首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Electron paramagnetic resonance (epr) and ultraviolet difference spectroscopy of vanadyl conalbumin indicate a binding capacity of two vanadyl ions, VO2+, per protein molecule in the pH 8–11 range; the binding capacity drops in the pH 6–8 range with an apparent pKa′ = 6.6. Iron-saturated conalbumin does not bind vanadyl ions, which suggests common binding sites for iron and vanadium. Ultraviolet difference spectroscopy indicates 2–3 tyrosines are involved in the binding of each metal ion; pH titrations show that three protons are released per vanadyl ion bound by conalbumin. Room and liquid nitrogen temperature X-band (ca. 9.2–9.5 gHz) epr spectra show that the vanadyl ion binds in three magnetically distinct environments (A, B, and C) that arise from interconvertible metal site configurations. These configurations are probably examples of conformational substrates of the protein. Q-band (ca 34 gHz) epr spectra resolve the spectral features more clearly and show that two configurations (A and B) have axially symmetric epr parameters but angles of noncoincidence of 12° and 8°, respectively, between the z components of the g and nuclear hyperfine tensors. The third (C) configuration has rhombic magnetic symmetry and a 6° angle of noncoincidence. These observations demonstrate that the metal sites are of low symmetry and are flexible in their geometry about the metal.The isotropic g and nuclear hyperfine tensor values and the line widths used in computer-simulated epr spectra are consistent with four oxygen or three oxygen and one nitrogen donor atoms binding equatorially to the VO2+ group. The apparent stability constant indicates that vanadyl ion binds to conalbumin approximately twelve orders of magnitude more weakly than iron to human serotransferrin but still sufficiently strongly to overcome hydrolysis.  相似文献   

2.
A variety of metal ions can bind to the iron-transport protein, transferrin, at two specific sites. For each metal ion, a carboxylate anion is concomitantly bound. Six metal ions which were examined fall into two classes based on proton release and ultraviolet spectral changes which accompany binding to the protein. Class II ions, which include Cu2+ and Zn2+, release approximately 2 H+/metal bond. Class III ions, which include Fe3+, Ga3+, Al3+, and VO2+, release approximately 3 H+/metal bound. The increase in absorbance near 242 nm, characteristic of tyrosine ionization, has the ratio 0.55–0.75 for class II:class III ions. Both Fe3+ and Cu2+ form metal-transferrin-oxalate complexes in the presence of excess C2O42?. Fe3+ releases close to 3 H+/metal whether forming oxalate or bicarbonate complexes with transferrin. Binding of Cu2+ to transferrin releases 2 H+/metal in the presence of C2O2?4 or HCO3?. Since equal numbers of H+/metal are released for both anions, it is likely that the bicarbonate ion does not lose its proton, and remains as HCO3? in transferrin. These results are interpreted in terms of possible combinations of ligands at the metal binding sites.  相似文献   

3.
The requirement for metal ions by glutamine synthetase of Escherichia coli in catalyzing the γ-glutamyl transfer reaction has been investigated. In order of decreasing V at pH 7.0, Cd2+, Mn2+, Mg2+, Ca2+, Co2+, or Zn2+ will support the activity of the unadenylylated enzyme in the presence of ADP. With AMP substituted for ADP to satisfy the nucleotide requirement, only Mn2+ or Cd2+ will support the activity of the unadenylylated enzyme. Kinetic and equilibrium binding measurements show a 1:1 interaction between the nonconsumable substrate ADP and each enzyme subunit of the dodecamer. (To obtain this result, each enzyme subunit must be active in catalyzing γ-glutamyl transfer.) The stability constant of the unadenylylated subunit for ADP-Mn is 3.5 × 105m?1, or ~2.86 × 107m?1 under assay conditions, with arsenate, Mn2+, and glutamine being responsible for this large affinity increase. Saturation of two Mn2+ ion-binding sites per enzyme subunit is absolutely required for activity expression. While apparently not affecting the affinity of the first Mn2+ bound (K′ = 1.89 × 106 M?1), glutamine increases the stability constant for the second Mn2+ bound from 2 × 104 to 5.9 × 105m?1. Reciprocally, increasing Mn2+ concentrations decreases the apparent Km′ value for glutamine. Glutamine (by producing a net uptake of protons in binding to the enzyme) is responsible for changing the proton release from 3 to about 1 for 2 Mn2+ bound per enzyme subunit, with ~0.5 H+ displaced in both fast and slow processes. The uv spectral change induced by the binding of the first Mn2+ to each enzyme subunit remains unchanged by the presence of glutamine. However, glutamine reduces the half-time of the spectral change or slow proton release from ~30 to ~20 sec at 37 °C. Binding and kinetic results indicate a mechanism involving a random addition of Mn2+ to two subunit sites. Saturation of the high-affinity site with Mn2+ induces a conformational change to an active configuration, while activity expression depends also on the saturation of a second Mn2+ binding site (at or near the catalytic site). Once the first Mn2+ binding site of the subunit is saturated, an active enzyme complex can be formed either by the sequential binding of Mn2+ and ADP at the second site or by the binding of ADP-Mn complex directly to this site if the concentration of ADP-Mn is greater than 10?8m in the assay. Some additional observations on the binding of Mg2+, Ba2+, Ca2+, and Zn2+ to the enzyme are presented.  相似文献   

