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1.
《BBA》1986,848(2):224-229
Evidence is presented for the presence of divalent cation binding sites in purified F1-ATPase from Micrococcus lysodeikticus (Micrococcus luteus). Electron paramagnetic resonance studies of native F1-ATPase indicate that the enzyme binds Mn2+ and Cu2+. Scatchard-type plot for Mn2+ binding to the enzyme indicates the presence of 3–4 independent and identical sites with a dissociation constant of 18.3 · 10−6 M. Cu2+ binds to the enzyme at only one kind of site(s). This Cu2+ binding site(s) is characterized by a moderately ionic ligand field provided by the protein and by a tetragonal symmetry of nitrogen and/or oxigen ligands. Competition studies indicate that Mg2+ binds at these Mn2+ and Cu2+ binding sites.  相似文献   

2.
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   

3.
Binding of fructose-6-P and Pi to rabbit liver fructose bisphosphatase has been analyzed in terms of four negatively cooperative binding sites per enzyme tetramer. The association of fructose-6-P occurs in the absence of divalent metal ion, although the extent of binding is increased in the order Mg2+ < Zn2+ < Mn2+. The binding of Pi shows an absolute requirement for divalent metal ion with Mn2+ being more effective than Mg2+. The interaction of the enzyme with the substrate analog, (α + β) methyl-d-fructofuranoside-1,6-P2 in the presence of Mn2+ closely resembles that found for fructose-1,6-P2 in the absence of Mn2+, although the measured constants are on average an order of magnitude smaller. Combination experiments with the three ligands show that the binding follows an identical ordered sequence, i.e., the tighter sites are initially occupied regardless of the ligand's identity. The binding of Pi or fructose-6-P is not altered by the presence of the other. Comparison of binding constant with Ki values obtained from steady-state assays permits identification of the catalytic sites expressed in the latter. The association of Mn2+ at the catalytic site can be induced by fructose-6-P or the substrate analog suggesting that a 1-phosphoryl group enhances but is not necessary for Mn2+ binding at this site. The binding of AMP is decreased in the presence of substrate analog relative to fructose-1,6-P2, suggesting that the 2-hydroxyl serves as a “molecular signal.” From the single and combined binding experiments, a calculation of the equilibrium constant for the overall hydrolysis reaction on the enzyme surface in the presence of Mn2+ has been carried out and an estimate made for the Mg2+ case.  相似文献   

4.
The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg2+-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca2+ + Mg2+-ATPase) of relatively low activity. Mg2+-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca2+ + Mg2+)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg2+-ATPase activity revealed apparent Km values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg2+-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg2+-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca2+ + Mg2+)-ATPase was 13.7 nmol 32P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol 32P/mg/min. Characterization of (Ca2+ + Mg2+)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent Kms for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca2+ + Mg2+)-ATPase activity was stimulated by potassium with an apparent Km of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca2+ + Mg2+)-ATPase are similar to those of the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca2+ + Mg2+)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.  相似文献   

5.
The (K+,Mg2+)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N2 without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co2+ > Mg2+ > Mn2+ > Zn2+ > Ca2+) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg2+, the enzyme was further activated by monovalent cations (K+, NH4+, Rb+ Na+, Cs+, Li+). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a Km of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K+ approached simple Michaelis-Menten kinetics, with a Km of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K+, and preferred Mn2+ to Mg2+. The results demonstrate that the (K+,Mg2+)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.  相似文献   

6.
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 favorsE 2 conformations of the enzyme, increased apparent affinity for K+, whereas oligomycin, which favorsE 1 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)E 2 conformations, are effective, whereas Ca2+, favoringE 1, 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 favorsE 1 conformations. K+ increases theK m 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   

7.
The binding of Mg2+ to intracellular 2,3-bisphosphoglycerate in the human red blood cell is significant to the function of the cell. We have studied interactions of Mg2+ and Mn2+ with 2,3-bisphosphoglycerate by magnetic resonance spectroscopy. The results of this study reveal the presence of two independent divalent metal cation binding sites of similar affinity (KD = 3.0 ± 0.5 mM) in the 2,3-bisphosphoglycerate molecule, one on each phosphoryl group, contrary to the assumption of one metal ion binding site made in the previous literature. Over the range of their intracellular concentrations, ATP and ADP, however, possess only one metal ion site in spite of the presence of multiple phosphoryl groups. These results are consistent with the chemistry of metal-chelation which requires the formation of 5- or 6-membered rings for the stability of chelate structures.  相似文献   

