盐胁迫对玉米根尖质膜上受钙激活的蛋白激酶的影响

以02mal／L的NaCl对玉米根尖进行盐胁迫,导致质膜上一种受钙激活的蛋白激酶（该激酶表现为钙依赖蛋白激酶的特性）的活性迅速增加。盐胁迫至15min时激酶的活性达到最大,较对照高30％,以后逐渐降低,盐胁迫至切50min时激酶活性仍略高于对照。10％PEG6000处理玉米很尖也可诱导质膜上受钙激活的蛋白激酶活性增加。用外源ABA处理玉米根尖,未导致上述蛋白激酶活性的变化。放射自显影显示质膜上33kD和58kD两种蛋白可能是质膜受钙激活的蛋白激酶的内源底物。     

盐胁迫对玉米根尖质膜上受钙激活的蛋白激酶的影响

以０．２ｍｏｌ／Ｌ的ＮａＣｌ对玉米根尖进行盐腔迫,导致质膜上一种受钙激活的蛋白激酶的活必迅速增加。盐腔迫至１５ｍｉｎ时激酶的活性达到最大,较对照高３０％,以后逐渐降低,盐胁迫至５０ｍｉｎ时激酶活性仍略高于对照。     

两相分配法制备玉米根质膜及其纯度鉴定

用Ｄｅｘｔｒａｎ Ｔ５００,ＰＥＧ３３５０两相体系制备玉米根质膜,首先在高盐浓度下选用五种不同的聚合物浓度,研究了玉米根系膜在两相体系中的分配情况,在此基础上研究了ＮａＣｌ浓度对玉米根质膜的纯度及得率的影响。结果表明,制备了玉米根质膜选用６．２％聚合物浓度,７．５ｍｍｏｌ／Ｌ ＮａＣｌ的两相体系比较合适。     

两相分配法制备玉米根质膜及其纯度鉴定

用ＤｅｘｔｒａｎＴ５００,ＰＥＧ３３５０两相体系制备玉米根质膜．首先在高盐浓度（２２ｍｍｏｌ／ＬＮａＣｌ）下选用五种不同的聚合物浓度（５．８％、６．０％、６．２％、６．３％、６．４％,Ｗ／Ｗ）,研究了玉米根质膜在两相体系中的分配情况,在此基础上进一步研究了Ｎａ－Ｃｌ浓度（２、４、５、１１、２２ｍｍｏｌ／Ｌ）对玉米根质膜的纯度及得率的影响．结果表明,制备玉米根质膜选用６．２％（Ｗ／Ｗ）聚合物浓度,７．５ｍｍｏｌ／ＬＮａＣｌ的两相体系比较合适．标志酶鉴定及低ｐＨ值磷钨酸染色电镜检测均表明获得了高纯度密实的正向型的质膜囊泡,质膜标志酶ＶＯ３－４－ＡＴＰａｓｅ的活性潜势达８８．９％．     

玉米细胞两种质膜囊泡的制备和鉴别

细胞质膜是细胞进行物质交换、能量转换及信息传递等重要生命活动的关键部位,现已成为细胞生物学、植物生理学和生物化学的重要研究领域。但目前对植物细胞质膜。：研究远远落后于对其它材料质膜的研究,其中一个重要原因就是制备高度纯化的具有特定朝向的密实质股囊泡比较困难,而不同的研究目的分别需要正向型（ROV）和内侧外翻型（IOV）两种质膜囊泡。我们在前人方法基础上加以改进,成功地制备了基本上为两种取向的密实质膜囊泡,并已用于质膜十”一泵和铁氰化钾还原酶的研究［’j。材料与方法1材料玉米（he＿）农大65的种子。奎叮…     

钙依赖蛋白激酶（CDPKs）在植物钙信号转导中的作用

CDPKs在植物钙信号转导中起重要作用。本文介绍了植物钙信号转导及CDPKs的结构与生化性质,在此基础上,重点总结了CDPKs在植物钙信号转导中的潜在调节作用,包括基因表达、代谢、离子和水分的跨膜运输、细胞骨架的动态变化、气孔运动和生长发育等,并提出了在CDPKs研究中已达成的共识和需要解决的问题。     

铝和铝＋钙对小麦幼苗根尖质膜,液泡膜微囊ATP酶和膜流动性？…

研究了铝和铝＋＿钙对小麦功苗根尖质膜、液泡膜微囊Ｈ＾＋－ＡＴＰ酶、Ｃａ＾２＋－ＡＴＰ酶、Ｍｇ＾２＋－ＡＴＰ酶活性及共动力学参数和膜流动性的影响。在质膜和液泡膜微囊制剂中加入１．０ｍｍｏｌ／Ｌ的ＡＩ＾３＋（ＡＩＣＩ３）时,Ｈ＾２＋－ＡＴＰ囊制剂中加入１．０ｍｍｏｌ／Ｌ的ＡＩ＾３＋（ＡＩＣＩ３）时,Ｈ＾＋－ＡＴＰ酶、Ｃａ＾２＋－ＡＴＰ酶、Ｍｇ＾２＋－ＡＴＰ酶活笥和酶促反应的Ｖｍａｘ及膜流动性下降,而酶     

