镧对镉胁迫下豌豆幼苗根部细胞内离子平衡及膜质子泵的影响

以水培豌豆(PisumsativumL)幼苗为材料,研究了La3+对Cd2+胁迫下豌豆幼苗生长、叶部与根部细胞对Cd2+的吸收、根部细胞内无机离子平衡体系及膜H+-ATPase,H+-PPase活性的影响。结果表明:Cd2+胁迫下喷施适宜浓度的La3+(0.50mg·L-1)可使豌豆幼苗相对生物量、苗长、叶绿素含量以及光合速率受到的毒害明显缓解,有效减少叶部与根部细胞对Cd2+的吸收,显著减轻了Cd2+胁迫导致的离子平衡系统的紊乱,明显缓解Cd2+对质膜及液泡膜H+-ATPase,H+-PPase活性的抑制作用。10mg·L-1Cd2+胁迫下La3+的保护效应比30mg·L-1Cd2+胁迫下更为显著。样品中La3+的测定和分析结果还表明,外源La3+处理后,La3+主要结合于细胞膜外,进入原生质体的仅占很小部分,但30mg·L-1Cd2+处理样品中La3+进入细胞的量则明显多于10mg·L-1Cd2+处理的样品。     

A microtiter plate assay for the detection of inhibitors of the Na +, K + -ATpase

A rapid microtiter plate assay for the detection of inhibitors of the Na⁺, K⁺-ATPase has been developed. The assay is based on the measurement of inorganic phosphate released from the substrate, ATP, and has been designed to be carried out in the individual wells of a microtiter plate. Since the production of inorganic phosphate is determined colorimetrically, multiple samples can be tested simultaneously using a microtiter plate reader. This microtiter plate assay is particularly useful for screening large numbers of samples, such as microbial culture supernatants.     

Synthesis of marine guanidine alkaloids and their application as chemical/biological tools

Ptilomycalin A and crambescidins, novel marine guanidine alkaloids, have a unique pentacyclic guanidine structure, and exhibit a considerable array of biological activities. The first method developed for the synthesis of the pentacyclic guanidine core structure involved successive 1,3-dipolar cycloaddition reactions and resulted in the first total synthesis of crambescidin 359. The synthesis of other pentacyclic guanidine derivatives has been based on this methodology and applied as tools for studying biological activities, and as chemical reaction catalysts.     

Involvement of Proton Transfer for Carbon Dioxide Reduction Coupled with Extracellular Electron Uptake in Shewanella oneidensis MR‐1

Microbial biosynthesis of hydrocarbon from CO₂ reduction driven by electron uptake process from the cathodic electrode has gained intensive attention in terms of potential industrial application. However, a lack of a model system for detailed studies on the mechanism of the CO₂ reduction hinders the improvement in efficiency for microbial electrosynthesis. Here, we examined the mechanism of microbial CO₂ reduction at the cathode by a well‐described microbe for extracellular electron uptake, Shewanella oneidensis MR‐1, capable of reducing gaseous CO₂ to produce formic acid. Using whole‐cell electrochemical assay, we observed stable cathodic current production at ?0.65 V vs Ag/AgCl KCl sat. associated with the introduction of CO₂. The observed cathodic current was enhanced by the addition of 4 μM riboflavin, which specifically accelerates the electron uptake process of MR‐1 by the interaction to its outer‐membrane c‐type cytochromes. The significant impact of an uncoupler agent and a mutant strain of MR‐1 lacking sole F‐type ATPase suggested the importance of proton import to the cytoplasm for the cathodic CO₂ reduction. The present data suggest that MR‐1 potentially serves as a model system for microbial electrosynthesis from CO₂.     

Caseinolytic Proteins (Clp) in the Genus Klebsiella: Special Focus on ClpK

Caseinolytic proteins (Clp), which are present in both prokaryotes and eukaryotes, play a major role in cell protein quality control and survival of bacteria in harsh environmental conditions. Recently, a member of this protein family, ClpK was identified in a pathogenic strain of Klebsiella pneumoniae which was responsible for nosocomial infections. ClpK is linked to the thermal stress survival of this pathogen. The genome wide analysis of Clp proteins in Klebsiella spp. indicates that ClpK is present in only 34% of the investigated strains. This suggests that the uptake of the clpk gene is selective and may only be taken up by a pathogen that needs to survive harsh environmental conditions. In silico analyses and molecular dynamic simulations show that ClpK is mainly α-helical and is highly dynamic. ClpK was successfully expressed and purified to homogeneity using affinity and anion exchange chromatography. Biophysical characterization of ClpK showed that it is predominantly alpha-helical, and this is in agreement with in silico analysis of the protein structure. Furthermore, the purified protein is biologically active and hydrolyses ATP in a concentration- dependent manner.     