8.
ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na+ + K+)-ATPase in the absence of Mg2+ (EDTA present) with a homogeneous but 15-fold different affinity, the Kd values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na+ + K+)-ATPase, respectively. The Kd value for ATP is equal to the Km for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na+ + K+)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg2+, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The Kd and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg2+ concentration. The Kd increases from 0.06 mM at 0.5 mM Mg2+ to a maximum of 0.26 mM at 2 mM Mg2+ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg2+ to 3.3 nmol/mg protein at 4 mM Mg2+. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg2+ concentration yields a maximal binding capacity at infinite Mg2+ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na+ + K+)-ATPase. The Kd for Mg2+ at the sites, where it exerts this effect, is 0.8 mM. The Kd for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg2+ to a maximum of 4.2 μM at 2 mM Mg2+ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg2+ concentration. Hence, Mg2+ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na+ + K+)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.  相似文献   

9.
Adenosine kinase (ATP:adenosine 5′-phosphotransferase, EC 2.7.1.20) from Lupinus luteus seeds has been obtained with good yield in almost homogeneous state by ammonium sulfate fractionation, chromatography on aminohexyl-Sepharose, and gel filtration. Active enzyme is a single polypeptide chain with a molecular weight of about 38,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel nitration. Estimated molecular activity is 156. The enzyme exhibits a strict requirement for divalent metal ions. Among several ions tested the following appeared to be active as cofactors: Co2+ ? Mn2+ > Mg2+ = Ca2+ ? Ni2+ > Ba2+. The optimal metal ion concentrations were as follows: Mn2+, 0.5 mm, Mg2+ and Ca2+, 1 mm, Co2+, 1.5 mm. The adenosine kinase shows optimum activity at pH 7.0–7.5. Km values for adenosine and ATP are 1.5 × 10?6 and 3 × 10?4m, respectively. Lupin adenosine kinase is completely inhibited by antisulfhydryl reagents. ATP is the main phosphate donor and among other nucleoside triphosphates ITP, dATP, GTP, and XTP can substitute it but less effectively. Among the ribo- and deoxyribonucleosides occurring in nucleic acids adenosine is phosphorylated effectively and 2′-deoxyadenosine at a lower rate. Of other adenosine analogs tested all adenine d-nucleosides and purine derivative ribosides, besides those with a hydroxyl group at C-6, were found to be substrates for lupin adenosine kinase. Pyrimidine ribo- and deoxyribonucleosides were not phosphorylated.  相似文献   

10.
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   

11.
The effect of metal ions on human activated Factor X (Factor Xa) hydrolysis of the chromogenic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222) was studied utilizing initial rate enzyme kinetics. The divalent metal ions Ca2+, Mn2+, and Mg2+ enhanced Factor Xa amidolytic activity with Km values of 30 μm, 20 μm, and 1.4 mm, respectively. Na+ activation of Factor Xa amidolytic activity was also found. The Km for Na+ activation was 0.31 m. Both the divalent metal ions and Na+ increased the affinity of Factor Xa for S2222 and had no effect on the maximal velocity of the reaction. Other monovalent cations were unable to activate Factor Xa. However, K+ was a competitive inhibitor of the Na+ activation (Ki = 0.14 m). Lanthanide ions inhibited Factor Xa amidolytic activity. Gd3+ inhibition of Factor Xa hydrolysis of S2222 was noncompetitive and had a Ki of 3 μm. The lanthanide ion inhibition could not be reversed by Ca2+ even when Ca2+ was present in a 1000-fold excess over its Km indicating nonidentity of the Factor Xa lanthanide and Ca2+ binding sites. It is concluded that the Factor Xa Ca2+ binding sites have characteristics different from those previously described for the Factor X molecule and that Mg2+, Na+, and K+ may be physiological regulators of Factor Xa activity.  相似文献   

