共查询到20条相似文献,搜索用时 31 毫秒
1.
Regulation of Ion Fluxes,Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells
Gap junctional communication between granulosa cells seems to play a crucial role for follicular growth and atresia. Application of the double whole-cell patch-clamp- and ratiometric fura-2-techniques allowed a simultaneous measurement of gap junctional conductance (G
j) and cytoplasmic concentration of free Ca2+ ([Ca2+]i) in a rat granulosa cell line GFSHR-17. The voltage-dependent gating of G
j varied for different cell pairs. One population exhibited a bell-shape dependence of G
j on transjunctional voltage, which was strikingly similar to that of Cx43/Cx43 homotypic gap junction channels expressed in pairs of oocytes of Xenopus
laevis. Within 15–20 min, gap junctional uncoupling occurred spontaneously, which was preceded by a sustained increase of [Ca2+]i and accompanied by shrinkage of cellular volume. These responses to the whole-cell configuration were avoided by absence of extracellular Ca2+, blockage of K+ efflux, or addition of 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) to the pipette solution. Even in the absence of extracellular Ca2+ or blockage of K+ efflux, formation of whole-cell configuration generated a Ca2+ spike that could be suppressed by the presence of 8-Br-cGMP. We propose that intracellular cGMP regulates Ca2+ release from intracellular Ca2+ stores, which activates sustained Ca2+ influx, K+ efflux and cellular shrinkage. We discuss whether gap junctional conductance is directly affected by cGMP or by cellular shrinkage and whether gap junctional coupling and/or cell shrinkage is involved in the regulation of apoptotic/necrotic processes in granulosa cells. 相似文献
2.
Many organic anions bind free Ca2+, the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (Kapp) describing the Ca2+ affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca2+ electrodes coupled to either a 3 M KCl or a Na+ selective electrode were used to redetermine Kapp. All solutions contained 130 mM Na+, whereas the concentration of the studied anion was varied from 15 to 120 mM, replacing Cl− that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3 M KCl reference electrode, leading to a systematic underestimation of Kapp if left uncorrected. Because the Na+ concentration in all solutions was constant, a Na+ electrode was used to directly measure the changes in the LJP at the 3 M KCl reference, which were under 5 mV but twice those predicted by the Henderson equation. Determination of Kapp either after correction for these LJP changes or via direct reference to a Na+ electrode showed that SCFAs do not bind Ca2+ and that the Kapp for the binding of Ca2+ to gluconate at pH 7.4, ionic strength 0.15 M, and 23 °C was 52.7 mM. 相似文献
3.
Ischemia is known to inhibit gap junction (GJ) mediated intercellular communication. However the detail mechanisms of this inhibition are largely unknown. In the present study, we determined the vulnerability of different cardiac GJ channels formed of connexins (Cxs) 43, 40, and 45 to simulated ischemia, by creating oxygen glucose deprived (OGD) condition. 5 minutes of OGD decreased the junctional conductance (Gj) of Cx43, Cx40 and Cx45 by 53±3%, 64±1% and 85±2% respectively. Reduction of Gj was prevented completely by restricting the change of both intracellular calcium ([Ca2+]i) and pH (pHi) with potassium phosphate buffer. Clamping of either [Ca2+]i or pHi, through BAPTA (2 mM) or HEPES (80 mM) respectively, offered partial resistance to ischemic uncoupling. Anti-calmodulin antibody attenuated the uncoupling of Cx43 and Cx45 significantly but not of Cx40. Furthermore, OGD could reduce only 26±2% of Gj in C-terminus (CT) truncated Cx43 (Cx43-Δ257). Tethering CT of Cx43 to the CT-truncated Cx40 (Cx40-Δ249), and Cx45 (Cx45-Δ272) helped to resist OGD mediated uncoupling. Moreover, CT domain played a significant role in determining the junction current density and plaque diameter. Our results suggest; OGD mediated uncoupling of GJ channels is primarily due to elevated [Ca2+]i and acidic pHi, though the latter contributes more. Among Cx43, Cx40 and Cx45, Cx43 is the most resistant to OGD while Cx45 is the most sensitive one. CT of Cx43 has major necessary elements for OGD induced uncoupling and it can complement CT of Cx40 and Cx45. 相似文献
4.
