低浓度尼日利亚菌素或氯化铵促进ATP形成机理的再探

在低盐介质中,含有垛叠类囊体（基粒）与不垛叠类囊体（间质片层）结构的叶绿体进行的光系统Ｉ电子传递与磷酸化反应（ＰＳＰ）都为低浓度的尼日利亚菌素所促进,低浓度的氯化铵对基粒结构叶绿体的系统Ｉ与包括两个系统的电子传递以及与之相偶联的磷酸化反应有促进效应,而对时间质片偶联的磷酸反应有促进效应,尼日利亚菌素或氯化铵对ＰＳＰ的促进效应在高盐介质中消失,且它们对高能态（Ｚ）形成的抑制效应在低盐介质中较高盐介质     

低浓度尼日利亚菌素或氯化铵促进ATP形成机理的再探

在低盐介质中,含有垛叠类囊体（基粒）与不垛叠类囊体（间质片层）结构的叶绿体进行的光系统Ｉ电子传递与磷酸化反应（ＰＳＰ）都为低浓度的尼日利亚菌素所促进,低浓度的氯化铵对基粒结构叶绿体的系统Ｉ与包括两个系统的电子传递以及与之相偶联的磷酸化反应有促进效应,而对间质片层膜上进行的ＰＳＰ反应无影响。尼日利亚菌素或氯化铵对ＰＳＰ的促进效应在高盐介质中消失,且它们对高能态（Ｚ）形成的抑制效应在低盐介质中较高盐介质中大。当存在吡啶时,在低盐介质中它们对Ｚ形成的影响是抑制作用,在高盐介质中则是促进效应。这都表明不同结构的膜上均存在区域化质子。     

金霉素促进光合磷酸化机理的再探

在叶绿体经TPCK—trypsin光下修饰后,电子传递加速、磷酸化解联、膜上偶联因子Mg~(2+)—ATP酶活力促进的条件下,用金霉素处理叶绿体,能降低TPCK—trypsin 对磷酸化的解联程度,部分降低膜上Mg~(2+)—ATP酶的激活。在NEM及TPCK—trypsin共同存在时,金霉素处理仍能部分恢复磷酸化活力。进一步证明了金霉素是作用在偶联因子上的γ亚单位或其邻近部位,使之减少能量耗散而提高磷酸化活力.     

水稻白化苗质体中光合磷酸化偶联因子的存在

用聚丙烯酰胺凝胶电泳分离水稻白化苗质体EDTA提取物的蛋白,在偶联因子的位置有一带出现;白化苗质体具膜上(Mg~(++)—Ca~(++))—ATP酶活力,质体膜与菠菜类囊体残缺膜重组后有光合磷酸化活力。分部离心和电镜观察的结果表明,活力检测中所用的600～1500×g之间的沉降部分确属质体部分,证明白化苗质体中有光合磷酸化偶联因子的存在。     

阳离子及ATP对绿豆线粒体膨胀与收缩的影响

本文报道了一价阳离子 K~+、Na~+及两价阳离子 Mg~(++)、Ca~(++)以及 ATP 对绿豆线粒体膨胀和收缩的影响。K~+、Na~+在低渗条件下引起线粒体瞬时的迅速膨胀。在同样离子强度下K~+引起的膨胀大于 Na~+。ATP 和 Mg~(++)能诱发低渗条件下膨胀线粒体的收缩,但对等渗和高渗 KCl 或 Nacl 溶液中膨胀的线粒体无明显作用。生理浓度的 Mg~(++)、Ca~(++)在低渗条件下引起线粒体缓慢的但幅度较大的膨胀,5mmol/l ATP 引起这种膨胀线粒体的部分收缩。1mmol/lca~(++)在含0.125mmol/l KCl 或在含0.25mol/l甘露醇的等渗介质中几乎不引起膨胀,而ATP 促进大幅度膨胀,10mmol/l MgCl_2引起这种膨胀线粒体的部分收缩。2mmol/l MgCl_2在含有0.25mol/l 甘露醇的等渗介质中引起明显膨胀,ATP 促进这种膨胀。0.125mol/lKCl+2mmol/l MgCl_2为肌动蛋白从单体聚合成多聚体所必须的条件。在此条件下,线粒体几乎不膨胀,而加入 ATP 后则促进大幅度膨胀。在电子显微镜下观察了等渗及低渗条件下线粒体形态变化。     

