氧化脂质及对大鼠心肌肌质网Ca~（2＋）－ATPase磷酸化微区运动状态的影响

用ＴＢＡ法测定了三尖杉酯碱的膜脂氧化效应;用纳秒荧光偏振技术研究了氧化膜脂对ＤＰＨ标记大鼠心肌肌质网膜脂、ＡＮＭ标记心肌肌质网Ｃａ２＋－ＡＴＰａ功能及磷酸化微区运动状态的影响。随膜脂中氧化磷脂的增加,肌质网膜脂双层的微粘度增加,磷脂分子摆动角减小：ＤＰＨ的荧光强度减弱,荧光寿命缩短。Ｃａ２＋－ＡＴＰａｓｅ的ＡＴＰ水解活性降低。ＡＮＭ标记Ｃａ２＋－ＡＴＰａｓｅ磷酸化微区的ｒ（ｔ）曲线半衰期减至６８±４ｎｓｅｃ。结果提示,膜脂中氧化磷脂的含量影响膜脂双层的流动性及Ｃａ２＋－ＡＴＰａｓｅ的ＡＴＰ水解活性和磷酸化微区的微细结构。     

消炎痛对猪胃H＋／K＋—ATP酶质子转运功能的抑制

作为猪胃Ｈ＋／Ｋ＋－ＡＴＰａｓｅ的非竞争性抑制剂,消炎痛明显抑制Ｈ＋／Ｋ＋－ＡＴＰａｓｅ泡囊的质子转运功能,造成质子泄漏。在０．１５ｍｇ／ｍｌ蛋白深度下,４％的消炎痛结合于Ｈ＋／Ｋ＋－ＡＴＰａｓｅ泡囊上。它能渗入膜脂相并显著降低膜的流动性。并使Ｈ＋／Ｋ＋－ＡＴＰａｓｅ内源荧光受到淬灭。从实验结果看来,消炎痛对猪胃Ｈ＋／Ｋ＋－ＡＴＰａｓｅ质子转运功能的抑制来自对酶蛋白和膜结构影响两个方面,而非仅抑制     

不同磷脂和糖脂对莱氏衣原体膜上Mg~（2＋）－ATPase活性的影响

莱氏衣原体膜上Ｍｇ~（２＋）－ＡＴＰａｓｅ用ＤＯＣ溶解后,经Ｓｅｐｈａｒｏｓｅ－６Ｂ和ＤＥＡＥ－ＣｅｌｌｕｌｏｓｅＤＥ－５２离子交换柱,得到了部分纯化的Ｍｇ~（２＋）ＡＴＰａｓｅ,并将此ＡＴＰａｓｅ与不同极性头部的磷脂和膜糖脂重组,研究了不同的极性头部的磷脂和膜糖脂对ＡＴＰａｓｅ活性的影响。此酶的活性不依赖酸性磷脂,ＰＧ、ＤＰＧ、大豆磷脂等明显抑制酶活性,中性磷脂ＤＭＰＣ、ＰＥ、ＰＣ则能增加酶活性,其中尤以非双层脂ＰＥ的作用最为明显。从莱氏衣原体膜上提取的糖脂（ＭＧＤＧ,ＤＧＤＧ）单独和ＡＴＰａｓｅ重组时,酶活性增加并不明显,当ＭＧＤＧ和ＤＧＤＧ以等比例混合时,能大大地增加酶活性。这表明Ｍｇ~（２＋）－ＡＴＰａｓｅ的活性很大程度上与磷脂的表面电荷及磷脂的组成相关。     

低pH对鲤鳃组织Na~ -K~ -ATPase酶活性的影响

Ｎａ Ｋ ＡＴＰａｓｅ酶几乎存在于所有动物的细胞中,是组成Ｎａ Ｋ 泵活性的主要部份,它不仅参与能量代谢、物质运送、氧化磷酸化的重要生化过程,而且它与膜上磷脂的结合状态,将影响膜的流动性,从而还会影响膜的其它功能。从人们发现ＤＤＴ对Ｎａ Ｋ ＡＴＰａｓｅ、ＭｇＡＴＰａｓｅ的抑制作用以来,广泛开展了多种污染物和生物体ＡＴＰａｓｅ之间关系的研究,并证实了有机农药、多氯联苯、金属、石油废水等污染物对ＡＴＰａｓｅ的影响。该研究国外已有不少的报道[1],而我国则报道甚少。特别是低ｐＨ对鲤鳃组织Ｎａ Ｋ ＡＴＰａｓｅ酶活性的…     