Daniela Uhríková Mária Hanulová Raylja S. Khusainova Pavol Balgavý 《生物化学与生物物理学报:生物膜》2005,1713(1):15-28
The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca2+ and Mg2+ cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA = 1:1 mol/base and in the range of concentration of the cation2+ 0-76.5 mM, the diffractograms show the coexistence of two lamellar phases: Lx phase with repeat distance dLx ∼ 8.26-7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and LDOPC phase with repeat distance dDOPC ∼ 6.45-5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated LDOPC phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC + DNA + Ca2+ aggregates was investigated in the range 20-80 °C. The transversal thermal expansivities of both phases were evaluated. 相似文献
5.
Amino-terminus and carboxyl-terminus of connexins have been proposed to be responsible for the transjunctional voltage-dependent gating (Vj-gating) and the unitary gap junction channel conductance (γj). To better understand the molecular structure(s) determining the Vj-gating properties and the γj of Cx50, we have replaced part of the amino-terminus of mCx50 by the corresponding domain of mCx36 to engineer a chimera Cx50-Cx36N, and attached GFP at the carboxyl-terminus of mCx50 to construct Cx50-GFP. The dual whole-cell patch-clamp technique was used to test the resulting gap junction channel properties in N2A cells. The Cx50-Cx36N gap junction channel lowered the sensitivity of steady-state junctional conductance to Vj (Gj/Vj relationship), slowed Vj-gating kinetics, and reduced γj as compared to Cx50 channel. Cx50-GFP gap junction channel showed similar Vj-gating properties and γj to Cx50 channel. We further characterized a mutation, Cx50N9R, where the Asn (N) at the ninth position of Cx50 was replaced by the corresponding Arg (R) at Cx36. The Gj/Vj relationship of Cx50N9R channel was significantly changed; most strikingly, the macroscopic residual conductance (Gmin) was near zero. Moreover, the single Cx50N9R channel only displayed one open state (γj = 132 ± 4 pS), and no substate could be detected. Our data suggest that the NT of Cx50 is critical for both the Vj-gating and the γj, and the introduction of a positively charged Arg at the ninth position reduced the Gmin with a correlated disappearance of the substate at the single channel level. 相似文献
6.
The relationship between single-channel and whole-cell conductance in the T-type Ca2+ channel CaV3.1
In T-type Ca2+ channels, macroscopic IBa is usually smaller than ICa, but at high Ca2+ and Ba2+, single-channel conductance (γ) is equal. We investigated γ as a function of divalent concentration and compared it to macroscopic currents using CaV3.1 channels studied under similar experimental conditions (TEAo and Ki). Single-channel current-voltage relationships were nonlinear in a way similar to macroscopic open-channel I/Vs, so divalent γ was underestimated at depolarized voltages. To estimate divalent γ, concentration dependence, iDiv, was measured at voltages <−50 mV. Data were well described by Langmuir isotherms with γmax(Ca2+) of 9.5 ± 0.4 pS and γmax(Ba2+) of 10.3 ± 0.5 pS. Apparent KM was lower for Ca2+ (2.3 ± 0.7 mM) than for Ba2+ (7.9 ± 1.3 mM). A subconductance state with an amplitude 70% that of the main state was observed, the relative occupancy of which increased with increasing Ca2+. As predicted by γ, macroscopic GmaxCa was larger than GmaxBa at 5 mM (GmaxCa2+/Ba:2+1.43 ± 0.14) and similar at 60 mM (GmaxCa2+/Ba:2+1.10 ± 0.02). However, over the range of activation, ICa was larger than IBa under both conditions. This was a consequence of the fact that Vrev was more negative for IBa than for ICa, so that the driving force determining IBa was smaller than that determining ICa over the range of potentials in standard current-voltage relationships. 相似文献
7.