4.
We found that anion channel blockers such as phosphotungstate and 4,4′-diisothiocyanatostilbene-2-2′-disulfonate (DIDS)_enhanced HCO3?-induced activation on porcine epididymal sperm. In the presence of these compounds, HCO3? increased the motility, respiration rate and especially the cAMP content of the sperm to a greater extent than did HCO3? alone. The enhancing effects were not observed in the absence of HCO3?, but were evident when the concentration of HCO3? was low. These compounds did not significantly alter the intracellular pH and did inhibit the adenylate cyclase activity of the sperm plasma membrane. When these compounds were added to sperm homogenate with ATP, the cAMP formed was reduced compared to the control. In addition, these compounds inhibited both the SO42? influx and efflux of the sperm. From these results, we conclude that the anion channel blockers tested principally inhibit the efflux of endogenous HCO3? derived from metabolic CO2, so that HCO3? accumulates intracellularly and stimulates the adenylate cyclase of the sperm.  相似文献   

5.
The binding of VIVO2+ to human serum transferrin (hTF) at the FeIII binding sites is addressed. Geometry optimization calculations were performed for the binding of VIVO2+ to the N-terminal lobe of hTF (hTFN), and indicate that in the presence of CO3 2? or HCO3 ?, VIV is bound to five atoms in a distorted geometry. The structures of VIVO–hTFN species optimized at the semiempirical level were also used to calculate the 51V and 14N A tensors by density functional theory methods, and were compared with the reported experimental values. Globally, of all the calculated VIVO–hTF structures, the one that yields the lowest calculated heats of formation and minimum deviations from the experimental values of the 51V and 14N A tensor components is the structure that includes CO3 2? as a synergistic anion. In this structure the V=O bond length is approximately 1.6 Å, and the vanadium atom is also coordinated to the phenolate oxygen atom of Tyr188 (at approximately 1.9 Å), the aspartate oxygen atom of Asp63 (at approximately 1.9 Å), the His249 Nτ atom (at approximately 2.1 Å), and a carbonate oxygen atom (at approximately 1.8 Å). The Tyr95 phenolic ocygen atom is approximately 3.3 Å from the metal center, and thus is very weakly bound to VIV. All of these oxygen atoms are able to establish dipolar interactions with groups of the protein.  相似文献   

6.
In this study, we presented a new approach for quantification of bicarbonate (HCO3?) molecules bound to PSII. Our method, which is based on a combination of membrane-inlet mass spectrometry (MIMS) and 18O-labelling, excludes the possibility of “non-accounted” HCO3? by avoiding (1) the employment of formate for removal of HCO3? from PSII, and (2) the extremely low concentrations of HCO3?/CO2 during online MIMS measurements. By equilibration of PSII sample to ambient CO2 concentration of dissolved CO2/HCO3?, the method ensures that all physiological binding sites are saturated before analysis. With this approach, we determined that in spinach PSII membrane fragments 1.1 ± 0.1 HCO3? are bound per PSII reaction center, while none was bound to isolated PsbO protein. Our present results confirmed that PSII binds one HCO3? molecule as ligand to the non-heme iron of PSII, while unbound HCO3? optimizes the water-splitting reactions by acting as a mobile proton shuttle.  相似文献   