氧化脂质及对大鼠心肌肌质网Ca^2＋—ATPase磷酸化微…

用ＴＡＢ法测定了三尖杉酯碱的膜脂氧化效应;用纳秒荧光偏振技术研究了氧化膜脂对ＤＰＨ标记大鼠心肌肌质网膜脂、ＡＮＭ标记心肌肌质网Ｃａ＾２＋－ＡＴＰａ功能及磷酸化微区运动状态的影响。随膜脂中氧化磷脂的增加,肌质网膜脂双层的微粘度增加,磷脂分子摆动角减小;ＤＰＨ的荧光强度减弱,荧光寿命缩短。Ｃａ＾２＋－ＡＴＰａｓｅ的ＡＴＰ水解活性降低。ＡＮＭ标记Ｃａ＾２＋－ＡＴＰａｓｅ磷酸化微区的ｒ（ｔ）曲线半衰期减至     

跨膜Ca~(2+)梯差对大豆下胚轴质膜H~+-ATPase活力的影响

采用两相法得到高纯度封闭的大豆下胚轴质膜微囊,研究了跨膜Ｃａ２＋梯差对质膜Ｈ＋－ＡＴＰａｓｅ质子转运和ＡＴＰ水解活力的影响。结果表明,在１０００：０．１,１０００：０．５,１０００：１及１０００：１０（μｍｏｌ／Ｌ：μｍｏｌ／Ｌ）几种梯差下,随着跨膜钙梯差的减小,质膜Ｈ＋－ＡＴＰａｓｅ质子转运活力逐步降低。然而,上述几种梯差对Ｈ＋－ＡＴＰａｓｅ水解活力的影响却很小。进一步研究发现,１０００：０．１及１０００：１（μｍｏｌ／Ｌ：μｍｏｌ／Ｌ）两种梯差对Ｋｍ值没有影响,但Ｋ＋对Ｈ＋－ＡＴＰａｓｅ的激活作用在两种梯差下存在显著差别。ＭＣ５４０荧光、ＤＰＨ荧光偏振结果表明,跨膜钙梯差影响着膜脂的聚集状态和流动性。本文对跨膜Ｃａ２＋梯差对于大豆下胚轴质膜Ｈ＋－ＡＴＰａｓｅ水解与质子转运活力影响的可能机制进行了讨论。     

耐低钾水稻的根质膜ATPase和H^＋分泌特性

对耐低钾和不耐低钾的水稻（ＯｒｙｚａｓａｔｉｖａＬ．）“威优４９”和“远诱１号”的根原生质膜ＡＴＰａｓｅ性质的研究表明,这两种水稻的质膜ＡＴＰａｓｅ活性的最适ｐＨ均为６０,均在底物ＡＴＰ浓度为３ｍｍｏｌ／Ｌ时达到最大反应速度,Ｋｍ值均在０．８５左右。Ｋ＋对这两种水稻的质膜ＡＴＰａｓｅ活性均具有促进作用。当介质中［Ｋｍ］≤５０ｍｍｏｌ／Ｌ时随［Ｋ＋］的增大,对耐低钾品种根质膜ＡＴＰａｓｅ活性的促进作用明显大于不耐低钾品种;当介质［Ｋ＋］在１００～２００ｍｍｏｌ／Ｌ之间时,Ｋ＋对两种水稻品种质膜ＡＴＰａｓｅ活性的刺激效应的差别减小。这两个水稻品种的基础Ｈ＋分泌没有明显差别,但钾刺激的Ｈ＋分泌存在差异,Ｋ＋对耐低钾品种Ｈ＋分泌的刺激效应大于不耐低钾品种。抑制剂实验表明在耐低钾和不耐低钾的水稻品种中,Ｋ＋刺激的根质膜ＡＴＰａｓｅ活性,Ｋ＋刺激的Ｈ＋分泌和Ｋ＋吸收之间存在着紧密的联系。对Ｋ＋刺激的质膜ＡＴＰａｓｅ活性和Ｈ＋分泌的抑制会减少根对Ｋ＋的吸收量。推测耐低钾水稻的质膜ＡＴＰａｓｅ和Ｈ＋分泌对Ｋ＋更为敏感,特别是在低浓度Ｋ＋存在时,可能是耐低钾水稻更能利用低浓度Ｋ＋、在低钾环境中能更好生长的一个原因。     