The Mg2+ ion-assisted activation mechanism of the active site Tyr8 of a human hematopoietic prostaglandin D2 synthase (H-PGDS) was studied by ultraviolet resonance Raman (UVRR) spectroscopy. Addition of Mg2+ to the native H-PGDS at pH 8.0 resulted in the Y8a Raman band of Tyr8 shifting from 1615 cm−1 to 1600 cm−1. This large shift to lower energy of the tyrosine Y8a vibrational mode is caused by the deprotonation of the tyrosine phenol group promoted by binding of Mg2+. Upon subsequent addition of glutathione (GSH), the Mg2+/H-PGDS solution showed the Tyr8 Raman band shifted to 1611 cm−1, which is 11 cm−1 higher than the frequency of the Mg2+ complex of H-PGDS, but 4 cm−1 lower than the Mg2+ free enzyme. These UVRR observations suggest that the deprotonated Tyr8 in the presence of Mg2+ is re-protonated by the abstraction of H+ from the thiol group of GSH, and that the re-protonated Tyr8 species forms a hydrogen bond with the thiolate anion of GSH. Density functional theory calculations on several model complexes of p-cresol were also performed, which suggested that the pKa and vibrational frequencies of the Tyr8 phenol group are affected by the degree and structure of hydration of the Tyr8 residue. 相似文献
8.
Rafael Mejía-Alvarez Claudia Kettlun Eduardo Ríos Michael Stern Michael Fill 《The Journal of general physiology》1999,113(2):177-186
Single canine cardiac ryanodine receptor channels were incorporated into planar lipid bilayers. Single-channel currents were sampled at 1–5 kHz and filtered at 0.2–1.0 kHz. Channel incorporations were obtained in symmetrical solutions (20 mM HEPES-Tris, pH 7.4, and pCa 5). Unitary Ca2+ currents were monitored when 2–30 mM Ca2+ was added to the lumenal side of the channel. The relationship between the amplitude of unitary Ca2+ current (at 0 mV holding potential) and lumenal [Ca2+] was hyperbolic and saturated at ∼4 pA. This relationship was then defined in the presence of different symmetrical CsCH3SO3 concentrations (5, 50, and 150 mM). Under these conditions, unitary current amplitude was 1.2 ± 0.1, 0.65 ± 0.1, and 0.35 ± 0.1 pA in 2 mM lumenal Ca2+; and 3.3 ± 0.4, 2.4 ± 0.2, and 1.63 ± 0.2 pA in 10 mM lumenal Ca2+ (n > 6). Unitary Ca2+ current was also defined in the presence of symmetrical [Mg2+] (1 mM) and low [Cs+] (5 mM). Under these conditions, unitary Ca2+ current in 2 and 10 mM lumenal Ca2+ was 0.66 ± 0.1 and 1.52 ± 0.06 pA, respectively. In the presence of higher symmetrical [Cs+] (50 mM), Mg2+ (1 mM), and lumenal [Ca2+] (10 mM), unitary Ca2+ current exhibited an amplitude of 0.9 ± 0.2 pA (n = 3). This result indicates that the actions of Cs+ and Mg2+ on unitary Ca2+ current were additive. These data demonstrate that physiological levels of monovalent cation and Mg2+ effectively compete with Ca2+ as charge carrier in cardiac ryanodine receptor channels. If lumenal free Ca2+ is 2 mM, then our results indicate that unitary Ca2+ current under physiological conditions should be <0.6 pA. 相似文献
9.
Christine PeineltHans-Jürgen Apell 《Biophysical journal》2002,82(1):170-181
Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca2+ and H+ on binding of the other ion in the E1 and P-E2 states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg2+ a strictly competitive binding sequence, H2E1?HE1?E1?CaE1?Ca2E1, was found with two Ca2+ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K1/2(2 Ca) = 34 nM, K1/2(H) = 1 nM and K1/2(H2) = 1.32 μM. Up to 2 Mg2+ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K1/2(Mg) = 165 μM, K1/2(Mg2) = 7.4 mM). In the P-E2 state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca2+ and H+ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E2CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca2+ (0.4 mM and 25 mM) and H+ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E1 and P-E2 conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position. 相似文献
10.