12.
Low concentrations of Mn2+ supported the basal adenylate cyclase activity in crude and purified sarcolemmal membranes from cardiac muscle more effectively than did relatively high concentrations of Mg2+; at saturating concentrations the cyclase activities obtained with Mg2+ or Mn2+ were similar. In contrast, Mg2+ supported the basal cyclase activities of crude membrane fractions and purified sarcolemmal membranes from skeletal muscle far more effectively than did Mn2+; at saturating concentrations of either metal ion the Mg2+-supported cyclase activities were 5- to 10-fold greater than Mn2+-supported activities. Further, compared to Mg2+, Mn2+ supported the cyclase activities very poorly in all the primary subcellular fractions of skeletal muscle, whereas this cation was at least as effective as Mg2+ in all fractions of cardiac muscle. The apparent affinities of the cyclase for Mn2+ in heart as well as skeletal muscle appeared to be greater compared to those for Mg2+. The skeletal muscle cyclase displayed greater apparent affinity for MnATP2? (app. Km 0.10 mm) compared to MgATP2? (app. Km 0.32 mm) whereas the heart enzyme displayed greater apparent affinity for MgATP2? (app. Km 0.07 mm) compared to MnATP2? (app. Km 0.19 mm). Following preactivation with guanyl-5′-yl imidodiphosphate and isoproterenol, Mn2+ (0.15 to 2 mm) supported the cyclase activity of skeletal muscle even more effectively than did optimally effective concentrations of Mg2+. With the heart enzyme the relatively greater potency of Mn2+ persisted following preactivation. Significant enhancement in the Mn2+-sensitivity of skeletal muscle cyclase was also observed when assayed in the presence of GTP and isoproterenol or in the presence of NaF. Preactivation of both heart and skeletal muscle cyclases caused selective enhancement in the enzyme's apparent affinity for free Me2+ (Mg2+ or Mn2+) without influencing the apparent Km for MeATP2? (MgATP2? or MnATP2?). Evidences were obtained to show that the poor effectiveness of Mn2+ in supporting the basal activity of skeletal muscle cyclase is not related to (a) potentiation by Mn2+ of adenosine-mediated inhibition of the cyclase, (b) Mn2+-induced lability of the cyclase, (c) indirect effects of Mn2+ on ATP-regenerating system, or (d) the presence of different cation-specific molecular forms of the cyclase. It is also shown that the onset of enhanced Mn2+ sensitivity of the skeletal muscle enzyme following preactivation is not accompanied by a general loss of cation specificity of the cyclase. These results suggest that cations support the catalytic activity of adenylate cyclase by interacting with an enzymeregulatory free metal binding site and that the differential cation sensitivity of nonactivated (basal) cyclases from heart and skeletal muscle is likely due to differences in the properties of such an allosteric metal site. Furthermore, the metal site appears to undergo a conformational change following interaction of the cyclase system with the guanyl nucleotide and isoproterenol since the cation sensitivity of the cyclase and the relative potency of cations depend on the conformational status of the enzyme.  相似文献   

13.
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   

14.
Hexokinase was partially purified from the leaves of Dendrophthoe falcata. The optimum pH for the enzyme was 8.5. The enzyme was sensitive to p-CMB and the inhibition could be reversed by 2-mercaptoethanol. The optimum temperature was 40° and energy of activation 6900 cal/mol. The enzyme had an absolute requirement for a divalent metal ion. Although Mg2+ was the preferred metal, it could be partially replaced by Mn2+ and Ca2+. ATP was the most effective phosphoryl donor. Glucose was the best substrate, the Km values of 0.14 and 0.26 mM were obtained at saturated and sub-saturated ATP concentration. Phosphorylation coefficients show the following order of reactivity of sugars: glucose mannose 2-deoxy D-glucose fructose glucosamine galactose ribose. The Km value for ATP was 0.16 mM, which increased to 0.35 mM in the presence of 0.5 mM ADP. ADP and 5′-AMP were competitive inhibitors with respect to ATP, and Ki values were 0.4 and 1.2 mM respectively.  相似文献   

15.
(1) The effects of calmodulin binding on the rates of Ca2+-dependent phosphorylation and dephosphorylation of the red-cell Ca2+ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg2+-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg2+-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca2+ + Mg2+)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca2+ affinity for sustaining ATP hydrolysis is raised at least 20-fold, Km(Ca) ? 10 μM (—calmodulin) and Km(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca2+ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (Km(Ca) ? 3–4 · 10-8 M). (Ca2+ + Mg2+)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca2+ + Mg2+)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca2+ + Mg2+)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (Km(MgATP) ? 360 μM), and CaATP is an inhibitor (Ki(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca2+- pump active site, producing a high (Ca2+ + Mg2+)-ATPase activity, high Ca2+ affinity and regulation of activity by MgATP.  相似文献   

