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1.
Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis 总被引:1,自引:0,他引:1
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HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg2+ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP–RNA interactions. The best divalent cations were Mn2+, Zn2+ and Cd2+, followed by Mg2+, Co2+ and Ni2+, while Cu2+, Yb2+ and Hg2+ were ineffective. In the HutP–RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein–RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg2+, Mn2+ and Ba2+), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition. 相似文献
2.
Dhanasekaran Shanmugam Bo Wu Ursula Ramirez Eileen K. Jaffe David S. Roos 《The Journal of biological chemistry》2010,285(29):22122-22131
Apicomplexan parasites (including Plasmodium spp. and Toxoplasma gondii) employ a four-carbon pathway for de novo heme biosynthesis, but this pathway is distinct from the animal/fungal C4 pathway in that it is distributed between three compartments: the mitochondrion, cytosol, and apicoplast, a plastid acquired by secondary endosymbiosis of an alga. Parasite porphobilinogen synthase (PBGS) resides within the apicoplast, and phylogenetic analysis indicates a plant origin. The PBGS family exhibits a complex use of metal ions (Zn2+ and Mg2+) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ∼320-kDa octamer, and low levels of dimers but no hexamers were also observed. The enzyme displays a broad activity peak (pH 7–8.5), with a Km for aminolevulinic acid of ∼150 μm and specific activity of ∼24 μmol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg2+ but not Zn2+ and shows two Mg2+ affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg2+-binding PBGS, however, metal ions are not required for TgPBGS octamer stability. A mutant enzyme lacking the C-terminal 13 amino acids distinguishing parasite PBGS from plant and animal enzymes purified as a dimer, suggesting that the C terminus is required for octamer stability. Parasite heme biosynthesis is inhibited (and parasites are killed) by succinylacetone, an active site-directed suicide substrate. The distinct phylogenetic, enzymatic, and structural features of apicomplexan PBGS offer scope for developing selective inhibitors of the parasite enzyme based on its quaternary structure characteristics. 相似文献
3.
Ribulose 1,5-diphosphate-dependent oxygenase activity was demonstrated for crystallized Fraction 1 protein (RuDP2 carboxylase EC 4.1.1.39) from tobacco. The kinetic properties of this oxygenase function were examined polarographically in air-equilibrated medium. Optimum activity was obtained at pH 8.4–8.6, and required 4–8 mm MgCl2. Higher Mg2+ concentrations decreased activity and slightly shifted the pH optimum to 8.2–8.3. The apparent Km (RuDP) and Km (Mg2+) were 22 μm and 0.5 mm, respectively. Oxygenase activity was inhibited by bicarbonate and indirectly by KCN. Kinetic studies suggest that the active inhibitory substance is the cyanohydrin derivative formed from the reaction of KCN with RuDP.Changes in oxygenase kinetics were observed upon addition of RuDP, as previously reported for the carboxylase function of this enzyme. Oxygenase activity required preincubation of the enzyme with both Mg2+ and low concentrations of bicarbonate. Activities were enhanced about 20 and 70% when FDP (0.1 mm) and NADPH (0.5 mm), respectively, were included during preincubation. 相似文献
4.
5.
Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca2+,Mg2+-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca2+ + Mg2+) > Mn2+ = (Ca2+ + Mn2+) > (Mg2+ + EGTA) > Ca2+. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn2+ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca2+,Mn2+-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca2+ + Mn2+) > (Ca2+ + Mg2+) > Mn2+ > (Mg2+ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed. 相似文献
6.
The pH dependence of chlortetracycline fluorescence has been reinvestigated. Below pH 7.5, excitation at 400 nm results in an emission band at 530 nm. Above pH 7.5, in addition to the above band, a strong fluorescence is observed at 430 nm on excitation at 345 nm. pKa values of 3.5 and 7.7 have been determined for the 530- and 430 nm bands, permitting their assignment to the ring A and ring BCD chromophores, respectively. Both bands are shown to be sensitive to divalent metal binding. Excitation energy transfer from the 430-bband to the 530-nm band is observed in metal complexes, with transfer efficiency being greater for the Mg2+ chelate than the Ca2+ chelate. The effect of solvent and lipid on band intensities and transfer efficiencies is reported. The interaction of chlortetracycline with BSA in the presence of Mg2+ leads to the observation of energy transfer from protein tryptophan residues to the 520-nm band of the complex via the intermediacy of the 430-nm band. 相似文献
7.
Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : V. Modulation of the Spinach Leaf Cytosolic Fructose 1,6-Bisphosphatase Activity in Vitro by Substrate, Products, pH, Magnesium, Fructose 2,6-Bisphosphate, Adenosine Monophosphate, and Dihydroxyacetone Phosphate 总被引:6,自引:5,他引:1
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How fructose 2,6-bisphosphate and metabolic intermediates interact to regulate the activity of the cytosolic fructose 1,6-bisphosphatase in vitro has been investigated. Mg2+ is required as an activator. There is a wide pH optimum, especially at high Mg2+. The substrate dependence is not markedly pH dependent. High concentrations of Mg2+ and fructose 1,6-bisphosphate are inhibitory, especially at higher pH. Fructose 2,6-bisphosphate inhibits over a wide range of pH values. It acts by lowering the maximal activity and lowering the affinity for fructose 1,6-bisphosphate, for which sigmoidal saturation kinetics are induced, but the Mg2+ dependence is not markedly altered. On its own, adenosine monophosphate inhibits competitively to Mg2+ and noncompetitively to fructose 1,6-bisphosphate. In the presence of fructose 2,6-bisphosphate, adenosine monophosphate inhibits in a fructose 1,6-bisphosphate-dependent manner. In the presence of adenosine monophosphate, fructose 2,6-bisphosphate inhibits in Mg2+-dependent manner. Fructose 6-phosphate and phosphate both inhibit competitively to fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate does not affect the inhibition by phosphate, but weakens inhibition by fructose 6-phosphate. Dihydroxyacetone phosphate and hydroxypyruvate inhibit noncompetitively to fructose 1,6-bisphosphate and to Mg2+, but both act as activators in the presence of fructose 2,6-bisphosphate by decreasing the S0.5 for fructose 1,6-bisphosphate. A model is proposed to account for the interaction between these effectors. 相似文献
8.
Divalent metal ion effects on a mutant histidinol phosphate phosphatase from Salmonella typhimurium 总被引:1,自引:0,他引:1
The effect of divalent metal ions on the activity of a mutant histidinol phosphate phosphatase has been studied. The enzyme was isolated from strain TA387, a mutant of Salmonella typhimurium with a nonsense lesion near the midpoint of the bifunctional hisB gene. Mn2+, Mg2+, Co2+, and Zn2+ shift the optimal pH of phosphatase activity to 6.5 while Be2+ and Ca2+ have no effect on the shape of the pH profile. In the absence of divalent metal ions, the pH optimum is 7.5. Four Me2+ ions, Mn2+, Co2+, Zn2+, and Fe2+ decreased the Km of histidinol phosphate at pH 6.5 from 5.5 mm (without Me2+) to 0.14 mm. Ni2+ and Be2+ increased the Km to 22.2 and 25.0 mm, respectively, and Ca2+ and Mg2+ had an intermediate effect. Changes in maximal velocity were substantially less, only about 2-fold changes being observed. It was shown that the maximal velocity at optimal pH was the same in the absence and presence of Mn2+. Kinetic analysis indicated that there was a rapid equilibrium-ordered addition of Mn2+ to the enzyme before the addition of the substrate, histidinol phosphate. A kimn2+ of 4.3 μm was calculated for the metal ion activation at both pH 6.5 and 7.5. Addition of ethyl-enediaminetetracetate (EDTA) strongly inhibited the phosphatase; inhibition could be reversed by addition of several Me2+ ions, Mg2+ being the most efficient followed by Mn2+. Prolonged incubation with EDTA led to irreversible inactivation. 相似文献
9.