植物细胞质膜H+-ATPase的结构与功能

植物细胞质膜Ｈ＋ＡＴＰａｓｅ属于Ｐ型质子泵。由该酶产生的跨膜电化学梯度是物质跨膜运输的原初动力。研究表明,质膜Ｈ＋ＡＴＰａｓｅ与植物的生长发育密切相关,被称为植物细胞的“主宰酶”。近年,关于该酶的生化特性,基因表达与调控以及结构与功能等方面的研究取得重要进展。对质膜Ｈ＋ＡＴＰａｓｅ的生化特性,分子结构,调节机制和生理功能等进行了综述     

牛磺酸对损伤的心肌肌膜ATPase活性影响的研究

本研究以异丙肾上腺素（Ｉｓｏｐｒｏｔｅｒｎｏｌ,Ｉｓｏ．）诱导的大鼠心肌损伤为模型,观察牛磺酸（Ｔａｕｒｉｎｅ,Ｔａｕ）对损伤心肌肌膜上Ｎ＿ａ￣＋—Ｋ＿－￣＋ＡＴＰａｓｅ、Ｃ＿ａ￣（２＋）－ＡＴＰａｓｅ、Ｍ＿ｇ￣（２＋）ＡＴＰＡＳＥ活性的影响。按Ｄａｈａｌｌａ方法分离及鉴定心肌肌膜同时分别测定三种ＡＴＰａｓｅ活性,结果：Ｉｓｏ组,与Ｔａｕ＋Ｉｓｏ组的三种ＡＴＰａｓｅ活性明显低于对照组与Ｔａｕ组,且同时Ｔａｕ＋Ｉｓｏ组明显高于Ｉｓｏ组但对照组与Ｔａｕ组之间差别无显著性。结果提示：牛磺酸能提高异丙肾上腺素诱导心肌损伤的心肌膜ＡＴＰａｓｅ活性,可能通过其保护心肌肌膜的结构及功能而实现的。     

低温处理对植物幼苗生长及根系ATPase同工酶的影响

过去所得的实验表明,植物中ＡＴＰａｓｅ活性变化与耐冷性有关。春小麦和番茄对低温是非常敏感的,当将这两种植物的幼苗在低温下培养时,质膜ＡＴＰａｓｅ活性下降甚至消失。相反,在冬小麦中,当幼苗在低温下处理时,质膜ＡＴＰａｓｅ活性增加．为了研究ＡＴＰａｓｅ同工酶变化与植物耐冷性之间的关系,以小麦等四种植物为材料,在０－１℃低温下处理２天、２周和４周,然后检测幼苗根系内的ＡＴＰａｓｅ同工酶,结果发现,在０－１℃培养时,幼苗根系内ＡＴＰａｓｅ同工酶谱带减少,ＡＴＰａｓｅ同工酶谱带变化与植物幼苗耐冷性呈一定相关性。ＡＴＰａｓｅ同工酶谱带变化可能是受冷害所致。     

High purity preparations of higher plant vacuolar H+-ATPase reveal additional subunits. Revised subunit composition

