Molecular aspects of activation mechanisms of CF₁

The Ca2( +) -dependent ATPase activity of spinach chloroplast coupling factor 1 (CF?) is activated by treatment with dithiothreitol (DDT). If excess of this reagent is eliminated by gel filtration, an Eadie-Hofstee biphasic plot is obtained. These results are consistent with the existence of two active forms of the enzyme governed by the redox state. We have observed that SDS-polyacrylamide gel electrophoresis pattern is affected by the pretreatment of the samples under those two different conditions. Spontaneous activation of the samples, due to a limited proteolytic process, has also been detected. In this case the electrophoretic pattern was also affected. The protease implied in this process could be a cystein protease co-isolated with CF?. These observations suggest that limited proteolysis, as well as redox-induced changes, are involved in the physiological regulation of the enzyme.     

Control of chloroplast ATP synthase (CF0CF1) activity by Δ pH

A biphasic decay of the thiol modulated ATPase activity is observed at fast deenergization of the thylakoids achieved by turning off the light and simultaneous injection of the uncoupler nigericin. Most likely the rapid phase (_1/2 = 5 s) represents an unstable, active E_f-form of the enzyme which decays to a less active, but more stable E_s-form. The two forms have different substrate affinities. Deactivation and reactivation kinetics indicate that the transition from the E_f- to the E_s-form is reversible, requires a low proton gradient (1 to 2 pH units) and most probably involves the release and binding, respectively, of two protons from the thylakoid lumen phase to sites which have an apparent pK of 6.6. The E_s-form decays to the inactive E_i-form with a half time of 90 s. Reactivation of the completely deactivated enzyme is a two-stage process comprising protonation of sites with a pK of 6.8 followed by protonation of sites of pK 4.9. The intermediate E_s'-form has a decay time which is similar to that of the E_s-form, but a different K_m for ATP. Therefore we conclude that activation is not the exact reversal of deactivation. The results are discussed in terms of a model of H+-linked activation/deactivation.     

The incidence of ΔF508 CF mutation,and associated haplotypes,in a sample of English CF families

Summary Data are presented for ΔF508 screening and KM19/XV2c haplotype analysis of 195 cystic fibrosis (CF) chromosomes from the British Caucasian population. We report the frequency of ΔF508 in this group to be 80% and find pronounced disequilibrium between the deletion and the KM 2, XV 1 haplotype. Haplotype analysis of 71 normal chromosomes is also presented. We report one individual who had meconium ileus and who does not have the ΔF508 mutation on either chromosome.     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

Direct dynamics simulations of the hydrogen abstraction reaction Cl + CF3CF2CH2OH

The mechanism and kinetics of 2,2,3,3,3-pentafluoropropanol (CF₃CF₂CH₂OH) reaction with Chlorine atom (Cl) is investigated in this work. Two hydrogen abstraction channels of the title reaction are identified. The geometries of all the stationary points in the potential energy surface are obtained at the BHandHLYP/6-311G** level, and the energies of the selected points along the minimum energy path (MEP) are improved by the CCSD(T) method. A dual-level direct dynamics method is employed to study the kinetic nature of the hydrogen-abstraction reaction channels. The calculated rate coefficients show that the hydrogen abstraction from the CH₂ group is the primary channel. The calculated total rate coefficients are in best agreement with the experimental values. The four-parameter rate coefficients expression of the title reaction between the temperatures 200 K and 1000 K is provided.     

Effect of pyridine homologues on proton flux through the CF0 · CF1 complex and photophosphorylation in chloroplasts