两相法分离玉米幼苗叶片生长部位质膜

以玉米幼叶生长部位为材料,通过水溶性多聚物ＰＥＧ４０００／Ｄｅｘｔｒａｎ Ｔ５００构成的两相系统,对玉米幼叶生长部位质膜进行了分离。结果表明,由ＰＥＧ４０００６．２％、Ｄｅｘｔｒａｎ Ｔ５００６．３％,ＫＣｌ５ｍｍｏｌ／Ｌ、磷酸缓冲液ｐＨ７．８４ｍｍｏｌ／Ｌ、蔗糖８．５％构成后两相系统最适宜分离玉米幼叶生长部位质膜。经过三次相分配后,质膜上相中的分配率达８５％以上。经－０．４ＭＰａ胁迫处理２４ｈ后     

大豆叶片质膜蛋白激酶的自身磷酸化反应

利用Ｆｅｒｒｅｌｌ和Ｍａｒｔｉｎ设计的测定印迹在ＰＶＤＦ膜上的蛋白激酶活性方法研究大豆叶片质膜蛋白激酶自身磷酸化反应活性,结果表明：与Ｍｇ－ＡＴＰ相比,Ｍｎ－ＡＴＰ是更有效的５７ｋＤ蛋白激酶自身磷酸化反应底物;钙离子可以促进该激酶的自身磷酸化反应活性,而且ＥＧＴＡ可以显著降低它在ＳＤＳ电泳中的迁移率,说明５７ｋＤ蛋白激酶为依赖于钙的蛋白激酶;     

Characteristics of Calcium activated Protein Kinase in the Plasma Membrane of Corn Root Tip Cells

Plasma membrane (PM) of high purity was prepared through a two-phase aqueous system from roots of 3 ～ 4 d old com ( Zea mays L. ) seedling, and within which calcium-activated protein kinase was identified. The kinase was mainly located in the inner side of the PM with a semi-activation concentration to free calcium ion of 50 μmol/L. Exogenous CaM had no agonistic effect on the protein kinase but CaM antagonist TFP could strongly inhibit its activity with a semi-inhibition concentration of 75 μmol/L. The protein kinase showed high specificity for the exogenous substrate, Histone Ⅲ S. These results indicated that this protein kinase could be a calcium-dependent and calmodulin-independent protein kinase (CDPK). Moreover, two proteins (33 kD and 58 kD) in PM might be the endogenous substrates of the CDPK.     

玉米根尖细胞液泡膜结合的蛋白激酶的存在及其性质

为了解液泡膜蛋白在植物细胞信号途径中的功能 ,用新型的非放射性同位素方法从玉米根细胞的高纯度液泡膜上鉴定出一种膜内在的蛋白激酶。这种蛋白激酶具有Ca2 依赖、CaM和磷脂酰丝氨酸不依赖等特性 ,与已在多种植物中报道的含有类似钙调素结构域的蛋白激酶CDPK相似。离体实验表明其活性的最适pH值为 6 .5 ,最适Ca2 浓度为 1 0 μmol/L。从最适pH值和去污剂的影响可以推测出其活性位点朝向胞质一侧。Zn2 对其活性没有明显的抑制作用 ,说明该激酶缺少某些哺乳动物的蛋白激酶常含有的锌指结构。当液泡膜蛋白在Ca2 和ATP存在的条件下被预磷酸化后 ,液泡膜H _ATPase的ATP水解和质子转运过程均被激活。激活的活性可以被碱性磷酸酶逆转。以上结果说明玉米根尖细胞的液泡膜中存在一种可能是CDPK的蛋白激酶。由它造成的Ca2 依赖的磷酸化作用激活了液泡膜H _ATPase的活性。这些结果将有助于深入研究CDPK在植物细胞信号转导中的功能。     

Identification of the Plasma Membrane Proteolipid Protein as a Constituent of Brain Coated Vesicles and Synaptic Plasma Membrane

We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S-1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes proteolipid adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1-2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.     