光合磷酸化偶联机制研究——Ⅷ、6-苄氨基嘌呤对光合磷酸化的促进作用

在反应介质pH 7.2条件下,观察到6-BA处理叶绿体后,能促进循环(+PMS)和非循环(+FeCy或MV)的光合磷酸化反应,提高叶绿体的偶联程度,增加P/e_2比值。此促进现象与叶片的生理状态密切有关。在对光合磷酸化有促进作用的情况下,6-BA可改善类囊体的能化状态,增加高能态的积累。对光激活的叶绿体膜上Mg~(2+)-ATP酶和偶联因子热活化的ATP酶活力均表现出促进作用。6-BA的作用部位可能与膜上的偶联因子有关。     

叶绿体结构和功能的研究——Ⅳ、红霉素对叶绿体能量转换的效应

研究了红霉素对叶绿体能量转换的效应,获知:10(-4)M红霉素抑制循环和非循环光合磷酸化,这种抑制是与反应底物ADP非竞争性的。在抑制ATP合成的浓度范围内,红霉素对基础电子传递的速率并无影响,但它抑制因偶联磷酸化而促进的那部分电子传递。红霉素还抑制膜上Mg~(2 )-ATP酶的活性。以上结果表明,红霉素似有光合磷酸化能量传递抑制剂的特点,它的作用部位可能接近膜上CF_2,或ATP酶的催化部位。     

菠菜能量转化复合体的人工组装

从菠菜叶绿体中分离了H~+-ATP酶复合体,其SDS-聚丙烯酰胺凝胶电泳呈九条亚单位带,为纯度较高的酶复合体。将H~+-ATP酶复合体重组于人工膜(脂质体)上,表现出PiATP交换活力。Mg~(++)-ATP酶活力也明显提高。并表现对DCCD,寡霉素的敏感性。 重组H~+-ATP酶复合体表现有ATP诱导的H~+转移。但脂质体也出现类似现象,为此对以pH改变为标准的检测方法提出商榷。     

增甘膦(Glyphosine)和调节膦(Krenite)对叶绿体光化学反应的效应

用2～12 mM增甘膦处理叶绿体,其循环磷酸化活力均高于对照,经增甘膦处理的叶绿体,再用两倍体积的缓冲液洗涤两次并离心,所得叶绿体的磷酸化活力仍比对照的要高。对照叶绿体的非循环磷酸化速度在处理时的较高温下降低得很多,而处理的叶绿体的活力变化不大或略有上升。增甘膦处理的叶绿体,其非循环磷酸化活力和非循环磷酸化条件下的铁氰化钾还原活力均比对照的要高,故其磷/氧值维持不变或略有下降。在5×10~(-5)～5x10~(-3)M,调节膦促进铁氰化钾还原,抑制非循环磷酸化,表现出明显的解联效应。调节膦也抑制循环磷酸化。这种抑制是与反应底物非竞争性的。在抑制磷酸化的有效浓度范围内,调节膦也抑制膜上ATPase活性和光诱导的叶绿体的质子吸收。增甘膦能促进电子传递从而也促进光合磷酸化。调节膦则具有光台磷酸化的解联剂的特征。     

草甘膦对叶绿体能量转换的影响

除草剂草甘膦抑制植物叶绿体光合磷酸化活力,促进希尔反应活力,表现出明显的解偶联效应。它对叶绿体膜上腺三磷酶(ATPase)活力也起抑制效应,说明ATP合成被抑制不是由ATP酶活力变化所引起。这种解偶联现象主要是因光下质子转移受到抑制,在较低浓度的草甘膦影响下,先抑制质醌转移的质子进入膜内腔,浓度增加到20 mM,对水释放质子也有抑制。所以草甘膦对叶绿体能量转换的影响主要反映在质子转移被抑制,引起磷酸化活力受抑制。     

Functional effects of chemical modification of unstacked and stacked chloroplasts with glutaraldehyde.