A fast protein liquid chromatography procedure for purification of the V-type H+-ATPase from higher plant vacuolar membrane to yield near-homogeneous enzyme with a specific activity of 20-25 mumol/mg.min is described. When precautions are taken to ensure the quantitative recovery of protein before sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the preparation is found to be constituted of seven major polypeptides of 100, 67, 55, 52, 44, 32, and 16 kDa, respectively, and two minor components of 42 and 29 kDa. The 52-, 44-, and 32-kDa polypeptides do not cross-react with antisera raised to the 67- and 55-kDa subunits of the enzyme, and two independent sample preparation procedures yield the same apparent subunit composition. The additional polypeptides are not breakdown products or aggregates of the previously identified subunits of the ATPase. The ATPase of tonoplast vesicles is subject to MgATP-dependent cold inactivation, and the conditions for inactivation are identical to those for the bovine chromaffin granule H+-ATPase (Moriyama, Y., and Nelson, N. (1989) J. Biol. Chem. 264, 3577-3582). Cold inactivation is accompanied by the detachment of five major polypeptides of 67, 55, 52, 44, and 32 kDa from the membrane, and all five components co-migrate with the corresponding polypeptides of the purified ATPase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 100- and 16-kDa polypeptides of the ATPase are not removed from the membrane during cold inactivation, but the latter can be purified to homogeneity by chloroform:methanol extraction of the fast protein liquid chromatography-purified enzyme. It is concluded that the tonoplast H+-ATPase is constituted of 6-7 major polypeptides organized into a peripheral sector comprising the 67-, 55-, 52-, 44-, and 32-kDa components and an integral sector consisting of the 100- and 16-kDa polypeptides. The V-type H+-ATPase from animal endomembranes and higher plant vacuolar membranes therefore have remarkably similar subunit compositions and gross topographies.     

Chloride-ion stimulation of the tonoplast H+-translocating ATPase from Hevea brasiliensis (rubber tree) latex. A dual mechanism.

The effect of Cl- and other anions on the tonoplast H+-translocating ATPase (H+-ATPase) from Hevea brasiliensis (rubber tree) latex was investigated. Cl- and other anions stimulated the ATPase activity of tightly sealed vesicles prepared from Hevea tonoplast, with the following decreasing order of effectiveness: Cl- greater than Br- greater than SO4(2-) greater than NO3-. As indicated by the changes of the protonmotive potential difference, anion stimulation of tonoplast H+-ATPase was caused in part by the ability of these anions to dissipate the electrical potential. This interpretation assumes not a channelling of these anions against a membrane potential, negative-inside, but a modification of the permeability of these ions through the tonoplast membrane. In addition, Cl- and the other anions stimulated the ATPase activity solubilized from the tonoplast membrane. Consequently, the tonoplast H+-pumping ATPase can be considered as an anion-stimulated enzyme. These results are discussed in relation to various models described in the literature for the microsomal H+-ATPase systems claimed as tonoplast entities.     

The Tonoplast H+-ATPase of Acer pseudoplatanus Is a Vacuolar-Type ATPase That Operates with a Phosphoenzyme Intermediate

The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After solubilization, the purification procedure included size-exclusion and ion-exchange chromatography. The H+-ATPase consists of at least eight subunits, of 95, 66, 56, 54, 40, 38, 31, and 16 kD, that did not cross-react with polyclonal antibodies raised to the plasmalemma ATPase of Arabidopsis thaliana. The 66-kD polypeptide cross-reacted with monoclonal antibodies raised to the 70-kD subunit of the vacuolar H+-ATPase of oat roots. The functional molecular size of the tonoplast H+-ATPase, analyzed in situ by radiation inactivation, was found to be around 400 kD. The 66-kD subunit of the tonoplast H+-ATPase was rapidly phosphorylated by [[gamma]-32P]ATP in vitro. The complete loss of radio-activity in the 66-kD subunit after a short pulse-chase experiment with unlabeled ATP reflected a rapid turnover, which characterizes a phosphorylated intermediate. Phosphoenzyme formed from ATP is an acylphosphate-type compound as shown by its sensitivity to hydroxylamine and alkaline pH. These results lead us to suggest that the tonoplast H+-ATPase of A. pseudoplatanus is a vacuolar-type ATPase that could operate with a plasmalemma-type ATPase catalytic mechanism.     