Shigehiko Miyamoto 《生物化学与生物物理学报:生物膜》2007,1768(7):1848-1854
The LolCDE complex of Escherichia coli releases outer membrane-specific lipoproteins from the inner membrane. Lipoproteins with Asp at + 2 remain in the inner membrane since this residue functions as a LolCDE avoidance signal depending on phosphatidylethanolamine. We examined the effects of other phospholipids on lipoprotein sorting in proteoliposomes reconstituted with LolCDE and various synthetic phospholipids. The lipoprotein release and ATP hydrolysis were both low at 2 mM Mg2+ but very high at 10 mM Mg2+ in proteoliposomes containing cardiolipin alone. However, the Lol avoidance function was abolished at 10 mM Mg2+, and the release of lipoproteins with Asp at + 2 was as efficient as that of outer membrane-specific lipoproteins. The addition of phosphatidylethanolamine to cardiolipin stimulated the ATP hydrolysis and increased the Lol avoidance function of Asp at + 2 at 2 mM Mg2+. The addition of phosphatidylglycerol to cardiolipin nearly completely inhibited the release of lipoproteins with Asp at + 2 even at 10 mM Mg2+, while that of outer membrane-specific lipoproteins was not. Taken together, these results indicate that three major phospholipids of E. coli differently affect lipoprotein sorting and the activity of LolCDE. 相似文献
11.
The membrane sector (F0) of H+-ATPase was prepared by trypsin and urea treatment of F1-F0 and reconstituted with purified F1. The oligomycin sensitivity of the reconstituted F1-F0 complex obtained by treating F1 or F0 with Mg2+ before binding is much higher than that obtained without Mg2+ treatment. The greater change in the intrinsic fluorescence of the reconstituted F1-F0 complex obtained by Mg2+ treatment suggests that conformational changes may occur during the reconstitution. We deduce that Mg2+ binds to membrane lipids, thus decreasing membrane fluidity and changing the physical state of the lipids to provide a suitable microenvironment for conformational changes in F0. The data also suggest that the conformational change in the F0 portion of the F1-F0 complex can be transmitted to the F1 portion, the conformation of which is in turn altered, resulting in the formation of an F1-F0 complex with high oligomycin sensitivity. On the other hand, Mg2+ may act on F1 directly to induce a suitable conformational change which is then trnsmitted to F0, resulting in the formation of an H+-ATPase with greater sensitivity to oligomycin.Abbreviations STED
0.25 M sucrose, 10 mM Tris-SO4, 0.2 mM EDTA, and 1 mM dithiothreitol, pH 8.0
- NADH
nicotinamide adenine dinucleotide, reduced form
- olig.
oligomycin
- OSCP
oligomycin sensitivity conferring protein
- F6
coupling factor 6
- F1
coupling factor one (or F1-ATPase)
- F1
+Mg
2+ and F1
–Mg
2+
the F1 treated and untreated with 1 mM Mg2+ respectively
- F0
the membrane sector proteins of the H+-ATPase
- TUF0
trypsin-urea – F0
- EUF0
EDTA-urea – F0
- F0
+Mg
2+ and F0
–Mg
2+
the F0 treated and untreated with 1 mM Mg2+ respectively
- (F1 · F0)+Mg
2+ and (F1 · F0)–Mg
2+
the reconstituted F1 · F0 complex containing Mg2+-treated F1 and F0 and untreated F1 and F0 respectively
- F1 · F0
+Mg
2+ and F1 · F0
–Mg
2+
the reconstituted H+-ATPase complex derived from the binding of purified F1 to the F0 treated and untreated with Mg2+ respectively
- F1
+Mg
2+ · F0 and F1
–Mg
2+ · F0
the reconstituted H+-ATPase derived from the binding of F0 to the purified F1 treated and untreated with Mg2+ respectively 相似文献
12.
Proper assembly of RNA into catalytically active three-dimensional structures requires multiple tertiary binding interactions, individual characterization of which is crucial to a detailed understanding of global RNA folding. This work focuses on single-molecule fluorescence studies of freely diffusing RNA constructs that isolate the GAAA tetraloop-receptor tertiary interaction. Freely diffusing conformational dynamics are explored as a function of Mg2+ and Na+ concentration, both of which promote facile docking, but with 500-fold different affinities. Systematic shifts in mean fluorescence resonance energy transfer efficiency values and line widths with increasing [Na+] are observed for the undocked species and can be interpreted with a Debye model in terms of electrostatic relaxation and increased flexibility in the RNA. Furthermore, we identify a 34 ± 2% fraction of freely diffusing RNA constructs remaining undocked even at saturating [Mg2+] levels, which agrees quantitatively with the 32 ± 1% fraction previously reported for immobilized constructs. This verifies that the kinetic heterogeneity observed in the docking rates is not the result of surface tethering. Finally, the KD value and Hill coefficient for [Mg2+]-dependent docking decrease significantly for [Na+] = 25 mM vs. 125 mM, indicating Mg2+ and Na+ synergy in the RNA folding process. 相似文献
13.