Although glutaraldehyde alkylates protein NH₂ groups to the same extent in unstacked and stacked thylakoids, the photosynthetic electron transport of the stacked membranes is always more inhibited. Inhibition of photosystem II electron transport, measured in the presence of lipophilic Hill oxidants, is 20–30% in unstacked and 60–70% in stacked thylakoids. Photosystem I electron transport is nearly completely inhibited in both preparations, but in the case of stacked thylakoids maximal inhibition occurs at a lower glutaraldehyde level than in unstacked thylakoids. In contrast, the photooxidation of the reaction center chromophore of photosystem I (P700) is unaffected by the glutaraldehyde treatment of either stacked or unstacked chloroplasts. The results are discussed with regard to the accessibility of membrane sites to exogenous electron transport cofactors, in view of the observation that N-methylphenazonium methosulfate, a quencher of electronically excited chlorophyll a, partitions more easily into the pigment domains of the glutaraldehyde-fixed unstacked thylakoids.     

A study on the lateral distribution of the plastoquinone pool with respect to Photosystem II in stacked and unstacked spinach chloroplasts

The quenching of Photosystem II (PS II) chlorophyll fluorescence by oxidised plastoquinone has been used in an attempt to determine their relative distribution in the partition zone and stroma-exposed thylakoid membranes. Thus, the PS II-plastoquinone interaction was determined in stacked (2.5 mM MgCl₂) and largely unstacked (0.25 mM MgCl₂) membranes. A method to correct for spillover or other quenching changes at the different MgCl₂ concentrations, which would compete with the plastoquinone-induced quenching, was devised utilising the quinone dibromothymoquinone. This compound is demonstrated to behave as an ideal (theoretically) PS II quencher at both high and low MgCl₂ concentrations, which indicates that it distributes itself homogeneously between partition zone and stroma-exposed membrane regions. In passing from the stacked to the unstacked configuration, the PS II-plastoquinone interaction decreases less than the PS II-dibromothymoquinone interaction. This is interpreted to mean that plastoquinone is present in both the partition zone and stroma-exposed membranes, with somewhat higher concentrations in the stroma-exposed membranes. Thus, plastoquinone is well placed to transport reducing equivalents from the partition zones to the stroma-exposed membranes.     

Characterization of Energy-Transduction in Thermal Pretreated Chloroplast from Spinach

Characterization of energy-transduction on the chloroplast thylakoid membranes from spinach (Spinacia oleracea L.) after thermal pretreatment was investigated. The related reactions of energy-transduction in chloroplasts were seriously affected by thermal pretreatment. The results were obtained as following: (1) The rate of cyclic photophosphorylation declined when the pretreatment temperature increased in the range of 25 to 45 ℃. (2) The thermal pretreatment led to a decrease of the activity of thylakoid membrane-bounded ATPase. (3) Proton uptake of chloroplasts and the fluorescence quenching of 9-aminoacridine (9-AA) in thylakoid membrane decreased after the thermal pretreatment, but addition of dicyclohexylcarbodiimide (DCCD) could partially restore the fluorescence quenching of 9-AA. (4) Both the rates of fast phase in electrochroism absorption change at 515 nm and the millisecond delayed light emission (ms-DLE) of chloroplast showed a progressive decrease upon raising the temperature of pretreatment. (5) Immunbloting analysis showed that the thermal pretreatment caused the changes of protein content and the electrophoresis mobility of thylakoid membrane-bound ATPase and its α-subunit. (6) If the temperature of pretreatment were higher than 33 ℃, oxygen uptake of PSⅠ -mediated in the samples was rapidly inhibited, but addition of sinapine into the reaction medium could partially restore the ability of oxygen uptake in the samples. These results are briefly discussed in relation to the change of permeability of thylakoid membranes, the dissociation of coupling factor complex as well as accumulation of the radicals in the thylakoid membranes after thermal pretreatment.     