At concentrations below 1 mM, hydrophobic pyridine homologues decrease the rate of photophosphorylation and light-stimulated hydrolysis of ATP and light-activated exchange of the tightly bound nucleotides in chloroplasts, but increase the rate of the Hill reaction. Unlike uncoupling agents, the presence of the organic base at such low concentrations decreases the rate of light-dependent leakage and has no effect on the efficiency of two-stage photophosphorylation in broken chloroplasts. By assuming that the organic base is bound to independent equivalent sites in the thylakoid membrane, a simple expression can be derived which relates the observed rates of photophosphorylation and light-stimulated hydrolysis of ATP quantitatively to the concentration of the organic base in solution and gives dissociation equilibrium constants which are on the order of the relative hydrophobicities of the pyridine homologues. A possible mechanistic model for the CF₀ · CF₁ complex is proposed which could serve as the basis for a unified interpretation of the kinetics of proton translocation in illuminated chloroplasts, the steady-state rate of photophosphorylation, the light-stimulated ATPase activity, and the light-activated exchange of tightly bound adenine nucleotides.Abbreviations AMPPNP adenylylimidodiphosphate - Chl chlorophyll - CF₀ · CF₁ the coupling factor complex of chloroplasts - DCCD N,N-dicyclohexylcarbodiimide - DTT dithiothreitol - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - TCA trichloroacetic acid - Tricine N-tris-(hydroxymethyl)methylglycine     

The effect of chloroplast coupling factor ATP synthetase (CF1 · CF0) reconstitution on fluidity properties of isolated thylakoid lipid vesicles

The reconstitution of chloroplast coupling factor ATP synthetase (CF₁ · CF₀) with thylakoid lipids by cholate dialysis produced vesicles that displayed higher steady-state anisotropy (r_s) values for both 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenyl hexatriene fluorescence than the pure lipid alone. This is interpreted as meaning that the insertion of protein into the lipid bilayer brings about an increase in the ordering of acyl chains. This ordering effect became more obvious as the protein-to-lipid ratio was increased. Time-resolved decay analyses of DPH fluorescence anisotropy confirmed the conclusion drawn from the steady-state measurements, but further indicated that the dynamic motion of the probe was also slightly restricted after CF₁ · CF₀ incorporation. The restriction of DPH motion and the change in the half-angle for its cone of rotation was observed at relatively low protein-to-lipid ratios as compared with other reconstituted or biological membranes, suggesting that perhaps lipid-protein interactions occur with the inserted CF₁ · CF₀ complex.     

Determination of the Group Electronegativity of CF3 Group in 3′-O-CF3-Thymidine by 1-NMR

Abstract

The interplay of enthalpy of the gauche effect (ΔH°_GE) of the [X3′-C3′-C4′-O4′] fragment in various 3′-substituted (X) 2′,3′-dideoxythymidine derivatives 1–7 and the inherent anomeric effect drives the two-state North ? South equilibrium in the constituent sugar moiety. The group electronegativity of 3′-OCF₃ substituent in Marriott's, Inamoto's and Mullay's scales has been determined from simple calibration graphs correlating the group electronegativity of various 3′-substituents (X) in 2′,3′-dideoxythymidine derivatives 1–7 with the experimental strength (ΔH°_GE) of the [X3′-C3′-C4′-O4′] gauche effect. ΔH°_GE has been experimentally determined from pseudorotational analyses of temperature-dependent ³J_HH coupling constants, and can be used as an unambiguous tool for direct experimental estimation of the group electronegativity of a specific substituent covalently attached to 3′-carbon of 2′,3′-dideoxythymidine, which can be compared, in turn, with the theoretical estimation carried out according to Marriott's or Inamoto's procedure. Inconsistency found between theoretical values in Marriott's and Inamoto's scales, on the one hand, and between our experimental estimate and the theoretical value in Marriott's scale, on the other, have been solved by refining the electronegativity scale using our experimental data for 1–7.     

Accurate ab initio calculation of the Ar–CF4 intermolecular potential energy surface

