H-ATPase in the plasma membrane of Arabidopsis pollen cells is involved in extracellular calmodulin-promoted pollen germination

In this paper, the role of the plasma membrane (PM) H⁺-ATPase in extracellular calmodulin (CaM)-promoted pollen germination and in tube growth of Arabidopsis thaliana was investigated. Pollen germination, pollen tube growth rate, free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) and Ca²⁺ channel activity in the PM of pollen cells were measured. In response to fusicoccin or CaM treatment, in vitro pollen germination and pollen tube growth rate, [Ca²⁺]_cyt and activity of a hyperpolarization-activated Ca²⁺-permeable channel increased. Sodium vanadate inhibited the promotion of these parameters by extracellular CaM. The results suggest that H⁺-ATPase may be involved in extracellular CaM-regulated pollen germination and in tube growth by modulation of the hyperpolarization-activated Ca²⁺ channel in the PM of A. thaliana pollen cells.     

Actin and myosin during pollen germination

Actin and myosin from pollen tubes of Lilium davidii were studied by using immunoblotting, Dot_Blot and myosin Ca 2+_ATPase analysis. On immunoblotting of the total soluble pollen tube proteins, anti_α_actin antibody labelled a polypeptide approximately 43 ku, which is considered to be the actin of lily. The mRNA encoding actin in ungerminated pollen and germinated pollen were both undetectable in our experiments. A myosin exhibited Ca 2+_ATPase activity, with a native molecular weight of 460 ku has been identified by using immunoblotting. A polypeptide of about 205 ku and a polypeptide of about 20 ku were the heavy chain and a set of light chain of the myosin, which can crossreact with anti_skeletal muscle myosin heavy chain monoclonal antibody and anti_skeletal muscle myosin light chain (20 ku) monoclonal antibody, respectively. The Ca 2+_ATPase activities of myosin in crude extracts of germinated pollen were positively related to the growth rates of pollen tubes.     

钙调磷酸酶B亚基钙结合位点的突变对B亚基结构和功能的影响

应用远紫外CD光谱、紫外差光谱、内源荧光光谱以及ANS荧光光谱，探究了钙调磷酸酶B亚基钙结合位点的突变对B亚基结构和功能的影响．结果显示:钙结合位点突变体Y105W激活CNA的能力强于CNB；而E110Q活化CNA的能力弱于CNB；Y105W和E110Q与Ca2+亲和性低于CNB；CNB和它的突变体二级结构趋于一致，但三级结构存在明显差异，这种差异可能是它们功能差异的结构基础．      

Purification and characterization of myosin from wheat mitochondria

Myosin was purified from wheat mitochondria using DE-52 anion exchange chromatography and Sephacryl S-300 gel filtration. The molecular weight of its heavy chain is about 210 ku, similar to that of muscle myosin Ⅱ(205 ku), and it could be recognized by the polyclonal antibodies against human skeletal muscle myosin Ⅱ. The ATPase activity of the mitochondrial myosin stimulated by F-actin from chicken muscle is 202.5 nmoles Pi/min·mg. The mitochondrial myosin could be activated by Ca²⁺ and was not inhibited by Ca²⁺ at high concentration. The results demonstrate that the myosin of wheat mitochondria shares some similarities with the skeletal muscle myosin Ⅱ.     

Immunohistochemical localization of Ca2+/calmodulin-dependent kinase in tobacco

The existence of Ca²⁺/calmodulin-dependent kinase (CaM kinase, CaMK) in tobacco is verified immuno- logically and its distribution in different tissues of tobacco is studied. It has been demonstrated that CaMK is mainly distributed in early developing anthers, developing ovules and embryos, lateral root primordium, apical meristem and leaf primordium of buds and mesophyll cells and developing vascular bundles of leaves. There is enormous CaM kinase distributed in leaf epidermis fair cells and guard cells of stomas too. Little kinase is found in mature stem or root cells. The distribution properties of CaM kinase in tobacco are consistent with those of CaM, suggesting that there exists the Ca²⁺ signal transduction pathway mediated by CaM kinase in tobacco and it plays an important role in the plant growth and development.     