Golgi cisternal unstacking stimulates COPI vesicle budding and protein transport

The Golgi apparatus in mammalian cells is composed of flattened cisternae that are densely packed to form stacks. We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae. We established an assay to measure protein transport to the cell surface in post-mitotic cells in which the Golgi was unstacked. Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes. Vesicle budding from unstacked cisternae in vitro was significantly increased compared to stacked membranes. These results suggest that Golgi cisternal stacking can directly regulate vesicle formation and thus the rate of protein transport through the Golgi. The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.     

Photosystem II solubilizes as a monomer by mild detergent treatment of unstacked thylakoid membranes*

We studied the aggregation state of Photosystem II in stacked and unstacked thylakoid membranes from spinach after a quick and mild solubilization with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by analysis by diode-array-assisted gel filtration chromatography and electron microscopy. The results suggest that Photosystem II (PS II) isolates either as a paired, appressed membrane fragment or as a dimeric PS II-LHC II supercomplex upon mild solubilization of stacked thylakoid membranes or PS II grana membranes, but predominantly as a core monomer upon mild solubilization of unstacked thylakoid membranes. Analysis of paired grana membrane fragments reveals that the number of PS II dimers is strongly reduced in single membranes at the margins of the grana membrane fragments. We suggest that unstacking of thylakoid membranes results in a spontaneous disintegration of the PS II-LHC II supercomplexes into separated PS II core monomers and peripheral light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mg2+-ATPase activity in wheat root plasma membrane vesicles: Time-dependence and effect of sucrose and detergents

Purified plasma membrane vesicles were isolated in the presence of 250 mM sucrose from 7-day-old roots of Triticum aestivum L. cv. Drabant by aqueous polymer two-phase partitioning. When added to a low-salt medium containing 9-aminoacridine (9-AA), the vesicles caused a much larger total decrease in 9-AA fluorescence when sucrose was absent than when sucrose was present. A slow component of the decrease was also larger in the absence of sucrose. Triton X-100 reduced the decrease in 9-AA fluorescence upon vesicle addition and abolished completely the slow component of the decrease. There was no correlation between the time-dependence of 9-AA fluorescence and that of the Mg²⁺-ATPase described below. The time course of Mg²⁺-ATPase activity was followed by sampling at short intervals (down to 10 s) and analyzing for P, released. In the absence of detergent, the rates of P, release were linear from zero minutes, whether 250 mM sucrose was present or not, but the rate was 10^?50% higher in the absence of sucrose than in its presence. Sucrose (250 mM) added during a minus-sucrose assay lowered Mg²⁺-ATPase activity within 2 min to the level observed with 250 mM sucrose present from the start. The effect of 25-1 100 mM sucrose was tested and there was little or no effect below KM) mM. Above 100 mM sucrose the rate of P, release decreased drastically; at 1 100 mM sucrose the rate was ca 20% the rate at 25 mM sucrose. The inhibitory effect of sucrose was not alleviated by increased concentrations of Mg²⁺ and/or ATP. nor was it affected by the presence or absence of Triton X-100. We conclude that sucrose somehow inhibits the Mg²⁺-ATPase directly or affects the conformation of the plasma membrane in such a way as to inhibit the enzyme. The presence of detergents increased Mg²⁺-ATPase activity in the order Triton X-100 (4–5-fold) > Zwittergent 3–14 = Na-cholate = octylglucoside > digitonin (2-fold). In all cases optimal activity was observed at detergent concentrations at or below the critical micellar concentration. The detergent concentration curves could be simulated by the sum of a stimulatory and an inhibitory reaction. At the optimal concentration, digitonin gave a linear time-course of P, release, whereas all the other detergents showed a distinct lag of 1–3 min before maximal rates were attained. The problems of using detergents in polarity assays are discussed.     

Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro

Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.     