竹红菌甲素对红细胞膜上几种酶光敏失活作用的研究

本文比较了竹红菌甲素对人红细胞膜AchE,GPDH,Na~ -K~ ATPase和Ca~(2 )-Mg~(2 )ATPase的光敏失活能力,结果表明甲素对Ca~(2 )-Mg~(2 )ATPase作用最强,Na~ -K~ ATPase次之,GPDH再次之,AchE最不敏感,甲素还引起膜蛋白巯基氧化,膜脂质过氧化。其中,巯基氧化可能是ATPase光敏失活的主要原因,而脂质过氧化对ATPase活力损伤作用不大。游离GPDH不如与膜结合的GPDH敏感。GSH,NAD分别对ATPase,GPDH有保护作用。膜蛋白的电泳及内源荧光证据表明:在GPDH活力受到严重损伤时,酶结构并未发生剧烈改变。     

Probing the catalytic subunit of the tonoplast H+-ATPase from oat roots. Binding of 7-chloro-4-nitrobenzo-2-oxa-1,3,-diazole to the 72-kilodalton polypeptide

The purified tonoplast H+-ATPase from oat roots (Avena sativa L. var. Lang) consists of at least three different polypeptides with masses 72, 60, and 16 kDa. We have used covalent modifiers (inhibitors) and polyclonal antibodies to identify the catalytic subunit of the H+-pumping ATPase. The inactivation of ATPase activity by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Nbd-Cl, an adenine analog) was protected by MgATP or MgADP, and showed kinetic properties consistent with active site-directed inhibition. Under similar conditions, [14C]Nbd-Cl preferentially labeled the 72-kDa polypeptide of the purified ATPase. This binding was reduced by MgATP or 2' (3')-)O-(2,4,6-trinitrophenyl) ATP. Nbd-Cl probably modified cysteinyl--SH or tyrosyl--OH groups, as dithiothreitol reversed both ATPase inactivation and [14C]Nbd-Cl binding to the 72-kDa subunit. The finding that N-ethylmaleimide inhibition of ATPase activity was protectable by nucleotides is consistent with the idea of sulfhydryl groups in the ATP-binding site. Polyclonal antibody made to the 72-kDa polypeptide specifically reacted (Western blot) with a 72-kDa polypeptide from both tonoplast-enriched membranes and the purified tonoplast ATPase, but it did not cross-react with the mitochondrial or Escherichia coli F1-ATPase. The antibody inhibited tonoplast ATPase and H+-pumping activities. We conclude from these results that the 72-kDa polypeptide of the tonoplast H+-ATPase contains an ATP- (or nucleotide-) binding site that may constitute the catalytic domain.     

Properties of the partially purified tonoplast H+-pumping ATPase from oat roots

Higher plant cells have one or more vacuoles important for maintaining cell turgor and for the transport and storage of ions and metabolites. One driving force for solute transport across the vacuolar membrane (tonoplast) is provided by an ATP-dependent electrogenic H+ pump. The tonoplast H+-pumping ATPase from oat roots has been solubilized with Triton X-100 and purified 16-fold by Sepharose 4B chromatography. The partially purified enzyme was sensitive to the same inhibitors (N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, and NO-3) as the native membrane-bound enzyme. The partially purified enzyme was stimulated by Cl- (Km(app) = 1.0 mM) and hydrolyzed ATP with a Km(app) of 0.25 mM. Thus, the partially purified tonoplast ATPase has retained the properties of the native membrane-bound enzyme. [14C]DCCD labeled a single polypeptide (14-18 kDa) in the purified tonoplast ATPase preparation. Two major polypeptides, 72 and 60 kDa, that copurified with the ATPase activity and the 14-18-kDa DCCD-binding peptide are postulated to be subunits of a holoenzyme of 300-600 kDa (estimated by gel filtration). Despite several catalytic similarities with the mitochondrial H+-ATPase, the major polypeptides of the tonoplast ATPase differed in mass from the alpha and beta subunits (58 and 55 kDa) and the [14C] DCCD-binding proteolipid (8 kDa) of the oat F1F0-ATPase.     

Fibrin menbrane endowed with biological function. II. Chloroplast adenosine triphosphatase embedded in fibrin membrane.