7.
A novel fluorescent probe‐based naphthalene Schiff, 1‐(C2‐glucosyl‐ylimino‐methyl)‐naphthalene‐2‐ol (L) was synthesized by coupling d ‐glucosamine hydrochloride with 2‐hydroxy‐1‐naphthaldehyde. It exhibited excellent selectivity and highly sensitivity for Al3+ in ethanol with a strong fluorescence response, while other common metal ions such as Pb2+, Mg2+, Cu2+, Co2+, Ni2+, Cd2+, Fe2+, Mn2+, Hg2+, Li+, Na+, K+, Fe3+, Cr3+, Zn2+, Ag+, Ba2+ and Ca2+ did not cause the same fluorescence response. The probe selectively bound Al3+ with a binding constant (Ka) of 5.748 × 103 M?1 and a lowest detection limit (LOD) of 4.08 nM. Moreover, the study found that the fluorescence of the L ? Al3+ complex could be quenched after addition of F? in the same medium, while other anions, including Cl?, Br?, I?, NO2?, NO3?, ClO4?, CO32?, HCO3?, SO42?, HSO4?, CH3COO?, PO43?, HPO42?, S2? and S2O32? had nearly no influence on probe behaviour. Binding of the [L ? Al3+] complex to a F? anion was established by different fluorescence titration studies, with a detection limit of 3.2 nM in ethanol. The fluorescent probe was also successfully applied in the imaging detection of Al3+ and F? in living cells.  相似文献   

8.
It has been known for some time that bicarbonate reverses the inhibition, by formate under HCO3 --depletion conditions, of electron transport in thylakoid membranes. It has been shown that the major effect is on the electron acceptor side of photosystem II, at the site of plastoquinone reduction. After presenting a historical introduction, and a minireview of the bicarbonate effect, we present a hypothesis on how HCO3 - functions in vivo as (a) a proton donor to the plastoquinone reductase site in the D1-D2 protein; and (b) a ligand to Fe2+ in the QA-Fe-QB complex that keeps the D1-D2 proteins in their proper functional conformation. They key points of the hypothesis are: (1) HCO3 - forms a salt bridge between Fe2+ and the D2 protein. The carboxyl group of HCO3 - is a bidentate ligand to Fe2+, while the hydroxyl group H-bonds to a protein residue. (2) A second HCO3 - is involved in protonating a histidine near the QB site to stabilize the negative charge on QB. HCO3 - provides a rapidly available source of H+ for this purpose. (3) After donation of a H+, CO3 2- is replaced by another HCO3 -. The high pKa of CO3 2- ensures rapid reprotonation from the bulk phase. (4) An intramembrane pool of HCO3 - is in equilibrium with a large number of low affinity sites. This pool is a H+ buffering domain functionally connecting the external bulk phase with the quinones. The low affinity sites buffer the intrathylakoid [HCO3 -] against fluctuations in the intracellular CO2. (5) Low pH and high ionic strength are suggested to disrupt the HCO3 - salt bridge between Fe2+ and D2. The resulting conformational change exposes the intramembrane HCO3 - pool and low affinity sites to the bulk phase.Two contrasting hypotheses for the action of formate are: (a) it functions to remove bicarbonate, and the low electron transport left in such samples is due to the left-over (or endogenous) bicarbonate in the system; or (b) bicarbonate is less of an inhibitor and so appears to relieve the inhibition by formate. Hypothesis (a) implies that HCO3 - is an essential requirement for electron transport through the plastoquinones (bound plastoquinones QA and QB and the plastoquinone pool) of photosystem II. Hypothesis (b) implies that HCO3 - does not play any significant role in vivo. Our conclusion is that hypothesis (a) is correct and HCO3 - is an essential requirement for electron transport on the electron acceptor side of PS II. This is based on several observations: (i) since HCO3 -, not CO2, is the active species involved (Blubaugh and Govindjee 1986), the calculated concentration of this species (220 M at pH 8, pH of the stroma) is much higher than the calculated dissociation constant (Kd) of 35–60 M; thus, the likelihood of bound HCO3 - in ambient air is high; (ii) studies on HCO3 - effect in thylakoid samples with different chlorophyll concentrations suggest that the left-over (or endogenous) electron flow in bicarbonate-depleted chloroplasts is due to left-over (or endogenous) HCO3 - remaining bound to the system (Blubaugh 1987).Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (common name: diuron) - PSII photosystem II - QA first plastoquinone electron acceptor of PSII - QB second plastoquinone acceptor of PS II  相似文献   

