光对花生叶片绿色细胞糖酵解代谢的影响

光下花生叶肉细胞悬浮液暗呼吸只有暗中的18％左右,丙酮酸含量下降,细胞质磷酸丙糖积累,叶绿体3—磷酸甘油醛脱氢酶活性上升,而非叶绿体的酶活性下降,叶绿体和细胞质的ATP/ADP比值同时增加。ATP/ADP>1时离体细胞质3—磷酸甘油醛脱氢酶活性下降,但叶绿体的酶不受影响。表明光下ATP/ADP比值上升影响细胞质3—磷酸甘油醛脱氢酶活性而使糖酵解受抑制。     

外源ATP对油菜幼苗耐寒性的影响

以“陇油7号”油菜为实验材料,研究了外源ATP对油菜幼苗耐寒性的影响。结果表明:与单独低温胁迫相比,外源ATP预处理再进行低温胁迫后,油菜幼苗MDA含量、O₂^-.含量均显著降低,而叶绿素含量、抗氧化酶活性(SOD、POD、CAT、APX)和RBOHD、RBOHF、CPK4、CPK5基因表达均增加;与外源ATP+低温相比,EGTA+外源ATP+低温处理下,MDA含量显著增加,总叶绿素含量、T-AOC酶活性、RBOHD、RBOHF基因表达均显著下降,DMTU+外源ATP+低温处理下,MDA含量显著增加,总叶绿素含量、Ca²⁺-ATPase酶活性、CPK4、CPK5基因表达均下降,表明外源ATP通过Ca²⁺和H₂O₂依赖性机制影响油菜幼苗的耐寒性。     

气体甲醛胁迫对蚕豆保卫细胞中过氧化氢的积累和气孔导度及开度的影响

以蚕豆为材料,考察气体甲醛（HCHO）胁迫对保卫细胞H2O2积累和叶片气孔导度、开度的影响。结果表明:气体HCHO胁迫增加了叶片中H₂O₂的积累,荧光显微分析发现在较低浓度(0.2⁰.4μmol·L^-1)气体HCHO胁迫下,保卫细胞中增加的H₂O₂主要分布在细胞质中,高浓度(0.8¹.6μmol·L^-1)气体HCHO胁迫不仅增加保卫细胞质中H₂O₂的积累,而且显著增加叶绿体中H₂O₂的含量及积累H₂O₂的叶绿体数量,这说明在高浓度气体HCHO胁迫下蚕豆保卫细胞中增加的H₂O₂主要来源于叶绿体和细胞质。保卫细胞中H₂O₂积累的增加显著降低蚕豆的气孔导度和开度,从而导致蚕豆HCHO吸收效率下降。气体HCHO胁迫下叶片中抗氧化酶活性的变化可能是H₂O₂积累增加的主要原因,气体HCHO胁迫显著增强叶片中CAT和SOD的活性,但只有低浓度HCHO胁迫诱导叶片POD活性,叶片APX对HCHO胁迫很敏感,低浓度的气体HCHO对叶片APX活性都有显著的抑制作用。     

小波变换及滚球算法在单分子荧光能量共振转移图像分析中的应用（英文）

单分子荧光共振能量转移技术是通过检测单个分子内的荧光供体及受体间荧光能量转移的效率来研究分子构象的变化.要得到这些生物大分子的信息就需要对大量的单分子信号进行统计分析,人工分析这些信息,既费时费力又不具备客观性和可重复性,因此本文将小波变换及滚球算法应用到单分子荧光能量共振转移图像中对单分子信号进行统计分析.在保证准确检测到单分子信号的前提下,文章对滚球算法和小波变换算法处理图像后的线性进行了分析,结果表明,滚球算法和小波变换算法不但能够很好地去除单分子FRET图像的背景噪声,同时还能很好地保持单分子荧光信号的线性.最后本文还利用滚球算法处理单分子FRET图像及统计15 bp DNA的FRET效率的直方图,通过计算得到了15 bp DNA的FRET效率值.     

外源γ-氨基丁酸调控甜瓜叶绿体活性氧代谢应对短期盐碱胁迫

以甜瓜品种‘金辉1号’为试材,采用深液流水培法,研究外源γ 氨基丁酸(GABA)对短期盐碱胁迫下甜瓜幼苗叶绿体活性氧代谢的调控作用.结果表明: 盐碱胁迫显著提高了甜瓜叶绿体内光合色素、丙二醛(MDA)和过氧化氢(H₂O₂)含量及超氧阴离子(O^-_·2)产生速率;增加抗坏血酸(AsA)和谷胱甘肽(GSH)等抗氧化物质含量;明显抑制H⁺-ATP酶(H⁺-ATPase)和H⁺ 焦磷酸酶(H⁺-PPiase)活性.外源叶面喷施GABA有效抑制了盐碱胁迫引起的叶绿体内O^-_·2、H₂O₂和MDA的积累,缓解了光合色素增加的趋势;显著提高SOD和AsA GSH循环各个酶的活性,增加了AsA和GSH库,降低了AsA/DHA和GSH/GSSH比值,增强了H⁺-ATPase和H⁺-PPiase 活性.表明外源GABA能加快叶绿体内活性氧代谢,促进AsA-GSH循环的运转,维持细胞膜的渗透性,进而缓解了盐碱胁迫引起的氧化伤害.     

