussed in the light of the three-dimensional structure. They emphasize the importance of the Thr-25-Mg2+ bond, although its absence is compatible with protein synthesis and thus with an active overall conformation of EF-Tu. (+info)
Copper, zinc, and magnesium levels in non-insulin dependent diabetes mellitus.
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A relationship has been reported between trace elements and diabetes mellitus. This study evaluated the role of such a relationship in 83 patients with non-insulin dependent diabetes mellitus (40 men and 43 women), with a mean duration of diabetes of 3.9 +/- 3.6 years. Patients with nephropathy were excluded. Thirty healthy non-diabetic subjects were studied for comparative analysis. Subjects were subdivided into obese and non-obese. Diabetic subjects were also subdivided into controlled and uncontrolled groups; control was based on fasting blood glucose and serum fructosamine levels. Plasma copper, zinc and magnesium levels were analysed using a GBC 902 double beam atomic absorption spectrophotometer. Plasma zinc and magnesium levels were comparable between diabetic and non-diabetic subjects, while copper levels were significantly elevated (p < 0.01) in diabetic patients. Age, sex, duration and control of diabetes did not influence copper, zinc, or magnesium concentrations. We conclude that zinc and magnesium levels are not altered in diabetes mellitus, but the increased copper levels found in diabetics in our study may merit further investigation of the relationship between copper and non-insulin dependent diabetes mellitus. (+info)
Potassium, magnesium, and fruit and vegetable intakes are associated with greater bone mineral density in elderly men and women.
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BACKGROUND: Osteoporosis and related fractures will be growing public health problems as the population ages. It is therefore of great importance to identify modifiable risk factors. OBJECTIVE: We investigated associations between dietary components contributing to an alkaline environment (dietary potassium, magnesium, and fruit and vegetables) and bone mineral density (BMD) in elderly subjects. DESIGN: Dietary intake measures were associated with both cross-sectional (baseline) and 4-y longitudinal change in BMD among surviving members of the original cohort of the Framingham Heart Study. Dietary and supplement intakes were assessed by food-frequency questionnaire, and BMD was measured at 3 hip sites and 1 forearm site. RESULTS: Greater potassium intake was significantly associated with greater BMD at all 4 sites for men and at 3 sites for women (P < 0.05). Magnesium intake was associated with greater BMD at one hip site for both men and women and in the forearm for men. Fruit and vegetable intake was associated with BMD at 3 sites for men and 2 for women. Greater intakes of potassium and magnesium were also each associated with less decline in BMD at 2 hip sites, and greater fruit and vegetable intake was associated with less decline at 1 hip site, in men. There were no significant associations between baseline diet and subsequent bone loss in women. CONCLUSION: These results support the hypothesis that alkaline-producing dietary components, specifically, potassium, magnesium, and fruit and vegetables, contribute to maintenance of BMD. (+info)
Regulation of homologous recombination: Chi inactivates RecBCD enzyme by disassembly of the three subunits.
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We report here an unusual mechanism for enzyme regulation: the disassembly of all three subunits of RecBCD enzyme after its interaction with a Chi recombination hot spot. The enzyme, which is essential for the major pathway of recombination in Escherichia coli, acts on linear double-stranded DNA bearing a Chi site to produce single-stranded DNA substrates for strand exchange by RecA protein. We show that after reaction with DNA bearing Chi sites, RecBCD enzyme is inactivated and the three subunits migrate as separate species during glycerol gradient ultracentrifugation or native gel electrophoresis. This Chi-mediated inactivation and disassembly of purified RecBCD enzyme can account for the previously reported Chi-dependent loss of Chi activity in E. coli cells containing broken DNA. Our results support a model of recombination in which Chi regulates one RecBCD enzyme molecule to make a single recombinational exchange ('one enzyme-one exchange' hypothesis). (+info)
Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments.
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Ca2+- and Mg2+-dependent endonucleases have been implicated in DNA fragmentation during apoptosis. We have demonstrated that particular nucleases of this type are inhibited by poly(ADP-ribosyl)ation and suggested that subsequent cleavage of PARP by caspase-3 might release these nucleases from poly(ADP-ribosyl)ation-induced inhibition. Hence, we purified and partially sequenced such a nuclease isolated from bovine seminal plasma and identified human, rat and mouse homologs of this enzyme. The extent of sequence homology among these nucleases indicates that these four proteins are orthologous members of the family of DNase I-related enzymes. We demonstrate that the activation of the human homolog previously specified as DNAS1L3 can induce Ca2+- and Mg2+-dependent DNA fragmentation in vitro and in vivo. RT-PCR analysis failed to detect DNAS1L3 mRNA in HeLa cells and nuclei isolated from these cells did not exhibit internucleosomal DNA fragmentation when incubated in the presence of Ca2+and Mg2+. However, nuclei isolated from HeLa cells that had been stably transfected with DNAS1L3 cDNA underwent such DNA fragmentation in the presence of both ions. The Ca2+ionophore ionomycin also induced internucleosomal DNA degradation in transfected but not in control HeLa cells. Transverse alternating field electrophoresis revealed that in nuclei from transfected HeLa cells, but not in those from control cells, DNA was cleaved into fragments of >1000 kb in the presence of Mg2+; addition of Ca2+in the presence of Mg2+resulted in processing of the >1000 kb fragments into 50 kb and oligonucleosomal fragments. These results demonstrate that DNAS1L3 is necessary for Ca2+- and Mg2+-dependent cleavage of DNA into both oligonucleosomal and high molecular mass fragments in specific cell types. (+info)
Coronary microvascular protection with mg2+: effects on intracellular calcium regulation and vascular function.