9.
The complexation of VO2+ ion with the high molecular mass components of the blood serum, human serum transferrin (hTf) and albumin (HSA), has been re-examined using EPR spectroscopy. In the case of transferrin, the results confirm those previously obtained, showing that VO2+ ion occupies three different binding sites, A, B1 and B2, distinguishable in the X-band anisotropic spectrum recorded in D2O. With albumin the results show that a dinuclear complex (VO)2dHSA is formed in equimolar aqueous solutions or with an excess of protein; in the presence of an excess of VO2+, the multinuclear complex (VO)xmHSA is the prevalent species, where x = 5-6 indicates the equivalents of metal ion coordinated by HSA. The structure of the dinuclear species is discussed and the donor atoms involved in the metal coordination are proposed on the basis of the measured EPR parameters. Two different binding modes of albumin can be distinguished varying the pH, with only one species being present at the physiological value. The results show that the previously named “strong” site is not the N-terminal copper binding site, and some hypothesis on the metal coordination is discussed, with the 51V Az values for the proposed donor sets obtained by DFT (density functional theory) calculations. Finally, preliminary results obtained in the ternary system VO2+/hTf/HSA are shown in order to determine the different binding strength of the two proteins. Due to the low VO2+ concentration used, the recording of the EPR spectra through the repeated acquisition of the weak signals is essential to obtain a good signal to noise ratio in these systems.  相似文献   

10.
The thermodynamic parameters, ΔH′, ΔG′, and ΔS′, and the stoichiometry for the binding of the substrate 2′-deoxyuridine-5′-phosphate (dUMP) and the inhibitor 5-fluoro-2′-deoxyuridine-5′-phosphate (FdUMP) to Lactobacillus casei thymidylate synthetase (TSase) have been investigated using both direct calorimetric methods and gel filtration methods. The data obtained show that two ligand binding sites are available but that the binding of the second mole of dUMP is extremely weak. Binding of the first mole of dUMP can best be illustrated by dUMP + TSase + H+?(dUMP-TSase-H+). [1] The enthalpy, ΔH1′, for reaction [1] was measured directly on a flow modification of a Beckman Model 190B microcalorimeter. Experiments in two different buffers (I = 0.10 m) show that ΔH1′ = ?28 kJ mol?1 and that 0.87 mol of protons enters into the reaction. Analysis of thermal titrations for reaction [1] indicates a free energy change of ΔG1′ = ?30 kJ mol?1 (K1 = 1.7 × 105 m?1). From these parameters, ΔS1′ was calculated to be +5 J mol?1 degree?1, showing that the reaction is almost totally driven by enthalpy changes. Gel filtration experiments show that at very high substrate concentrations, binding to a second site can be observed. Gel filtration experiments performed at low ionic strength (I = 0.05 m) reveal a stronger binding, with ΔG1′ = ?35 kJ mol?1 (K1 = 1.2 × 106 m?1), suggesting that the forces driving the interaction are, in part, electrostatic. Addition of 2-mercaptoethanol (0.10 m) had the effect of slightly increasing the dUMP binding constant. Binding of FdUMP to TSase is best illustrated by 2FdUMP + TSase + nHH+?FdUMP2 ? TSase ? (H+)nH. [2] The enthalpy for this reaction, ΔH2, was also measured calorimetrically and found to be ?30 kJ mol?1 with nH = 1.24 at pH 7.4 Assuming two FdUMP binding sites per dimer as established by Galivan et al. [Biochemistry15, 356–362 (1976)] our calorimetric results indicate different binding energies for each site. Based on the binding data, a thermodynamic model is presented which serves to rationalize much of the confusing physical and chemical data characterizing thymidylate synthetase.  相似文献   