植物中的气体信号分子硫化氢：无香而立，其臭如兰

硫化氢（H₂S）一直被认为是一种有毒气体,作为第三种气体信号分子,H₂S在生物体中的生理功能逐渐被揭示。植物中H2S信号研究在不到10年时间已取得了长足进步。植物体内H2S的生成酶比动物细胞丰富,定位于细胞质、线粒体和叶绿体等多个亚细胞部位,表达具有时空性。目前,植物领域H₂S的功能研究主要采用药理学方法。随着研究的深入,遗传学证据不断加强。内源H₂S的研究手段也在不断进步,从亚甲基蓝间接测定,发展到气/液相色谱、荧光探针、活体电极等直接检测手段。植物中H₂S的生理功能研究主要集中在对干旱、重金属等环境非生物胁迫的缓解作用及机理,也有一些植物生长发育调控方面的报道。目前了解到,H₂S可通过与植物激素、其它气体信号分子、活性氧等相互作用以及蛋白质巯基化修饰等方式发挥生理功能。虽然植物气体信号的研究有其特殊性,也遇到很多困难,但是H₂S信号的广泛而特殊的生理功能是一个具有重要科学意义和应用前景的研究领域。     

小波变换及滚球算法在单分子荧光能量共振转移图像分析中的应用

单分子荧光共振能量转移技术是通过检测单个分子内的荧光供体及受体间荧光能量转移的效率来研究分子构象的变化．要得到这些生物大分子的信息就需要对大量的单分子信号进行统计分析,人工分析这些信息,既费时费力又不具备客观性和可重复性,因此本文将小波变换及滚球算法应用到单分子荧光能量共振转移图像中对单分子信号进行统计分析．在保证准确检测到单分子信号的前提下,文章对滚球算法和小波变换算法处理图像后的线性进行了分析,结果表明,滚球算法和小波变换算法不但能够很好地去除单分子FRET图像的背景噪声,同时还能很好地保持单分子荧光信号的线性．最后本文还利用滚球算法处理单分子FRET图像及统计15 bp DNA的FRET效率的直方图,通过计算得到了15 bp DNA的FRET效率值．     

外源ATP对盐胁迫下油菜幼苗生长的影响

研究了外源ATP处理对盐胁迫下油菜幼苗生长的影响,探讨了过氧化氢(H₂O₂)和钙离子(Ca²⁺)作为信号分子在ATP对油菜幼苗耐盐性调控过程中的作用。结果表明:与单独Na Cl处理相比,ATP+Na Cl处理降低了油菜幼苗死细胞数量、ROS(■和H₂O₂)含量、离子(Ca²⁺、Na⁺、Cl^-)含量、MDA含量及Na⁺/K⁺比和相对电导率,增加了叶片中叶绿素、脯氨酸、可溶性糖含量和抗氧化酶(SOD、POD、CAT、APX)活性,提高了抗氧化酶基因(CAT、SOD、APX、GR)、NADPH氧化酶基因(RBOHD、RBOHF)、P5CS1基因、MAPK激酶基因(MAPK3、MAPK6)、耐盐基因(NHX1、SOS1)转录;与ATP+Na Cl处理相比,ATP+Na Cl+抑制剂(DPI、DMTU和EGTA)处理下油菜幼苗中相对电导率、MDA、叶绿素、脯氨酸、可溶性糖含量和抗...     