Ar–CF₄ intermolecular interaction potential is studied by ab initio calculations at the MP2 and CCSD(T) levels of theory containing the so-called bond functions ({3s3p2d1f} basis set was chosen) both with and without a correction for the basis-set superposition error. The calculations were performed with Dunning's correlation consistent basis sets (aug-cc-pVXZ, X = D, T, Q, 5) to extrapolate the Ar–CF₄ potential energy minimum and intermolecular distance to their complete basis set (CBS) limits. It is shown that the addition of bond functions results in a dramatic improvement in the convergence of the calculated interaction energies at the MP2/aug-cc-pVTZ level. The MP2/{3s3p2d1f}-aug-cc-pVTZ potential energy surface even approaches the CCSD(T)/aug-cc-pVQZ potential energy surface. The potential energy minima and the intermolecular distances are both significantly closer to the CBS limit when using the bond functions, and it implies that adding bond functions in the calculation has a great effect on the interaction energies. We also find that with bond functions included in the CCSD(T)/aug-cc-pVDZ model chemistry, the potential energy minima are extremely close to the CBS limit and are better than the CCSD(T)/aug-cc-pVQZ values. Several levels of theory described in the text were used to determine pairwise analytic potential energy surfaces for Ar+CF₄. The analytic potential energy surfaces are in very good agreement with the ab initio values.     

Deletion ΔF508 and haplotype analysis of CFTR gene region in Slovak CF patients

Summary Analysis of a sample of 50 unrelated cystic fibrosis (CF) patients and 46 nuclear families from Slovakia (Czechoslovakia) by the polymerase chain reaction and Southern hybridization revealed that the proportion of the F508 mutation was 58% in this population, and that the frequency of the B (i.e., KM19/XV2c [1–2]) haplotype was increased in both F508 and nonF508 CF chromosomes (98% and 46%, respectively). These results support the view that the trans-European gradient of the F508 frequency is of a geographical rather than of an ethnic origin, and that in Slavonic populations, there exists an as yet unidentified but frequent CF mutation other than F508, associated with the B haplotype.     

DNA酶缓解CF患者的呼吸困难

在治疗囊性纤维变性(CF)的Ⅰ期临床试验中,重组DNA酶能使肺功能提高10～20％,并显著改善16位受试患者中11位的呼吸状况。试验由国立心脏、肺和血液研究所(Belhesda,MD)的Ronald Crystal主持,将天然DNA剪切酶的Genentech Incis(S.San Francisco,CA)rDNA离体喷雾施于患者。 CF患者的肺易于感染,因为其中累积了厚厚的粘液。为抗感染,白细胞集结于肺部,其裂解后产生     

Substitutions of the Conserved Gly47 Affect the CF1 Inhibitor and Proton Gate Functions of the Chloroplast ATP Synthase ε Subunit

The conserved residue Gly47 of the chloroplast ATP synthase ε subunit was substituted with Leu, Arg, Ala and Glu by site-directed mutagenesis. This process generated the mutants εG47L, εG47R, εG47A and εG47E, respectively. All the ε variants showed lower inhibitory effects on the soluble CF1(-ε) Ca^2 -ATPase compared with wild-type ε. In reduced conditions, εG47E and εG47R had a lower inhibitory effect on the oxidized CF1(-ε) Ca^2 -ATPase compared with wild-type ε. In contrast, εG47L and εG47Aincreased the Ca^2 -ATPase activity of soluble oxidized CF1(-ε). The replacement of Gly47 significantly impaired the interaction between the subunit ε and γ in an in vitro binding assay. Further study showed that all ε variants were more effective in blocking proton leakage from the thylakoid membranes. This enhanced ATP synthesis of the chloroplast and restored ATP synthesis activity of the reconstituted membranes to a level that was more efficient than that achieved by wild-type ε. These results indicate that the conserved Gly47 residue of the ε subunit is very important for maintaining the structure and function of the ε subunitand may affect the interaction between the ε subunit, β subunit of CF1 and subunit Ⅲ of CF0, therebyregulating the ATP hydrolysis and synthesis, as well as the proton translocation role of the subunit Ⅲ of CF0.     