11.
Properties of partially purified NADP-malic enzyme (EC 1.1.1.40) from glumes of developing wheat grains were examined. The pH optimum for enzyme activity was influenced by malate and shifted from 7.3 to 7.6 when the concentration of malate was increased from 2 to 10 mM. The Km values, at pH 7.3, for various substrates were: malate, 0.76 mM; NADP, 20 μM and Mn2+, 0.06 mM. The requirement of Mn2+ cation for enzyme activity could be partially replaced by Mg2+ or Co2+. Mn2+ dependent enzyme activity was inhibited by Pb2+, Ni2+, Hg2+, Zn2+, Cd2+, Al3+ and Fe3+. During the reaction, substrate molecules (malate and NADP) reacted with enzyme sequentially. Activity of malic enzyme was inhibited by products of the reaction viz pyruvate, HCO3? and NADPH2. At a limiting fixed concentration of NADP, these products induced a positive cooperative response to increasing concentrations of malate.  相似文献   

12.
Unadenylylated glutamine synthetase (EC 6.3.1.2) was isolated and purified to homogeneity from Salmonella typhimurium. The enzyme molecule is a symmetrical aggregate of 12 subunits arranged in two hexagonal layers, as is evident from electron micrographs. The subunit molecular weight of the enzyme was found to be approximately 50,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate when compared to Escherichia coli glutamine synthetase and other protein standards. A long tube of glutamine synthetase was formed as a single-stranded coil resulting from incubation of the enzyme in a low ionic strength buffer. A study of Mn(II) binding to the unadenylylated enzyme at 25 °C was conducted as a function of pH. At pH 7.1 two classes of metal ion sites per subunit were found with KD values of 3.7 × 10?6 and 1.7 × 10?4m, while at pH 6.8 these values were 1.1 × 10?5 and 1.0 × 10?4m, respectively. Only one set of binding sites was observed at pH 6.2 with a KD value of 1.0 × 10?4m. The metal ion binding sites were further investigated by monitoring proton relaxation rates (prr) and the epr spectrum of enzyme-bound Mn(II). The longitudinal prr of water protons at pH 7.1 indicate that protons interacting with enzyme-Mn(II) at the “tight” site (KD = 3.7 × 10?6) are de-enhanced (?b1 = 0.42) and result from water protons beyond the inner coordination sphere. The second Mn(II) site has a value of ?b2 = 35 for the binary enhancement, suggesting that this site probably has two to three rapidly exchanging water molecules in its coordination sphere. The epr spectrum of enzyme-bound Mn(II) at the “tight” site is isotropic and is dramatically sharpened by adding the substrate analog methionine sulfoximine. Subsequent addition of ATP or the ATP analog, AMP-PCP (adenylyl methylene diphosphate) produced anisotropic spectra that were similar, suggesting that both ATP and AMP-PCP bind similarly on the enzyme surface. However, a marked change in the Mn(II) environment from anisotropic to near cubic results from the addition of ADP to the quaternary enzyme-Mn(II)-sulfoximine- (AMP-PCP) complex, indicating that ADP displaces AMP-PCP. No change in the anisotropic spectrum due to the enzyme-Mn(II)-sulfoximine-ATP complex is seen by the addition of ADP. This experimental result supports the experimental findings of Ronzio and Meister [Proc. Nat. Acad. Sci. USA59, 164 (1968)], who established that ATP phosphorylates methionine sulfoximine, thereby producing an inactive enzyme. The allosteric effectors, AMP and Trp, have little effect on the epr spectrum of the complex formed from Mn(II), enzyme, sulfoximine, and ADP, suggesting the absence of direct coordination of AMP or Trp to the bound Mn(II). The prr and epr results reported herein with glutamine synthetase from S. typhimurium when compared to those seen for the enzyme from E. coli [Villafranca et al., Biochemistry15, 544 (1976)] demonstrate some similarities but also many substantial differences between the enzymes from these two bacterial sources.  相似文献   

13.
Bicarbonate depletion of chloroplast thylakoids reduces the affinity of the herbicide, ioxynil, to its binding site in Photosystem (PS) II. This herbicide is found to be a relatively more efficient inhibitor of the Hill reaction when HCO?3 is added to CO2-depleted thylakoids in subsaturating rather than in saturating concentrations. The reason for this dependence of the inhibitor efficiency on the HCO?3 concentration is that the inactive HCO?3-deficient PS II reaction chains bind less ioxynil than the active PS II electron-transport chains that have bound HCO?3, and, thus, after addition of a certain amount of ioxynil the concentration of the free herbicide increases when the HCO?3 concentration decreases. Therefore, the inhibition of electron transport by ioxynil increases at decreasing HCO?3 levels. Measurements on the effects of modification of lysine and arginine residues on the rate of electron transport are also presented: the rate of modification is faster in the presence than in the absence of HCO?3. Therefore, we suggest that surface-exposed lysine or arginine residues are not involved in binding of HCO?3 (or CO2 or CO2?3) to its binding protein, but that HCO?3 influences the conformation of its binding environment such that the affinity for certain herbicides and the accessibility for amino acid modifiers are changed.  相似文献   