Toolsee J. Singh Akira Akatsuka Kuo-Ping Huang 《Archives of biochemistry and biophysics》1982,218(2):360-368
The stimulation of phosphorylase kinase by Mg2+ was studied. Both the nonactivated and activated kinases are stimulated by Mg2+ at concentrations that are 100- to 200-fold greater than ATP. This stimulation is observed at both pH 6.8 and 8.2 and results in a 10-fold increase in the activity of the nonactivated kinase. Mg2+ stimulation is additive with that observed by calmodulin. Both the Ca2+-dependent and -independent activities of the kinase are stimulated by high [Mg2+]. Kinetically this stimulation can be explained by a decrease in the Km for both phosphorylase b and ATP or an increase in V. The pH ratio (0.06) is unaffected by [Mg2+] between 5 and 20 mm, but increases when [Mg2+] is less than 5 mm or greater than 20 mm. The stimulation by high [Mg2+] is explained by a direct effect of this cation on the kinase molecule rather than on its protein substrate, phosphorylase. This activating effect of high [Mg2+] does not result in any permanent change in the kinase molecule and can be readily reversed by diluting [Mg2+] to a low value. 相似文献
10.
《Plant science》1986,43(1):51-56
Analysis of the pH 8.3 electrophoresis of the Phaseolus vulgaris seed lectins suggests that each of the five isolectins exists in five different forms. Incubations of the lectins in the presence of EDTA or of metal ions such as Mn2+, Mg2+, Co2+ and Ni2+ shows that their different electrophoretic behaviours are related to the binding of metal ions to the molecules.Determinations of the isolectin metal content by atomic absorption measurements demonstrate that at pH 8.3 their five electrophoretic forms are related to the number of subunits (0, 1, 2, 3 or 4) which contain metal ions in the tetrameric molecules. 相似文献
11.
Yang W Chen WY Wang H Ho JW Huang JD Woo PC Lau SK Yuen KY Zhang Q Zhou W Bartlam M Watt RM Rao Z 《Nucleic acids research》2011,39(22):9803-9819
Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced β-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5′-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg2+ or Mn2+ ions. 5′-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a ‘doughnut-shaped’ toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg2+ ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases. 相似文献
12.
An alkaline inorganic pyrophosphatase (IP) from leaves of an orchid, Aranda Christine 130 (Arachnis hookerana var. luteola × Vanda Hilo Blue) was purified by acetone precipitation and chromatography on Sephadex G-75 and DEAE-cellulose. The IP gave a single band on non-denaturing gel electrophoresis at pH 8.3 and its M, determined by gel filtration, was 28 000. The pH optimum was 9 and the IP required Mg2+ for its activity and stability. The IP exhibited high specificity for PPi and attained a maximum activity at a Mg2+: PPi ratio of 10:1. Other cations tested could not replace Mg2+ and they were also found to be inhibitory. The IP was also inhibited by EDTA and F? but not by iodoacetamide. 相似文献
13.
The capacity of various metal ions to support activation of bovine factor IX, by the coagulant protein of Russell's Viper venom, has been examined. The following metal ions, at concentrations which saturate their effect, promoted activation of factor IX, at approximately equal efficiency: Ca2+, Mn2+, Sr2+, and Co2+, Other metal ions, i.e., Ba2+, and Mg2+, at saturating concentrations, led to a maximum rate of activation of factor IX of 25%, compared to Ca2+, The lanthanides, Gd2+, and Tb3+, also promoted activation in this system, at maximal rates of approximately 15%, compared to Ca2+, In this study, it was also discovered that the esterase activity of bovine factor IXa was dependent upon the presence of metal ions. Utilizing α-N-benzoyl-l-arginine ethyl ester as the substrate, steady state kinetic analysis in the absence of metal ion indicated that the Km and Vmax for this substrate was 20 mm and 2.9 μmol substrate cleaved min?1 mg?1 of factor IXa, respectively, at pH 8.0 and 30 °C. In the presence of optimal concentrations of Ca2+, Mn2+, Mg2+, Sr2+, and Ba2+, the Vmax values for this same substrate increased to 6.7, 5.9, 5.0, 5.0, and 3.7 μmol cleaved min?1 mg?1 of factor IXa, respectively. None of these metal ions had an affect on the Km value of this substrate. 相似文献
14.