16.
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   

17.
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   

18.
The (Na++K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na++K+)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg2+: ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (Ea) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the Ea below this was very high (> 100 kcal/mol).Incresed Mg2+ concentrations at Mg2+: ATP ratios in excess of 1:1 inhibited the (Na++K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na++K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and Ea values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km for ATP.Since both of cholesterol and Mg2+ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context.  相似文献   

19.
Multiple-equilibrium equations were solved to investigate the individual and separate effects of Mg2+, Mn2+, Ca2+, ATP4–, and their complexes on the kinetics of brain adenylate cyclase. The effects of divalent metals and/or ATP4– (in excess of their participation in complex formation) were determined and, from the corresponding apparent affinity values, the following kinetic constants were obtained:K m(MgATP)=1.0 mM,K i(ATP4–)=0.27 mM,K m(MnATP)=0.07 mM, andK i(CaATP)=0.015 mM. MgATP, MnATP, ATP4–, and CaATP were shown to compete for the active site of the enzyme. Hence, it is proposed that endogenous metabolites with a strong ligand activity for divalent metals, such as citrate and some amino acids, become integrated into a metabolite feedback control of the enzyme through the release of ATP4– from MgATP. Ca2+ fluxes may participate in the endogenous regulation of adenylate cyclase by modifying the level of CaATP. The free divalent metals show an order of affinityK 0.5(Ca2+)=0.02 mM,K 0.5(Mn2+)=3.8 mM,K 0.5(Mg2+)=4.7 mM, and an order of activity Mn2+>Mg2+>Ca2+. The data indicate that Mn2+ and Mg2+ ions may compete for a regulatory site distinct from the active site and increaseV m without changingK m(MgATP),K m(MnATP), orK i(ATP4–). The interactions of ATP4– and CaATP, which act as competitive inhibitors of the reaction of the enzyme with the substrates MgATP and MnATP, and Mg2+ and Mn2+, which act as activators of the enzyme in the absence of hormones, are shown to follow the random rapid equilibrium BiBi group-transfer mechanism of Cleland with the stipulation that neither Mg2+ nor Mn2+, in excess of their respective participation in substrate formation, are obligatorily required for basal activity. ATP4– and CaATP are involved in dead-end inhibition. For MgCl2 saturation curves at constant total ATP concentration, the computer-generated curves based on the RARE BiBi model predict a change in the Hill cooperativityh from a basal value of 2.6, when Mg2+ is not obligatorily required, to 4.0 when the addition of hormones or neurotransmitters induces an obligatory requirement for Mg2+.Abbreviations used: Me, divalent metal; MeT (MgT or MnT), total Me (Me2+ and its complexes); ATPT, total ATP (ATP4– and its complexes).  相似文献   

20.
Mg2+-ATPase activity was identified in the cytosol of human erythrocytes. A partial purification of this activity was achieved by an initial DEAE-Sephadex column chromatography, followed by gel filtration on Sephadex G-100 and then a second DEAE-Sephadex chromatography procedure. The enzyme appeared in the void volume of the Sephadex G-100 column and was retained on an Amicon XM100A ultrafiltration membrane. The molecular weight of the enzyme was estimated to be 113 000 from SDS gels. The above purification protocol yielded an enzyme with an optimal pH between 7.6 and 8.2. The enzyme activity increased linearly between 30 and 44°C. It was stable for several months at ?20°C. Magnesium was essential for activity, but the rate attainable with Mn2+ was at least as great as that due to Mg2+. No other divalent cation was able to substitute for Mg2+ or Mn2+. Neither low nor high Ca2+ concentrations significantly affected the enzymatic activity. Substrate specificity studies showed that ATP was the preferred substrate followed by CTP (46% of the rate produced by ATP). Hydrolysis of GTP, UTP, ITP and ADP was less than 10% of the rate seen with ATP. No phosphatase, pyrophosphatase, phosphodiesterase, hexokinase, phosphofructokinase or adenylate cyclase activity could be detected in this enzyme preparation. Calmodulin, which stimulates the (Ca2+ + Mg2+)-ATPase of the human erythrocyte membrane, failed to enhance the Mg2+-ATPase activity. Of considerable interest, the activity of this Mg2+-ATPase was enhanced approximately 5-fold by low concentrations of mercuric ion, p-hydroxymercuribenzoate and DTNB, but was much less sensitive to iodoacetamide.  相似文献   

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