14.
We studied anionic inhibition of the reaction CO2 + OH?? HCO3? catalyzed by human red cell carbonic anhydrase B (I) and C (II), using iodide and cyanate. In the forward reaction with respect to CO2 as the substrate, inhibition was mixed but favoring noncompetitive; the back reaction, with HCO3? as the substrate, yielded strict competitive kinetics. Mean inhibition constants, KI, in the pH range 7.2–7.5 are: iodide, 0.5 mm for enzyme B and 16 mm for C; cyanate, 0.8 μm for B and 20 μm for C. When OH? was considered as the substrate for the forward reaction, cyanate and chloride behaved as competitive inhibitors. The true inhibition constant (KI0) for cyanate (calculated for infinitely low OH?) is 0.4 μm for enzyme B and 4 μm for C. Apart from the difference in anion affinity and some 10-fold higher activity of C > B, the isozymes showed similar patterns of inhibition. Data agree with generally proposed mechanisms describing the active site as ZnH2O with pKa of about 7.  相似文献   

15.
The green marine macroalga Ulva lactuca L. was found to be able to utilize HCO3? from sea water in two ways. When grown in flowing natural sea water at 16°C under constant dim irradiance, photosynthesis at pH8.4 was suppressed by acetazolamide but unaffected by 4,4′-diisothiocyanostilbene-2,2′-disulphonate. These responses indicate that photosynthetic HCO3? utilization was via extracellular carbonic anhydrase (CA) -mediated dehydration followed by CO2 uptake. The algae were therefore described as being in a ‘CA state’. If treated for more than 10 h in a sea water flow-through system at pH9.8, these thalli became insensitive to acetazolamide but sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulphonate. This suggests the involvement of an anion exchanger (AE) in the direct uptake of HCO3?, and these plants were accordingly described as being in an ‘AE state’. Such thalli showed an approximately 10-fold higher apparent affinity for HCO3? (at pH9.4) than those in the ‘CA state’, while thalli of both states showed a very high apparent affinity for CO2. These results suggest that the two modes of HCO3? utilization constitute two ways in which inorganic carbon may enter the Ulva lactuca cells, with the direct entry of HCO3?, characterizing the ‘AE state’, being inducible and possibly functioning as a complementary uptake system at high external pH values (e.g. under conditions conducive to high photosynthetic rates). Both mechanisms of entry appear to be connected to concentrating CO2 inside the cell, probably via a separate mechanism operating intracellularly.  相似文献   

16.
Action of Cl? + HCO3 ?1 ions on Mg2+-ATPase from brain plasma membranes of fish and rats has been studied. Maximal effect of the anions on the “basal” Mg2+-ATPase activity is revealed in the presence of 10 mM Cl? and 3 mM HCO3 ?1 at physiological values of pH of incubation medium. The studied Cl?, HCO3 ?-activated Mg2+-ATPases of both animal species, by their sensitivity to SH-reagents (5,5-dithio-bis-nitrobenzoic acid, N-ethylmaleimide), oligomycin, and orthovanadate, are similar to transport ATPase of the P-type, but differ from them by molecular properties and by sensitivity to ligands of GABAA-receptors. It has been established that the sensitive to GABAA-ergic ligands, Cl?, HCO3 ?-activated Mg2+-ATPase from brain of the both animal species is protein of molecular mass around 300 kDa and of Stock’s radius 5.4 nm. In fish the enzyme is composed of one major unit of molecular mass approximately 56 kDa, while in rats-of three subunits of molecular masses about 57, 53, and 45 kDa. A functional and structural coupling of the ATP-hydrolyzing areas of the studied enzyme to sites of binding of GABAA-receptor ligands is suggested.  相似文献   