木麻黄质膜离子泵在酸雨条件下对镧的响应

研究镧对酸雨(pH为4.5)胁迫下木麻黄幼苗早期生长和质膜离子泵ATP酶活性的影响。结果表明:较低浓度La3+浸种能使幼苗株高、根长、鲜重以及质膜质子泵活性明显增强,在200mg·L-1La3+处理时达到最高增强值,而高浓度的La3+浸种使之表现出抑制效应;幼苗质膜钙泵的活性受La3+抑制。质子泵活性与细胞的伸长生长呈显著正相关关系,镧对质子泵的活化作用有助于促进植物的生长,且适量的镧具有缓解酸雨胁迫下生长的植物细胞质酸化,维持细胞内环境稳定的间接作用。为抗酸雨和促生长,木麻黄经镧浸种8h的最适宜La3+浓度为50～200mg·L-1。     

ATPases as Multi-Response Sensing System for Various Organic and Inorganic Analytes

Summary. The possibility of using synaptic plasma membrane (SPM) enzymes Na⁺/K⁺-ATPase and Mg²⁺-ATPase, isolated from rat brain, as a biological component of multi-response sensing system for detection of different compounds (alkaline and heavy metal salts, organic compounds) was studied. The method is based on the spectrophotometric determination of inorganic ortho-phosphate (P_i) that serves as a measure of the enzymatic activity in the presence of various analytes. The concentration of P_i, liberated by enzyme catalysed hydrolysis of adenosinetriphosphate (ATP), was followed spectrophotometrically, by single exposure to analytes or in the mixture. P_i was dose dependent on the analyte concentration. Alkaline elements (Na, K, Mg), heavy metals (Pb, Cd, Hg, Cu, Fe, Co, Zn), toxic organic compounds (pyridine, urea, chlorpyrifos), and some drugs (digoxin, gitoxin) showed diverse effects, inducing the inhibition or stimulation of the enzymes activity. Development of simple test method for simultaneous detection of the investigated analytes based on the variation of medium assay composition was discussed.     

A Flow System Based on a Fluorometer and a Luminometer to Monitor the Correlation of Protein Conformation and Function

A flow system to monitor the reactions in solutions was constructed, which connected a fluorometer and a luminometer in series. Using a stream through it, the correlation of fluorescence and luminescence was measured. The system was tested with actin. The fluorescence emission gave the signal of polymerization of labeled actin and the luminescence gave that of ATP hydrolysis by actin. The observations showed the correlation between ATP hydrolysis and polymerization. Applications of other proteins to this system will give insights into the characteristics of proteins.     

Modeling the effect of external electric field and current on the stochastic dynamics of ATPase nano-biomolecular motors

The rotary motion of the F₀ part of the F₀F₁-ATPase motor that synthesizes the ATP molecules used by the intracellular stepping motors, such as kinesins, is modeled within a stochastically fluctuating medium via the application of the Langevin dynamics wherein the random intramembrane fluctuations are represented by a white noise. We have investigated the influence of an applied electric field and an applied electric current on this rotary motion, and the subsequent production rate of the ATP molecules. It is seen that the application of a field, or a current, changes both the elastic behavior of the cell membrane and the transmembrane potential. These changes in turn transform the dynamics of the F₀ part of the motor. We have found that at low fields, the role of transmembrane potential becomes significant in the production rate of the ATP molecules, whereas at high fields the changes induced in the surface tension of the membrane also contribute to the production rate of the ATP.     

应用胰凝乳蛋白酶消化法比较冬季和夏季鲢鱼肉肌球蛋白的构造特性

以夏季和冬季鲢为研究对象,利用胰凝乳蛋白酶能水解羧基端含芳香族氨基酸残基肽键的特性,根据其特异性酶切部位,结合电泳手段来分析肌球蛋白的内部构造差异性。结果表明,与夏季样品相比,冬季鲢的肌原纤维蛋白经酶切生成的肌球蛋白头部S-1较长,在高温下分子量为165 ku的重酶解肌球蛋白HMM容易被再降解成小片段的135 ku HMM,呈现出冬季肌球蛋白的结构不稳定性。在不同温度下加热夏季和冬季肌球蛋白,其ATPase失活速度和酶解肌球蛋白生成S-1的产生量的减少速度呈现一致性,说明酶解生成的S-1只来源于有活性的肌球蛋白。同时,冬季肌球蛋白热变性温度较夏季肌球蛋白要低6 ℃,表明冬季肌球蛋白的不稳定性。