癌细胞膜流动性与蛋白激酶C相关性的初步探讨

细胞膜流动性是细胞的重要物理性质,与细胞功能密切相关。为深入认识细胞膜与细胞功能的关系,研究癌症机理及寻找预防治疗的方法提供新的视角和实验数据,采用荧光漂白后恢复技术检测皮肤癌A431细胞和正常HACAT细胞的膜流动性,同时测定蛋白激酶C的活性及mRNA的表达。通过激光扫描共聚焦显微镜的荧光恢复曲线计算得知A431细胞的荧光恢复率和扩散系数均低于HACAT细胞,即A431细胞膜流动性低于HACAT细胞;而蛋白激酶C活性、mRNA表达量却高于正常细胞。以上差异都有统计学意义（P〈0．05）。由此推断癌细胞膜流动性与蛋白激酶C相互作用、相互影响,彼此之间存在密切的关系。     

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 microM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin-treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin-treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 microM reserpine, 30 microM NH4+, or 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP-induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules with digitonin-treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 microM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin-treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane-bound dopamine-beta-hydroxylase. The studies demonstrate that 20 microM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.     

大豆叶片质膜蛋白激酶的自身磷酸化反应

利用Ferrell和Martin（1991）设计的测定印迹在PVDF膜上的蛋白激酶活性方法研究大豆叶片质膜蛋白激酶自身磷酸化反应活性,结果表明：与Mg－ATP相比,Mn－ATP是更有效的57KD蛋白激酶自身磷酸化反应底物;钙离子可以促进该激酶的自身磷酸化反应活性,而且EGTA可以显著降低它在SDS电泳中的迁移率,说明57KD蛋白激酶为依赖于钙的蛋白激酶;预磷酸化反应实验证明57KD蛋白激酶具有多个自身磷酸化反应位点,其分子的自身磷酸化状态可调性暗示这一激酶可能具有重要的生理功能。     

Calmodulin Modulates Mitogen-Activated Protein Kinase Activation in Response to Membrane Depolarization in PC12 Cells

Abstract: In the absence of neurotrophic factors, chronic depolarization of plasma membrane has been shown to maintain several populations of primary neurons in culture. We report that in the PC12 cell line, depolarization causes Ca²⁺ influx through voltage-gated Ca²⁺ channels, which is able to stimulate extracellular-regulated kinase (ERK) activity. We studied which mediators were responsible for ERK activation resulting from increased levels of Ca²⁺ in the cytoplasm and found that calmodulin was involved in this process. The addition of W13, a calmodulin inhibitor, to the culture medium, prevented ERK activation when PC12 cells were depolarized. In addition, we show that high K⁺ treatment did not induce Trk A phosphorylation, thus excluding the possibility of Ca²⁺ operating through this receptor to activate the ERK signal transduction pathway. Moreover, although high K⁺ treatment is able to phosphorylate the epidermal growth factor receptor (EGFR) and thus to activate the ERK signal transduction pathway, we demonstrate that W13 did not alter the state of EGFR phosphorylation in conditions that almost completely blocked ERK activation. These data suggest that calmodulin mediates ERK activation induced by increases in intracellular Ca²⁺ concentration in PC12 cells by a mechanism that seems to be independent of Trk A and EGFR activation.     

Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin by Cytoskeletal-Associated Intermediate Filament Protein Kinase Activity in Astrocytes

These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

A 67-kDa plasma-membrane-bound Ca2+-stimulated protein kinase active in sink tissue of higher plants

A novel 67-kDa protein kinase (p67 ^cdpk ) was identified in the microsomal membrane fraction of apple (Malus domestica Borkh. cv. Braeburn) suspension cultures and subsequently found to be active in sink tissues. Microsomal proteins were blotted onto Nylon or polyvinylidenedifluoride membranes, and p67 ^cdpk assayed by in situ-labelling renatured proteins with [γ-³²P]ATP; thin-layer electrophoresis/thin-layer chromatography of acid hydrolysates of the ³²P-labelled protein band indicated that serine and threonine, but not tyrosine residues were phosphorylated. A detailed analysis of the ion-dependency of p67 ^cdpk revealed that it was a Ca²⁺-stimulated, Mg²⁺-dependent protein kinase. However, p67 ^cdpk was ten times more active in the presence of 10 mM Mn²⁺, and these assay conditions were used routinely to increase the sensitivity of the assay. Activity of p67 ^cdpk was found at high levels in the plasma membrane, and solubilisation experiments with a number of detergents suggested that p67 ^cdpk is an integral membrane protein. A homologous protein kinase with similar biochemical properties was also present in cell-suspension cultures of pear and maize. In maize (Zea mays L.) plants, sink tissues, such as young expanding leaves of both light-grown and etiolated plants, mature etiolated tissue and roots all had high levels of p67 ^cdpk activity. However, mature light-grown (source) tissues had barely detectable levels. In etiolated maize leaves and coleoptiles the kinase activity was highest in expanding tissue and decreased as the cells expanded. When etiolated maize plants were exposed to light, the activity of p67 ^cdpk was reduced to background levels after 8 h. Although p67 ^cdpk has biochemical properties similar to those of other plant calcium-dependent protein kinases, this is the first identification of a membrane-bound calcium-dependent protein kinase which is specifically active in sink tissues. Received: 14 July 1997 / Accepted: 25 September 1997