A. S. Rozanov S. N. Zagrebelny A. B. Beklemishev 《Applied Biochemistry and Microbiology》2009,45(1):31-37
The coding region of Escherichia coli K12 xylose (glucose) isomerase gene was inserted into the pRAC expression vector and cloned in E. coli BL21(DE3) cells. After induction of expression of the cloned gene, the proportion of recombinant xylose isomerase accounted for 40% of the total protein content. As a result of one-stage purification by affinity chromatography, a protein preparation of 90% purity was obtained. The recombinant enzyme catalyzed the isomerization of glucose to fructose and exhibited maximum activity (0.8 U/mg) at 45°C and pH 6.8. The enzyme required Mg2+ ions as a cofactor. When Mg2+ and Co2+ ions were simultaneously present in the reaction medium, the enzyme activity increased by 15–20%. Complete replacement of Mg2+ with Co2+ decreased the enzyme activity. In the presence of Ca2+ at concentrations comparable to the concentration of Mg2+, the enzyme was not inhibited, although published data reported inhibition of similar enzymes by Ca2+. The recombinant enzyme exhibited a very low thermostability: it underwent a slow inactivation when incubated at 45°C and was completely inactivated after incubation at 65°C for 1 h. 相似文献
15.
Magnesium-dependent adenosine triphosphatase, purified from sheep kidney medulla using digitonin, has been characterized in a series of kinetic and magnetic resonance studies. Kinetic studies of divalent metal activation using either Mg2+ or Mn2+ indicate a biphasic response to divalent cations. Apparent Km values of 23 μm for free Mg2+ and 3.3 μm for free Mn2+ are obtained at low levels of added metal, while Km values of 0.50 mm for free Mg2+ and 0.43 mm for free Mn2+ are obtained at much higher levels of divalent cations. In all cases the kinetic data indicate that the binding of divalent metals is independent of the substrate, ATP. Kinetic studies of the substrate requirements of the Mg2+-ATPase also yield biphasic Lineweaver-Burk plots. At low ATP concentrations, kinetic studies yield apparent Km values for free ATP of 6.0 and 1.4 μm with Mg2+ and Mn2+, respectively, as the activating divalent metals. At much higher levels of ATP the response of the enzyme to ATP changes so that Km values for free ATP of 8.0 and 2.0 mm are obtained for Mg2+ and Mn2+, respectively. In both cases, however, the binding of ATP is independent of added metal. ADP inhibits the Mg2+-ATPase and the kinetic data indicate that ADP competes with ATP at both the high and low affinity sites. Dixon plots of the data are consistent with competitive inhibition at both ATP sites, with Ki values of 10.5 μm and 4.5 mm. Electron paramagnetic resonance and water proton relaxation rate studies show that the enzyme binds 1 g ion of Mn2+ per 469,000 g of protein. The Mn2+ binding studies yield a KD for Mn2+ at the single high affinity site of 2 μm, in good agreement with the kinetically determined activator constant for Mn2+ at low Mn2+ levels. Moreover, the EPR binding studies also indicate the existence of 34 weak sites for Mn2+ per single high affinity Mn2+ site. The KD for Mn2+ at these sites is 0.55 mm, in good agreement with the kinetic activator constant for Mn2+ of 0.43 mm, consistent with additional activation of the enzyme by the large number of weaker metal binding sites. The enhancement of water proton relaxation by Mn2+ in the presence of the enzyme is also consistent with the tight binding of a single Mn2+ ion per 469,000 Mr protein and the weaker binding of a large number of divalent metal ions. Analysis of the data yields a value for the enhancement for bound Mn2+ at the single tight site, ?b, of 5 and an enhancement at the 34 weak sites of 11. The frequency dependence of water proton relaxation by Mn2+ at the single tight site yields a dipolar correlation time (constant from 8–60 MHz) of 3.18 × 10?9 s. The kinetics and metal binding studies, together with the effect of temperature on ATPase activity at high and low levels of ATP, are consistent with the existence in this preparation of a single Mg2+-ATPase, with high and low affinity sites for divalent metals and for ATP. Observations of both high and low affinities for ATP have been made with two other purified ATPases. The similarities of these systems to the Mg2+-ATPase described here are discussed. 相似文献
16.