We have investigated the effects of temperature on the conductance and voltage-dependent kinetics of cardiac gap junction
channels between pairs of seven-day embryonic chick ventricle myocytes over the range of 14–26°C. Records of junctional conductance
(G
j
) and steady-state unit junctional channel activity were made using the whole-cell double patch-clamp technique while the
bath temperature was steadily changed at a rate of about 4°C/min. The decrease inG
j
upon cooling was biphasic with a distinct break at 21°C. In 12 cell pairs,Q
10 was 2.2 from 26 to 21°C, while between 21 and 14°C it was 6.5. The meanG
j
at 22°C (G
j22
) was 3.0±2.1 nS, ranging in different preparations from 0.24 to 6.4 nS. At room temperature, embryonic cardiac gap junctions
contain channels with conductance states near 240, 200, 160, 120, 80 and 40 pS. In the present study, we demonstrate that
cooling decreases the frequency of channel openings at all conductance levels, and at temperatures below 20°C shifts the prevalence
of openings from higher to lower conductance states: all 240 pS openings disappear below 20°C; 200 pS openings are suppressed
at 17°C; below 16°C 160 and 120 pS events disappear and only 80 and 40 pS states are seen. Temperature also affected the voltage-dependent
kinetics of the channels. Application of a 6 sec, 80 mV voltage step across the junction (V
j80
) caused a biexponential decay in junctional conductance. Decay was faster at lower temperatures, whereas the rate of recovery
ofG
j
after returning toV
j0
was slowed. Cooling reduced the fast decay time constant, increased both recovery time constants, and decreased the magnitude
of GitGj decay, thus leaving a 10–16% larger residual conductance (G
ss/G
init,±80 mVV
j
) at 18 than at 22°C. From these results we propose that embryonic chick cardiac gap junctions contain at least two classes
of channels with different conductances and temperature sensitivities. 相似文献
14.
Ira R. Josephson Antonio Guia W. Jonathan Lederer Michael D. Stern 《Biochemical and biophysical research communications》2010,396(3):763-766
The contraction of adult mammalian ventricular cardiomyocytes is triggered by the influx of Ca2+ ions through sarcolemmal L-type Ca2+ channels (LCCs). However, the gating properties of unitary LCCs under physiologic conditions have remained elusive. Towards this end, we investigated the voltage-dependence of the gating kinetics of unitary LCCs, with a physiologic concentration of Ca2+ ions permeating the channel. Unitary LCC currents were recorded with 2 mM external Ca2+ ions (in the absence of LCC agonists), using cell-attached patches on K-depolarized adult rat ventricular myocytes. The voltage-dependence of the peak probability of channel opening (Po vs. Vm) displayed a maximum value of 0.3, a midpoint of −12 mV, and a slope factor of 8.5. The maximum value for Po of the unitary LCC was significantly higher than previously assumed, under physiologic conditions. We also found that the mean open dwell time of the unitary LCC increased twofold with depolarization, ranging from 0.53 ± 0.02 ms at −30 mV to 1.08 ± 0.03 ms at 0 mV. The increase in mean LCC open time with depolarization counterbalanced the decrease in the single LCC current amplitude; the latter due to the decrease in driving force for Ca2+ ion entry. Thus, the average amount of Ca2+ ions entering through an individual LCC opening (∼300-400 ions) remained relatively constant over this range of potentials. These novel results establish the voltage-dependence of unitary LCC gating kinetics using a physiologic Ca2+ ion concentration. Moreover, they provide insight into local Ca2+-induced Ca2+ release and a more accurate basis for mathematical modeling of excitation-contraction coupling in cardiac myocytes. 相似文献
15.