The binding of the anticoagulation factor Ⅰ from the venom of Agkistrodon acutus to activated factor Ⅹ

Anticoagulation factor Ⅰ (ACF Ⅰ) from the venom of Agkistrodon acutus prolonged plasma prothrombin time (PPT) with dose-dependent manner and exhibited marked anticoagulant activity only at the concentration higher than its critical concentration (12 nmol/L). It was discovered that ACFⅠ formed a 1:1 complex with activated coagulation factor (FⅩa) in the presence of Ca2+ ions by the method of polyacrylamide gel electrophoresis. Both native ACFⅠ and decalcified ACFⅠ failed to form complexes with FⅩa in the absence of Ca2+. Sr2+ ions were able to replace Ca2+ ions in the binding of ACFⅠ to FⅩa, but both Ba2+ ions and Tb3+ ions were ineffective. ACFⅠ was a new member of the Ⅸ/Ⅹ-bp family in the C-type lectin superfamily, and had a amino acid composition similar to the other members of this family. It was composed of 251 amino acid residues with a molecular weight of 29 603.6 u on non-reducing condition, determined by MALDI-TOF-MS, and a molecular weight of 14.7 ku on reducing condition, determined by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).     

新型LED红色荧光材料Ca_xSr_(1-x-y-z)MoO_4:yEu~(3+);zNa~+的制备和表征

利用Na+为电荷补偿剂制备CaxSr1-x-y-zMoO4:yEu3+,zNa+红色LED荧光粉体.研究了电荷补偿剂的用量、基质材料中Ca2+的含量、合成温度、反应时间及发光中心Eu3+摩尔分数对荧光材料晶体结构和发光性能的影响.结果表明:最佳工艺条件为电荷补偿剂摩尔分数为6%,Eu3+摩尔分数为8%,Ca2+摩尔分数为60%,反应温度900℃和反应时间2 h.光谱测试结果表明,该荧光材料可被311,395或465 nm有效激发,发射峰在616 nm.     

钙调神经磷酸酶

钙调神经磷酸酶是一种依赖Ca^2 －CaM的磷蛋白磷酸酶。主要存在于脑组织神经元中，由催化亚基A和调节亚基B1：1组成。钙调神经磷酸酶是一个侈底物的磷蛋白磷酸酶。它的活性还受到Ni^2 和Mn^2 等金属离子的调节。     

Identification of a DNase activated inXenopus egg extracts undergoing apoptosis

A cell-free apoptosis system was established by adding dATP and cytochrome c toXenopus laevis egg extracts S-150. Accompanied by an incubation process, an apoptosis-specific DNase was activated in egg extracts which depended on Mg²⁺ and inhibited by Zn²⁺. Two nucleases existing in egg extracts were revealed by in-gel nuclease assay. Further experiments showed that 27 ku nuclease which was different from other Ca²⁺ /Mg²⁺ -dependent nucleases was a possible candidate involved in apoptosis.     

八肋游仆虫中心蛋白的核磁共振研究

利用液体核磁共振方法对八肋游仆虫中心蛋白全长蛋白(EoCen),LC端结构域(EoCen-LC)和N端结构域(EoCen-N)进行了分析,并研究了蛋白与钙离子的相互作用情况.研究结果表明,未加入钙离子,全长蛋白,LC端结构域和N端结构域谱图重叠严重,峰型较差,说明未折叠成良好的三级结构,存在较大的构象交换或一定程度的聚集;与钙离子结合后全长蛋白,LC端结构域三级结构有所改善,但仍然无法用液体核磁共振方法进行进一步的结构研究.N端结构域与钙离子结合后谱峰分散性好,说明N端结构域具有较好的三级结构,能够应用核磁共振的方法进行结构解析.     

Phosphatidylglycerol effect on oxygen-evolving activity in Ca2+-depleted photosystem II

The effect of anionic phosphatidylglycerol (PG) on oxygen evolution in a photosystem II (PS II ) particle depleted of Ca²⁺ (designated d_CaPSII ) has been investigated. The major finding is the observation of a new role of PG in the PSII function. That is, PG restores nearly the lost oxygen evolution in d_caPS II particle owing to Ca²⁺ depletion to the levels in intact PS II. Furthermore, there is a stimulation of oxygen-evolving activity in the d_CaPSII complexed with PG in the presence of exogenous CaCl₂, which PG enhances increasingly oxygen evolution with increasing CaCl₂ concentration. It is suggested that PG-induced oxygen evolution recovery of d_Ca PS II particle results from resumption of normal structure in protein by PG effect, whereas the enhancement of oxygen evolution in complex subject to CaCl₂ is ascribed to the optimization of such a structure due to coordination complex formation of Ca²⁺ ions with PG.     