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The use of Mg2+-supplemented hyperkalemic cardioplegia preserves microvascular function. However, the mechanism of this beneficial action remains to be elucidated. We investigated the effects of Mg2+ supplementation on the regulation of intracellular calcium concentration ([Ca2+]i) and vascular function using an in vitro microvascular model. Ferret coronary arterioles (80-150 micrometer in diameter) were studied in a pressurized (40 mmHg) no-flow, normothermic (37 degrees C) state. Simultaneous monitoring of internal luminal diameter and [Ca2+]i using fura 2 were made with microscopic image analysis. The microvessels (n = 6 each group) were divided into four groups according to the content of MgCl2 (nominally 0, 1.2, 5.0, and 25.0 mM) in a hyperkalemic cardioplegic solution ([K+] 25.0 mM). After baseline measurements, vessels were subjected to 60 min of hypoxia with hyperkalemic cardioplegia (equilibrated with 95% N2-5% CO2) containing each concentration of Mg2+ ([Mg2+]) and were then reoxygenated. During hyperkalemic cardioplegia, [Ca2+]i increased in a time-dependent manner in all groups. In the lower [Mg2+] cardioplegia groups, [Ca2+]i was significantly increased at the end of the 60-min cardioplegic period (247 +/- 44 nM and 236 +/- 49 nM in [Mg2+] 0 and 1.2 mM groups, respectively; both P < 0.05 vs. baseline) with 19.6-17.2% vascular contraction. Conversely, there was no significant [Ca2+]i increase in the higher [Mg2+] cardioplegia groups and less vascular contraction (5.4-4.1%, both P < 0.05 vs. [Mg2+] 1.2 mM group). After reperfusion, agonist (U-46619, thromboxane A2 analog)-induced vascular contraction was significantly enhanced in the lower [Mg2+] cardioplegia groups (both P < 0.05 vs. control) but was normalized in the higher [Mg2+] cardioplegia groups. Intrinsic myogenic contraction was significantly decreased in the lower [Mg2+] cardioplegia groups (both P < 0.05 vs. control) but was preserved in the higher [Mg2+] cardioplegia groups. These results suggest that supplementation of the solution with >5.0 mM [Mg2+] may prevent hyperkalemic cardioplegia-related intracellular Ca2+ overloading and preserve vascular contractile function in coronary microvessels. (+info)
Nucleotide binding to the G12V-mutant of Cdc42 investigated by X-ray diffraction and fluorescence spectroscopy: two different nucleotide states in one crystal.
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The 2.5 A crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine-5'-diphospho-beta-amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42 x GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP-bound molecule, but not in the molecule bound to GDPNH2. The C-terminus containing the CaaX-motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively. (+info)
Extracellular heavy-metal ions stimulate Ca2+ mobilization in hepatocytes.
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Populations of hepatocytes in primary culture were loaded with fura 2 and the effects of extracellular heavy-metal ions were examined under conditions that allowed changes in fura 2 fluorescence (R340/360, the ratio of fluorescence recorded at 340 and 360 nm) to be directly attributed to changes in cytosolic free [Ca2+] ([Ca2+]i). In Ca2+-free media, Ni2+ [EC50 (concentration causing 50% stimulation) approximately 24+/-9 microM] caused reversible increases in [Ca2+]i that resulted from mobilization of the same intracellular Ca2+ stores as were released by [Arg8]vasopressin. The effects of Ni2+ were not mimicked by increasing the extracellular [Mg2+], by addition of MnCl2, CoCl2 or CdCl2 or by decreasing the extracellular pH from 7.3 to 6.0; nor were they observed in cultures of smooth muscle, endothelial cells or pituitary cells. CuCl2 (80 microM), ZnCl2 (80 microM) and LaCl3 (5 mM) mimicked the ability of Ni2+ to evoke Ca2+ mobilization. The response to La3+ was sustained even in the absence of extracellular Ca2+, probably because La3+ also inhibited Ca2+ extrusion. Although Ni2+ entered hepatocytes, from the extent to which it quenched fura 2 fluorescence the free cytosolic [Ni2+] ([Ni2+]i) was estimated to be <5 nM at the peak of the maximal Ni2+-evoked Ca2+ signals and there was no correlation between [Ni2+]i and the amplitude of the evoked increases in [Ca2+]i. We conclude that extracellular Ni2+, Zn2+, Cu2+ and La3+, but not all heavy-metal ions, evoke an increase in [Ca2+]i in hepatocytes by stimulating release of the hormone-sensitive intracellular Ca2+ stores and that they may do so by interacting with a specific cell-surface ion receptor. This putative ion receptor may be important in allowing hepatocytes to contribute to regulation of plasma heavy-metal ions and may mediate responses to Zn2+ released into the portal circulation with insulin. (+info)