Isolation and Characterization of Phosphoenolpyruvate Phosphatase from Germinating Mung Beans (Vigna radiata) 总被引:15,自引:7,他引:8
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A phosphoenolpyruvate (PEP) phosphatase was purified to homogeneity from germinating mung beans (Vigna radiata). It was found to be a tetrameric protein (molecular mass 240,000 daltons) made up of apparently identical subunits (subunit molecular mass 60,000 daltons). It was free from bound nucleotides. It did not show pyruvate kinase activity. The enzyme showed high specificity for PEP. Pyrophosphate and some esters (nucleoside di- and triphosphates) were hydrolyzed slowly and phosphoric acid monoesters were not hydrolyzed. The enzyme showed maximum activity at pH 8.5. At this pH, the Km of PEP was 0.14 millimolar and the Vmax was equal to 1.05 micromoles pyruvate formed per minute per milligram enzyme protein. Dialysis of the enzyme against 10 millimolar triethanolamine buffer (pH 6.5), led to loss of the catalytic activity, which was restored on addition of Mg2+ ions (Km = 0.12 millimolar). Other divalent metal ions inhibited the Mg2+ -activated enzyme. PEP-phosphatase was inhibited by ATP and several other metabolites. 相似文献
17.
Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal 总被引:3,自引:2,他引:3
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Katherine D. McMahon Michael A. Dojka Norman R. Pace David Jenkins Jay D. Keasling 《Applied microbiology》2002,68(10):4971-4978
A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like β-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg2+, and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms. 相似文献
18.
Glutamine synthetase of pea leaves: divalent cation effects, substrate specificity, and other properties 总被引:1,自引:0,他引:1
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Purified glutamine synthetase from pea seedlings was most active with Mg2+ as the metal activator, but Mn2+ and Co2+ were 45 to 60% and 30 to 45% as effective, respectively, when assayed at the optimal pH for each cation. The Mg2+ saturation curve was quite sigmoid, and evidence indicates that MgATP is the active ATP substance. Co2+ also gave a sigmoidal saturation curve, but when Mn2+ was varied only slightly sigmoidal kinetics were seen. Addition of Mn2+, Ca2+, or Zn2+ at low concentrations sharply inhibited the Mg2+ -dependent activity, partially by shifting the pH optimum. Addition of Co2+ did not inhibit Mg2+-dependent activity. The nucleotide triphosphate specificity changed markedly when Co2+ or Mn2+ replaced Mg2+. Using the Mg2+-dependent assay, the Michaelis constant (Km) for NH4+ was about 1.9 × 10−3 M. The Km for l-glutamate was directly proportional to ATP concentration and ranged from 3.5 to 12.4 mm with the ATP levels tested. The Km for MgATP also varied with the l-glutamate concentration, ranging from 0.14 mm to 0.65 mm. Ethylenediaminetetracetic acid activated the enzyme by up to 54%, while sulfhydryl reagents gave slight activation, occasionally up to 34%. 相似文献
19.
Noriyuki Koyama Kazuo Koshiya Yoshiaki Nosoh 《Archives of biochemistry and biophysics》1980,199(1):103-109
ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (?). The subunit structure is suggested to be α3β3γδ?. The enzyme is activated by Mg2+ and Ca2+. The pH optima of the enzyme with 0.1 and 2.0 mm Mg2+ are 9 and 6, and those with 1 and 10 mm Ca2+ are 8–9 and 7, respectively. Ca2+-ATPase hydrolyzes only ATP, whereas Mg2+-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and Km of the enzyme with ATP in the presence of 10 mm Ca2+ or 0.6 mm Mg2+ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 mm, respectively. The enzyme with Mg2+ is appreciably activated by HCO?3. Relationship of the ATPase to the active transport system in the bacterium is suggested. 相似文献
20.
The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His244 or His266 residue. The binding of Zn2+ to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co2+, Ca2+, Ni2+, Mg2+, and Mn2+. The strength of Ca2+, Co2+, Mg2+, Mn2+ and Ni2+ binding to the site close to His244 is stronger than that of these divalent metal ions binding to the site close to His266. 相似文献