碱析分离法处理造纸红液

采用碱析分离法处理浓缩造纸红液,研究了体系pH值、碱析剂加入量、反应温度和时间等因素对碱析反应的影响,确定了碱析反应的优化工艺条件,并对碱析反应机理进行了讨论。实验结果表明,pH值对木质素磺酸盐与钙离子间络合作用影响显著,氢离子可与木质素磺酸盐螯合,从而导致Ca²⁺与木质素磺酸盐的螯合作用降低,由此碱析分离法需在较高pH值下进行反应。以10mL造纸浓缩红液为处理对象,碱析反应的优化条件为：pH值为13,碱板剂Ca²⁺加入量为8g/L,反应温度为40℃,反应时间为30min时,木质素沉淀量最大为4.5g/10mL。     

Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin

Small-conductance Ca2+-activated K+ channels (SK channels) are independent of voltage and gated solely by intracellular Ca2+. These membrane channels are heteromeric complexes that comprise pore-forming alpha-subunits and the Ca2+-binding protein calmodulin (CaM). CaM binds to the SK channel through the CaM-binding domain (CaMBD), which is located in an intracellular region of the alpha-subunit immediately carboxy-terminal to the pore. Channel opening is triggered when Ca2+ binds the EF hands in the N-lobe of CaM. Here we report the 1.60 A crystal structure of the SK channel CaMBD/Ca2+/CaM complex. The CaMBD forms an elongated dimer with a CaM molecule bound at each end; each CaM wraps around three alpha-helices, two from one CaMBD subunit and one from the other. As only the CaM N-lobe has bound Ca2+, the structure provides a view of both calcium-dependent and -independent CaM/protein interactions. Together with biochemical data, the structure suggests a possible gating mechanism for the SK channel.     

Relationship between LTP and the nuclear protein induced by calcineurin

Results demonstrate that calcineurin plays a role in long term potentiatibn (LTP) in the hippocampus. A 45 ku enzyme was administered, which exhibits Ca²⁺ /calmodulin independent activity to rats, and the protein components of nuclear extracts from the cell-free system of rat hippocampus were tested. It is found that the level of a component significantly increased in nuclear extracts following the activation of calcineurin. The concentration of the component in the nucleus is also markedly increased following hippocampal LTP elicited by ginsenosides. These results suggest that the activation of calcineurin induces a preexisting protein component to translocate to the nucleus in the hippocampus. The nuclear translocation of the component may be required for LTP in the hippocampus.     

Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca²⁺-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

Effect of Ca on CO2 corrosion properties of X65 pipeline steel

The effect of Ca²⁺ on CO₂ corrosion to X65 pipeline steel was investigated in the simulated stratum water of an oil field containing different concentrations of Ca²⁺. It is found that Ca²⁺ can enhance the corrosion rate, especially in the Ca²⁺ concentration from 256 to 512 mg/L, which can be attributed to the growing grain size and loosing structure of corrosion scales with increasing Ca²⁺ concentration. X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) investigations reveal that a complex carbonate (Fe, Ca)CO₃ forms at high Ca²⁺ concentration due to the gradual replacement of Fe²⁺ in FeCO₃ by Ca²⁺.     

Effects of La3+ and Gd3+ on Ca2+ influx in rat hepatoma H-35 cells

Effects of La³⁺ and Gd³⁺ on Ca²⁺ influx were investigated in rat hepatoma H-35 cells by measuring the initial rate of⁴⁵Ca²⁺ uptake. It was found that the maximum initial rate of Ca²⁺ uptake was increased six-to ten-fold at low concentrations of La³⁺ and Gd³⁺. Kinetic analyses by measuring the initial rate of Ca²⁺ influx at different external Ca²⁺ concentrations indicated the existence of two intracellular exchangeable components in the basal Ca²⁺ system, with low and high affinities for Ca²⁺, and only one class of Ca²⁺ binding sites was observed in the La³⁺-or Gd³⁺-treated cells. For high affinity, La³⁺ and Gd³⁺ increased both kinetic parametersK _m andV _max of basai Ca²⁺ influx. La³⁺ and Gd³⁺ compete directly with Ca²⁺ for Ca²⁺ binding site for low affinity. The kinetics is competitive.     