荧光共振能量转移效率的实时定量测量

荧光共振能量转移（FRET）广泛用于研究分子间的距离及其相互作用,与荧光显微镜结合,可定量获取有关生物活体内蛋白质、脂类、DNA和RNA的时空信息。随着绿色荧光蛋白（GFP）的发展,FRET荧光显微镜有可能实时测量活体细胞内分子的动态性质。提出了一种定量测量FRET效率以及供体与受体间距离的简单方法,仅需使用一组滤光片和测量一个比值,利用供体和受体的发射谱肖除光谱间的串扰。该方法简单快速,可实时定量测量FRET的效率和供体与受体间的距离,尤其适用于基于GFP的供体－受体对。     

植物抗坏血酸过氧化物酶的表达调控以及对非生物胁迫的耐受作用

抗坏血酸过氧化物酶(Ascorbate peroxidase, APX)属于I型血红素过氧化物酶, 它催化H2O2依赖的L-抗坏血酸氧化作用, 对抗坏血酸表现出高度的专一性。植物APX基因家族由4个亚家族组成, 分别为细胞质、叶绿体、线粒体和过氧化物酶体基因亚家族, 每个亚家族中又含有不同的APX同工酶。作为植物抗坏血酸-谷胱甘肽循环中的一个关键组分, APX在细胞H₂O₂代谢过程中起着至关重要的作用。研究表明植物APX是氧化还原信号系统中调节细胞水平H₂O₂非常重要的一种酶, APX同工酶的表达机制非常复杂, 细胞质APX受多种信号调节表达, 两种叶绿体APX通过选择性剪接进行组织特异性调节。通过调控产生的APX可调节细胞中的氧化还原信号, 进而提高植物对非生物胁迫的耐受性。文章综述了植物APX的催化机制、表达调控机理以及响应植物非生物逆境胁迫的重要作用。     

Dynamic ligand-induced conformational rearrangements in P-glycoprotein as probed by fluorescence resonance energy transfer spectroscopy

P-glycoprotein (Pgp), a member of the ATP-binding cassette transporter family, functions as an ATP hydrolysis-driven efflux pump to rid the cell of toxic organic compounds, including a variety of drugs used in anticancer chemotherapy. Here, we used fluorescence resonance energy transfer (FRET) spectroscopy to delineate the structural rearrangements the two nucleotide binding domains (NBDs) are undergoing during the catalytic cycle. Pairs of cysteines were introduced into equivalent regions in the N- and C-terminal NBDs for labeling with fluorescent dyes for ensemble and single-molecule FRET spectroscopy. In the ensemble FRET, a decrease of the donor to acceptor (D/A) ratio was observed upon addition of drug and ATP. Vanadate trapping further decreased the D/A ratio, indicating close association of the two NBDs. One of the cysteine mutants was further analyzed using confocal single-molecule FRET spectroscopy. Single Pgp molecules showed fast fluctuations of the FRET efficiencies, indicating movements of the NBDs on a time scale of 10-100 ms. Populations of low, medium, and high FRET efficiencies were observed during drug-stimulated MgATP hydrolysis, suggesting the presence of at least three major conformations of the NBDs during catalysis. Under conditions of vanadate trapping, most molecules displayed high FRET efficiency states, whereas with cyclosporin, more molecules showed low FRET efficiency. Different dwell times of the FRET states were found for the distinct biochemical conditions, with the fastest movements during active turnover. The FRET spectroscopy observations are discussed in context of a model of the catalytic mechanism of Pgp.     

Polarized light spectroscopy of photosynthetic membranes in magneto-oriented whole cells and chloroplasts. Fluorescence and dichroism

1. The wavelength dependence of the fluorescence polarization (FP) ratio and dichroism has been studied with magneto-oriented (10–13 kG) whole cells of Chlorella pyrenoidosa, Scenedesmus obliquus, Euglena gracilis and spinach chloroplasts suspended in their aqueous growth media (or Tris-buffered sucrose solution in the case of the chloroplasts) under physiological conditions. The FP ratio is defined as the fluorescence intensity polarized parallel divided by the intensity polarized perpendicular to the membrane planes.

2. The FP ratio is typically in the range of 1.2–1.9 in Chlorella, 1.20–1.25 in Scenedesmus and 1.4–1.5 in spinach chloroplasts at fluorescence wavelengths above 690 nm. Below 690 nm the FP ratio decreases steadily with decreasing wavelength and may be as low as approx. 1.05 at 660 nm. These results are interpreted in terms of the orientation of the Q_y transition moment vectors of the different spectroscopic forms of chlorophyll. For the chlorophyll a 680 form these vectors are inclined at angles of 30° or less (in Chlorella) with respect to the membrane planes, while the shorter wavelength chlorophyll a 670 forms appear to be not nearly as well oriented.

3. The Euglena fluorescence peak is red shifted to 714 nm (in the other algae and chloroplasts it is situated at 685 nm) and the FP ratio is approx. 1.20 in the 720–730 nm region and decreases with decreasing wavelength below 720 nm and is only 1.05 at 690 nm. This wavelength dependence is in good qualitative agreement with the fluorescence microscope studies of single chloroplasts of Euglena by Olson, R. A., Butler, W. H. and Jennings, W. H. ((1961) Biochim. Biophys. Acta, 54, 615–617).