Chloroplast ATPase (CF₁) was embedded in fibrin membrane, which is formed by fibrinogen-fibrin conversion in the presence of thrombin and is stabilized by blood coagulation factor XIII under physiological conditions. This immobilized chloroplast ATPase has the potent ATPase activity, 0.3 μmoles Pi/mm²/min and does not cause the cold inactivation of the enzymic activity.     

Does a proton-pumping ATPase exist in the tonoplast?

In order to account for the accumulation of metabolites in plant vacuoles, the existence of a proton-pumping ATPase has been widely suggested in the literature. The demonstration of such a tonoplast-bound ATPase was merely based on the characterization of a nitrate-sensitive microsomal fraction. In some examples, this ATPase activity has been evidenced on vacuole preparations obtained under conditions which were criticized by Boller. The application of the reverse phase high-performance liquid chromatography method (RP-HPLC) to the simultaneous separation of adenine nucleotides, in the presence of tonoplast vesicles isolated from Catharanthus roseus, showed results not necessarily correlated with the ATPase hypothesis. Moreover, in light of the H+-quenching of quinacrine fluorescence observed during ATP hydrolysis by vacuoles or tonoplast vesicles, the existence of a proton-pumping ATPase may be questioned.     

胡杨液泡膜微囊的纯化及其质子转运活性

 为进一步研究液泡膜及 H+ - ATP酶在胡杨抵御盐胁迫中所起的作用 ,比较了研磨、捣碎和超声破碎三种细胞破碎方法 ,从悬浮培养的胡杨细胞中制备液泡膜微囊的效果 ;并用差速离心和不连续蔗糖密度梯度离心纯化了胡杨液泡膜微囊 .通过测定 H+ - ATP酶对 NO-3 、VO3-4和 Na N3的敏感性 ,以及焦磷酸酶质子转运活性表明 ,液泡膜微囊主要分布在 0 %～ 2 5%的蔗糖界面上 .捣碎法破碎细胞结合差速离心和蔗糖密度梯度离心可获得正向微囊比例高、封闭性好和酶活性高的液泡膜微囊     

Purification of the N,N'-dicyclohexylcarbodiimide-binding proteolipid of a higher plant tonoplast H+-ATPase

The H+-ATPase of Beta vacuolar membrane (tonoplast) comprises at least three functionally distinct subunits of Mr = 67,000, 57,000, and 16,000, respectively (Manolson, M. F., Rea, P. A., and Poole, R. J. (1985) J. Biol. Chem. 260, 12273-12279). The hydrophobic carboxyl reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the enzyme with pseudo-first order kinetics, and the concentration dependence of the reaction indicates that DCCD interacts with a single site on the enzyme to exert its inhibitory effect. The apparent pseudo-first order rate constant (k0) is reciprocally dependent on membrane protein concentration, which is expected if a large fraction of the DCCD partitions into the lipid phase. k0 has a nominal value of 1000 M-1 min-1 at a protein concentration of 250 micrograms/ml, although when phase partitioning is taken into account, the true, protein concentration-independent value of k0 is calculated to be about an order of magnitude lower. [14C]DCCD primarily labels the Mr = 16,000 polypeptide of native tonoplast vesicles. Binding is venturicidin-insensitive and occurs at a rate similar to the rate of enzyme inactivation, implying that inhibition is a direct result of covalent modification of the Mr = 16,000 polypeptide. Labeling of the containing Mr = 8,000 subunit of mitochondrial F0F1-ATPase is, on the other hand, faster by a factor of 5 and totally abolished by venturicidin. These results confirm that the Mr = 16,000 polypeptide which copurifies with tonoplast H+-ATPase activity is a subunit of the enzyme. Most of the DCCD-reactive Mr = 16,000 subunit is extracted from acetone:ethanol-washed tonoplast vesicles by chloroform:methanol. [14C]DCCD bound to the Mr = 16,000 polypeptide is enriched in the chloroform:methanol extract by 5-fold compared with native tonoplast and the specific activity (nmol of [14C]DCCD/mg of protein) can be increased a further 37-fold by chromatography on DEAE-Sephadex. It is concluded that the Mr = 16,000 subunit of the tonoplast H+-ATPase is a proteolipid.