Cardiac protective role of a novel erythrocyte-derived depressing factor on rats and its Ca^2＋ mechanism

The cardiac protective role of a novel erythro-cyte-derived depressing factor (EDDF) on spontaneous hy-pertensive rats (SHR), calcium overload (CaO) rats and Wistar rats and its mechanism was evaluated. Mean artery pressure (MAP), heart rate (HR) and LVdp/dtmax were measured by physiological recorder. The effect of EDDF on the Ca2+-ATPase activity in myocardial sarcoplasmic reticu-lum (SR) of CaO rats was determined by inorganic phos-phate assay. Calcium transport in myocytes was measured by 45Ca2+ radioactive isotope measurement. The phosphoryla-tion levels of extracellular signal-regulated protein kinases (ERK1/2) in myocardial tissue of SHR and CaO rats were measured by Western blot method. And the ultrastructures of cardiac muscle cells were observed with the transmission electron microscope. The results indicated that EDDF could significantly decrease MAP, HR and LVdp/dtmax in a dose dependent manner (P < 0.05). It seems that the mechanism might relate with activating the Ca2+-APTase, enhancing the uptake and release of Ca2+ from SR (P < 0.05), decreasing the phosphorylation levels of ERK1/2 of myocytes (P < 0.01) and lightening the ultrastructural lesion of cardiac muscle cells. In CaO rats, the Ca2+-ATPase activity decreased clearly com-pared to control (64.99 7.16 vs 94.48 7.68 nmol·min-1 ·mg-1 protein, P < 0.01), while EDDF (100 mg/mL) could significantly increase the activity (87.93 ?9.54 vs 64.99 ?7.16, P < 0.05, n = 7). Both uptake and release rate of Ca2+ (祄ol 45Ca2+/g protein/min) from myocardial SR of CaO rats re-markably decreased compared to control (32.40 ?2.70 and 15.46 ?1.49 vs 61.09 ?10.89 and 25.47 ?4.29, P < 0.05); EDDF (100 mg/mL) could significantly stimulate their activi-ties (50.48 6.76 and 21.76 2.75 vs 32.40 2.70 and 15.46 1.49, P < 0.05). EDDF could evidently down-regulate the phosphorylation of ERK1/2 in myocardial tissue from SHR and CaO rats (P < 0.01), lighten the ultrastructural lesion of cardiac muscle cells of SHR as well. It is concluded that EDDF seems to play protective roles on both structure and function of heart, which closely related with amelioration of Ca2+ transport and inhibition of Ca2+-MAP kinase pathway.     

Heat and hyposmotic stimulation increase in [Ca^2＋]i by Ca^2＋ influx in rat synoviocytes

Rheumatoid arthritis （RA）, which is marked by inflammatory synovitis, is a common, chronic autoimmune-disease, whose pathogenesis is complex and still unclear. In order to explore the effects of heat and hyposmotic stimuli on synoviocytes in rheumatoid arthritis, the changes of [Ca^2＋]i induced by heat, hyposmotic and 4α-PDD stimuli were observed in synoviocytes. [Ca^2＋]i elevation induced by heat 28℃, hyposmotic and 4α-PDD stimuli is found to be positively relative to increasing temperature, decreasing osmolality and rising concentration of 4α-PDD. Results show that there is reciprocity among these stimuli and desensitization, and that [Ca^2＋]i elevation depends on Ca^2＋ influx, but not necessarily links to Ca^2＋ release from intracellular stores and voltage-dependent Ca^2＋ channel in synoviocytes. The above characteristics of Ca^2＋ influx are similar to those of TRPV4. A probable mechanism has been suggested that heat and hyposmotic stimulation might increase the level of [Ca^2＋]i by activating the TRPV4-like channel and Ca^2＋ influx in the synoviocytes.