Biochemical,Mössbauer,and EPR studies of the diiron cluster of phenol hydroxylase from Pseudomonas sp. strain CF 600

Phenol hydroxylase of Pseudomonas sp. strain CF600 comprises three components: DmpP is an FAD- and [2Fe-2S]-containing reductase; DmpM is a cofactorless activator protein; and DmpLNO is the oxygenase. Single turnover experiments established that DmpLNO contains the active site, but requires DmpM for efficient turnover: the steady-state turnover rate reaches a maximum at 1.5 DmpM:1 DmpLNO. Chemical cross-linking experiments showed that DmpM interacts with the large subunit of the DmpLNO oxygenase complex. M?ssbauer studies revealed that the active site of the oxygenase can accommodate two types of diiron clusters, each of these cluster types having two equivalent sites. Cluster form I, representing typically around 85% of total Fe, has DeltaE(Q) = 1.73 mm/s and delta = 0.54 mm/s, while cluster II exhibits DeltaE(Q) = 0.79 mm/s and delta = 0.48 mm/s. Studies in strong applied magnetic fields suggest that the two iron sites of cluster I are bridged by an oxo group while sites in cluster II appear to be hydroxo-bridged. Reduction of the samples with dithionite yields the diferrous forms of the clusters. Air oxidation of the reduced samples leads to an increase of the cluster II fraction, accompanied by a corresponding decrease in catalytic activity. The reduced oxygenase samples exhibit at X-band an integer spin EPR signal centered, in parallel mode, at g = 16.6. Quantitative analysis showed that 19% of the clusters contribute to the EPR signal, suggesting that cluster II is the EPR-active species. Incubation with dithiothreitol (DTT) inactivated the oxygenase by a mechanism apparently involving H(2)O(2) generation. In addition, M?ssbauer studies of DTT-inactivated enzyme showed that all ferric iron belonged to one diamagnetic diferric cluster with parameters that indicate that DTT coordinates to the cluster.     

镁,钙子对组装于人工膜上CF0—CF1质子传导的影响

在电压箝位下,研究Ｍｇ＾２＋、Ｃａ＾２＋对重组于平板脂双层上ＣＦ０－ＣＦ１质子传导的影响,表明：（１）跨膜质子梯度０时（ΔｐＨ＝０）,在３０￣１５０ｍＶ范围内有大量的金Ｃａ＾２＋引起的正电流比在无金属离子溶液中的一定幅度的增加。在５０ｍＶ以上,Ｍｇ＾２＋比Ｃａ＾２＋有更大的促进正向电流的作用;（２）当ΔｐＨ＝３时,Ｍｇ＾２＋比Ｃａ＾２＋更有显著增加正向膜电流的作用,电压在０ｍＶ时就已显示出来;（３     

technologica CF Imager叶绿素英光成像系统

正高分辨率检测器纯黑机箱,方便暗适应Fluorlmager软件,具超强数据处理功能1600个LED光源116 cm~2均匀光照成像范围116 cm2均匀光照成像范围1600个LED光源仪器特点:〉1600个LED光源116 cm2均匀光照成像范围预先校准扫描检测器CCD 2/3"〉分辨率1392x1040〉Binning技术,灵敏度更高自动识别、分析96孔板上、培养皿或盆栽的个体或群落最多可对250个样品进行实时分析     

Airway epithelial cells—Hyperabsorption in CF?

Airway epithelial cells transport electrolytes and are central to the disease cystic fibrosis (CF), which is an inherited transport defect affecting smaller airways and a number of other epithelial organs. Clinically, CF is dominated by a chronic lung disease, the main cause of morbidity and mortality. Airway obstruction by thick mucus and chronic infection by Pseudomonas aeruginosa eventually lead to loss of pulmonary function. Loss of function of CFTR Cl^? channels was found to be the cause for CF. However, intensive research on the detailed mechanism of CF lung disease for more than 25 years produced a bewildering number of hypotheses and an endless discussion whether reduced Cl^? secretion, primarily located in airway submucosal glands, or dehydration of the airways, driven by a hyperabsorption of Na⁺ ions, is the primary cause of the disease. Recent results suggest a fine-tuned regulation of the airway fluid layer, but how significant really are Cl^? and Na⁺ transport?     

CF—1型侧侧消化道吻合器通过鉴定

今年4月4日至6日,上海手术器械六厂、上海第一医学院附属中山医院共同研制成功的CF—1型侧侧消化道吻合器通过鉴定。来自全国二十四个省、市、自治区和部队医疗单位的120余名专家、教授应邀参加了这次鉴定会。该器械是在GF—1型管形消化道吻合器、XF—1型消化道缝合器试制成功的基础