Lateral mobility of the light-harvesting complex in chloroplast membranes controls excitation energy distribution in higher plants

Chloroplast thylakoid protein phosphorylation produces changes in light-harvesting properties and in membrane structure as revealed by freeze-fracture electron microscopy. Protein phosphorylation resulted in an increase in the 77 °K fluorescence signal at 735 nm relative to that at 685 nm. In addition, a decrease in connectivity between Photosystem II centers (PS II) and a dynamic quenching of the room temperature variable fluorescence was observed upon phosphorylation. Accompanying these fluorescence changes was a 23% decrease in the amount of stacked membranes. Microscopic analyses indicated that 8.0-nm particles fracturing on the P-face moved from the stacked into the unstacked regions upon phosphorylation. The movement of the 8.0-nm particles was accompanied by the appearance of chlorophyll b and 25 to 29 kD polypeptides in isolated stroma lamellae fractions. We conclude that phosphorylation of a population of the light-harvesting chlorophyll $\text{a}\text{b}$ protein complexes (LHC) in grana partitions causes the migration of these pigment proteins from the PS II-rich appressed membranes into the Photosystem I (PS I) enriched unstacked regions. This increases the absorptive cross section of PS I. In addition, we suggest that the mobile population of LHC functions to interconnect PS II centers in grana partitions; removal of this population of LHC upon phosphorylation limits PS II → PS II energy transfer and thereby favors spillover of energy from PS II to PS I.     

Insights into the complex 3-D architecture of thylakoid membranes in the unicellular cyanobacterium Cyanothece sp. ATCC 51142

In cyanobacteria and chloroplasts, thylakoids are the complex internal membrane system where the light reactions of oxygenic photosynthesis occur. In plant chloroplasts, thylakoids are differentiated into a highly interconnected system of stacked grana and unstacked stroma membranes. In contrast, in cyanobacteria, the evolutionary progenitors of chloroplasts, thylakoids do not routinely form stacked and unstacked regions, and the architecture of the thylakoid membrane systems is only now being described in detail in these organisms. We used electron tomography to examine the thylakoid membrane systems in one cyanobacterium, Cyanothece sp. ATCC 51142. Our data showed that thylakoids form a complicated branched network with a rudimentary quasi-helical architecture in this organism. A well accepted helical model of grana-stroma architecture of plant thylakoids describes an organization in which stroma thylakoids wind around stacked granum in right-handed spirals. Here we present data showing that the simplified helical architecture in Cyanothece 51142 is lefthanded in nature. We propose a model comparing the thylakoid membranes in plants and this cyanobacterium in which the system in Cyanothece 51142 is composed of non-stacked membranes linked by fret-like connections to other membrane components of the system in a limited left-handed arrangement.Key words: cyanobacteria, Cyanothece 51142, thylakoid membrane, electron tomography, chloroplast     

The extent to which the spatial separation between photosystems I and II associated with granal formation limits noncyclic electron flow in isolated lettuce chloroplasts

Uncoupled noncyclic electron flow in stacked (granal) chloroplasts with a lateral heterogeneity in the distribution of the two photosystems has been compared with that in unstacked (agranal) chloroplasts with a near-uniform distribution. Chloroplasts were maintained in either structural state in the same assay medium so as to equalize effects of ionic composition which may influence reaction rates. The assay medium, an ion-deficient solution, was capable of supporting high rates of electron flow from water to methyl viologen. At high irradiance, unstacked chloroplasts exhibited an uncoupled rate which was 30% (in chloroplasts isolated from lettuce grown in low light) or 55% (in chloroplasts isolated from lettuce grown in high light) higher than that of stacked chloroplasts; the percentage remained relatively constant in the temperature range 7 to 22 degrees C for both high-light and low-light chloroplasts. At low irradiance, stacked low-light chloroplasts, despite the spatial separation of the two photosystems, gave higher rates of electron flow than did unstacked low-light chloroplasts. The addition of MgCl2 to stacked chloroplasts increased the uncoupled rate of noncyclic electron flow, but only at relatively high irradiances. The differences observed for stacked and unstacked chloroplasts, and for high-light and low-light chloroplasts are discussed. The approach taken in this work should be useful in other comparisons of stacked and unstacked chloroplasts.