Brittany A. Kirk 《Journal of inorganic biochemistry》2010,104(2):207-210
In biological systems, enzymes often use metal ions, especially Mg2+, to catalyze phosphodiesterolysis, and model aqueous studies represent an important avenue of examining the contributions of these ions to catalysis. We have examined Mg2+ and Ca2+ catalyzed hydrolysis of the model phosphodiester thymidine-5′-p-nitrophenyl phosphate (T5PNP). At 25 °C, we find that, despite their different Lewis acidities, these ions have similar catalytic ability with second-order rate constants for attack of T5PNP by hydroxide (kOH) of 4.1 × 10−4 M−1s−1 and 3.7 × 10−4 M−1s−1 in the presence of 0.30 M Mg2+ and Ca2+, respectively, compared to 8.3 × 10−7 M−1s−1 in the absence of divalent metal ion. Examining the dependence of kOH on [M2+] at 50 °C indicates different kinetic mechanisms with Mg2+ utilizing a single ion mechanism and Ca2+ operating by parallel single and double ion mechanisms. Association of the metal ion(s) occurs prior to nucleophilic attack by hydroxide. Comparing the kOH values reveals a single Mg2+ catalyzes the reaction by 1800-fold whereas a single Ca2+ ion catalyzes the reaction by only 90-fold. The second Ca2+ provides an additional 10-fold catalysis, significantly reducing the catalytic disparity between Mg2+ and Ca2+. 相似文献
16.
Vrushali Dandavate Jayesh Jinjala Haresh Keharia Datta Madamwar 《Bioresource technology》2009,100(13):3374-3381
Burkholderia multivorans V2 (BMV2) isolated from soil was found to produce an extracellular solvent tolerant lipase (6.477 U/mL). This lipase exhibited maximum stability in n-hexane retaining about 97.8% activity for 24 h. After performing statistical optimization of medium components for lipase production, a 2.2-fold (14 U/mL) enhancement in the lipase production was observed. The crude lipase from BMV2 was partially purified by ultrafiltration and gel permeation chromatography with 24.64-fold purification. The Km and Vmax values for partially purified BMV2 lipase were found to be 1.56 mM and 5.62 μmoles/mg min. The metal ions Ca2+, Mg2+ and Mn2+ had stimulatory effect on lipase activity, whereas Cu2+, Fe2+ and Zn2+ strongly inhibited the lipase activity. EDTA and PMSF at 10 mM concentration strongly inhibited the lipase activity. Non-ionic and anionic surfactants stimulated the lipase activity. BMV2 lipase was proved to be efficient in synthesis of ethyl butyrate ester under non-aqueous environment. 相似文献
17.
Chunxia Zhao Xin Zhan Sidney W. Whiteheart Michael G. Fried 《Analytical biochemistry》2010,399(2):237-245
The hexahistidine (His6)/nickel(II)-nitrilotriacetic acid (Ni2+-NTA) system is widely used for affinity purification of recombinant proteins. The NTA group has many other applications, including the attachment of chromophores, fluorophores, or nanogold to His6 proteins. Here we explore several applications of the NTA derivative, (Ni2+-NTA)2-Cy3. This molecule binds our two model His6 proteins, N-ethylmaleimide sensitive factor (NSF) and O6-alklyguanine-DNA alkyltransferase (AGT), with moderate affinity (K ∼ 1.5 × 106 M−1) and no effect on their activity. Its high specificity makes (Ni2+-NTA)2-Cy3 ideal for detecting His6 proteins in complex mixtures of other proteins, allowing (Ni2+-NTA)2-Cy3 to be used as a probe in crude cell extracts and as a His6-specific gel stain. (Ni2+-NTA)2-Cy3 binding is reversible in 10 mM ethylenediaminetetraacetic acid (EDTA) or 500 mM imidazole, but in their absence it exchanges slowly (kexchange ∼ 5 × 10−6 s−1 with 0.2 μM labeled protein in the presence of 1 μM His6 peptide). Labeling with (Ni2+-NTA)2-Cy3 allows characterization of hydrodynamic properties by fluorescence anisotropy or analytical ultracentrifugation under conditions that prevent direct detection of protein (e.g., high ADP absorbance). In addition, fluorescence resonance energy transfer (FRET) between (Ni2+-NTA)2-Cy3-labeled proteins and suitable donors/acceptors provides a convenient assay for binding interactions and for measurements of donor-acceptor distances. 相似文献
18.