17.
Dehydrocyclopeptine epoxidase (DE) activity was determined in cell free preparations of Penicillium cyclopium. The enzyme transforms dehydrocyclopeptine into cyclopenin by a mixed function oxygenation. It needs molecular oxygen and uses NAD(P)H, ascorbate or d,l-6-methyl-5,6,7,8-tetrahydropteridine as cosubstrates. DE is inhibited by CN?, SCN?, 1,10-phenanthroline, EDTA, 2,2′-bipyridine, sodium diethyldithiocarbamate, dicoumarol, p-chloromercuribenzoate and ions of different heavy metals, but not by CO and the lead salt of diethyldithiocarbamate. These properties indicate a specific importance of Fe2+-ions, SH-groups and flavins. DE activity is increased by Fe2+ and FAD. The enzyme may be therefore a Fe2+ activated FAD containing flavoprotein. DE was enriched 268-fold by (NH4)2SO4 precipitation and chromatography on Sephadex G-200. Its MW estimated by Sephadex chromatography, exceeds 480 000.  相似文献   

18.
The binding of Mn2+ to the anthranilate synthetase-phosphoribosyltransferase enzyme complex from Salmonella typhimurium was examined by electron paramagnetic resonance studies. Two types of binding sites were observed: one to two tight sites with a dissociation constant of 3–5 μm and five to six weaker sites with a dissociation constant of 40–70 μm. The activator constant for Mn2+ was found to be 9 μm for the glutamine-linked anthranilate synthetase activity and 4 μm for the phosphoribosyltransferase activity. These values are both in the range of the dissociation constant for the tight sites. Water proton relaxation rate measurements showed that the binary enhancement values for both classes of sites were equivalent, ?b = 10.7 ± 2.0. The addition of chorismate to the Mn2+-enzyme complexes when predominantly the tight Mn2+ sites were occupied resulted in a large decrease in the observed enhancement (?T = 2.0). Addition of 5-phosphoribosyl-1-pyrophosphate to the enzyme-Mn2+ complexes caused large decreases in the water proton relaxation rate (?T = 1.5) when tight or tight plus weaker Mn2+ sites were occupied. No changes in the water proton relaxation rate were observed when glutamine, pyruvate, or anthranilate were added; a small decrease was observed when enzyme-Mn2+ was titrated with tryptophan. Tryptophan significantly altered the effect of the binding of chorismate but not of 5-phosphoribosyl-1-pyrophosphate. The effect of tryptophan on the water proton relaxation rate of a Mn2+-enzyme-chorismate complex using a variant enzyme complex which is tryptophan hypersensitive (P. D. Robison, and H. R. Levy, 1976, Biochim. Biophys. Acta. 445, 475–485) occurred at lower concentrations than for the normal enzyme complex. The uncomplexed anthranilate synthetase subunit was titrated with Mn2+ and found to have one to two binding sites with a dissociation constant of 300 ± 100 μm. This dissociation constant is much larger than the activator constant for Mn2+ for uncomplexed anthranilate synthetase which was determined to be 4 μm. These results indicate that the Mn2+-binding sites on anthranilate synthetase are altered when the enzyme complex is formed and that both chorismate and 5-phosphoribosyl-1-pyrophosphate interact closely with enzyme-bound Mn2+ or cause a large effect upon its environment.  相似文献   

19.
Activation by different anions of γ-glutamyltransferase obtained in a. particulate form from fruiting bodies of Lentinus edodes has been studied using either L-γ-glutamyl-p-nitroanlide or lentinic acid as substrate. The mushroom transferase was activated by SCN?, NO3?, Cl?, Br?, ClO3?, Bro3?, N3?, I? and F?, but not those alkali and earth cations previously believed to activate the animal transferase, nor by citrate, claimed to be effective for the kidney bean transferase. Among anions proved hardly to activate the transferase were ClO4?, NO2?, HCO3?, H2PO4?, SO32? and SO42?. A high concentration of these anions more or less impeded the halide activation. Kinetic studies revealed that halides function as activators of increasing Vmax while keeping Km constant. These observations appeared least compatible with the possibility that the anion activation might involve a non-specific effect of high solute concentration, viz. dissociation of the enzyme from the supporting structure in the particulates. The activating effect of halides described here probably extends also to the animal enzymes.  相似文献   

20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号