《Cell communication & adhesion》2013,20(4-6):233-237
Cx45 channel sensitivity to CO2, transjunctional voltage (Vj) and inhibition of calmodulin (CaM) expression was tested in oocytes by dual voltage-clamp. Cx45 channels are very sensitive to Vjand close preferentially by the slow gate, likely the same as the chemical gate. With CO2-induced drop in junctional conductance (Gj), the speed of Vj-dependent inactivation of junctional current (Ij) and Vjsensitivity increased. With 40 mV Vj, the τ of single exponential Ijdecay reversibly decreased by ~40% with CO2, and Gj steady state/Gj peakdecreased multiphasically, indicating that kinetics and Vjsensitivity of chemical/slow-Vjgating are altered by changes in [H+]iand/or [Ca2+]i. With 15 min exposure to CO2, Gjdropped to 0% in controls and by ~17% following CaM expression inhibition; similarly, Vjsensitivity decreased significantly. This indicates that the speed and sensitivity of Vj-dependent inactivation of Cx45 channels are increased by CO2, and that CaM plays a role in gating. Cx32 channels behaved similarly, but the drop in both Gj steady state/Gj peakand τ with CO2matched more closely that of Gj peak. In contrast, sensitivity and speed of Vjgating of Cx40 and Cx26 channels decreased, rather than increased, with CO2application. 相似文献
19.
A cDNA clone (GenBank Accession No. AY835398) encoding a sesquiterpene synthase, (E)-β-farnesene synthase, has been isolated from Artemisia annua L. It contains a 1746-bp open reading frame coding for 574 amino acids (66.9 kDa) with a calculated pI = 5.03. The deduced amino acid sequence is 30-50% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, β-farnesene, from farnesyl diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the Km- and kcat-values for farnesyl diphosphate, is 2.1 μM and 9.5 × 10−3 s−1, respectively resulting in the efficiency 4.5 × 10−3 M−1 s−1. The enzyme exhibits substantial activity in the presence of Mg2+, Mn2+ or Co2+ but essentially no activity when Zn2+, Ni2+ or Cu2+ is used as cofactor. The concentration required for maximum activity are estimated to 5 mM, 0.5 mM and <10 μM for Mg2+, Co2+ or Mn2+, respectively. Geranyl diphosphate is not a substrate for the recombinant enzyme. 相似文献
20.
Singh Jaipaul Hustler Brenda I. Waring John J. Howarth Frank C. 《Molecular and cellular biochemistry》1997,176(1-2):127-134
This study employs both dietary and physiological studies to investigate the relationship between calcium (Ca2+) and magnesium (Mg2+) signalling in the mammalian myocardium. Rats maintained on a low Mg2+ diet (LMD; 39 mg Kg-1 Mg2+ in food) consumed less food and grew more slowly than control rats fed on a control Mg2+ diet (CMD; 500 mg Kg-1 Mg2+ in food). The Mg2+ contents of the heart and plasma were 85 ± 3% and 34 ± 6.5%, respectively relative to the control group. In contrast, Ca2+ contents in the heart and plasma were 177 ± 5% and 95 ± 3%. The levels of potassium (K+) was raised in the plasma (129 ± 16%) and slightly decreased in the heart (88 ± 6%) compared to CMD. Similarly, sodium (Na+) contents were slightly higher in the heart and lowered in the plasma of low Mg2+ diet rats compared to control Mg2+ diet rat. Perfusion of the isolated Langendorff's rat heart with a physiological salt solution containing low concentrations (0-0.6 mM) of extracellular magnesium [Mg2+]0 resulted in a small transient increase in the amplitude of contraction compared to control [Mg2+]0 (1.2 mM). In contrast, elevated [Mg2+]0 (2-7.2 mM) caused a marked and progressive decrease in contractile force compared to control. In isolated ventricular myocytes the L-type Ca2+ current (ICa,L was significantly (p < 0.001) attenuated in cells dialysed with 7.1 mM Mg2+ compared to cells dialysed with 2.9 µM Mg2+. The results indicate that hypomagnesemia is associated with decrease levels of Mg2+ and elevated levels of Ca2+ in the heart and moreover, internal Mg2+ is able to modulate the Ca2+ current through the L-type Ca2+ channel which in turn may be involved with the regulation of contractile force in the heart. 相似文献