4. By means of a model calculation it is shown that the high FP ratios observed with Chlorella are entirely consistent with the low values of the degree of polarization (0.01–0.06) determined by previous workers with unoriented cell suspensions.

5. The influence of reabsorption and the resulting distortion in the wavelength dependence of the FP ratio are described. The possibility that the fluorescence is polarized by scattering artifacts, rather than being a result of the intrinsic orientation of chlorophyll, is considered.

6. Linear dichroism studies with Chlorella and spinach chloroplasts confirm the orientation of the Q_y transition moment vectors deduced from the FP ratio. Furthermore, it appears that the porphyrin rings are tilted out of the membrane plane and that the carotenoid molecules tend to lie with their long axes in the lamellar plane.

7. In Euglena, dichroism studies indicate that chlorophyll a 680 is unoriented, while chlorophyll a 695 appears to be oriented similar to chlorophyll a 680 in Chlorella or spinach chloroplasts, a result which is also in accord with the measured FP ratio of Euglena.

8. The possibility that the magnetic field gives rise to the reorientation of individual chlorophyll molecules is shown to be highly unlikely.     

A Comparative Study on Function and Membrane Proteins of Chloroplasts of Three Species of Cephalotaxus in Late Winter

Three species of evergreen Cephalotaxus (C. fortunei Hook. f., C. sinensis Li and C. harringtonia cv. Fastigiata) were used as materials to study some functional properties of chloroplasts. It is found that the oxygenevolving capacity of the chloroplasts from these plants is inhibited but partial reaction of PS-II and effect of Mg²⁺ on energy distribution between two photosystems are detectable during the winter. Seasonal effects on the functional properties of chloroplasts from evergreen Cephalotaxus are similar to that of conifer chloroplasts. The ratio between F₆₈₅, F₆₉₅ and F₇₃₅ of fluorescence emission spectra at 77°K of chloroplasts is different among these three species. It is found by using SDS-PAGE that the number of polypeptide resolved from thylakoid membrane of C. harringtonia cv. Fastigiata substantially differs from that of C. fortunei Hook.f. and C. sinensis Li. The result shows that the fluorescence emission spectrum feature and polypeptide composition of thylakoid membrane may be used as a tool for systematics of the genus Cephalotaxus.     

Signal/noise analysis of FRET-based sensors

Molecular sensors based on intramolecular Förster resonance energy transfer (FRET) have become versatile tools to monitor regulatory molecules in living tissue. However, their use is often compromised by low signal strength and excessive noise. We analyzed signal/noise (SNR) aspects of spectral FRET analysis methods, with the following conclusions: The most commonly used method (measurement of the emission ratio after a single short wavelength excitation) is optimal in terms of signal/noise, if only relative changes of this uncalibrated ratio are of interest. In the case that quantitative data on FRET efficiencies are required, these can be calculated from the emission ratio and some calibration parameters, but at reduced SNR. Lux-FRET, a recently described method for spectral analysis of FRET data, allows one to do so in three different ways, each based on a ratio of two out of three measured fluorescence signals (the donor and acceptor signal during a short-wavelength excitation and the acceptor signal during long wavelength excitation). Lux-FRET also allows for calculation of the total abundance of donor and acceptor fluorophores. The SNR for all these quantities is lower than that of the plain emission ratio due to unfavorable error propagation. However, if ligand concentration is calculated either from lux-FRET values or else, after its calibration, from the emission ratio, SNR for both analysis modes is very similar. Likewise, SNR values are similar, if the noise of these quantities is related to the expected dynamic range. We demonstrate these relationships based on data from an Epac-based cAMP sensor and discuss how the SNR changes with the FRET efficiency and the number of photons collected.     

Examination of the role of the clamp-loader and ATP hydrolysis in the formation of the bacteriophage T4 polymerase holoenzyme

Transient kinetic analyses further support the role of the clamp-loader in bacteriophage T4 as a catalyst which loads the clamp onto DNA through the sequential hydrolysis of two molecules of ATP before and after addition of DNA. Additional rapid-quench and pulse-chase experiments have documented this stoichiometry. The events of ATP hydrolysis have been related to the opening/closing of the clamp protein through fluorescence resonance energy transfer (FRET). In the absence of a hydrolysable form of ATP, the distance across the subunit interface of the clamp does not increase as measured by intramolecular FRET, suggesting gp45 cannot be loaded onto DNA. Therefore, ATP hydrolysis by the clamp-loader appears to open the clamp wide enough to encircle DNA easily. Two additional molecules of ATP then are hydrolyzed to close the clamp onto DNA. The presence of an intermolecular FRET signal indicated that the dissociation of the clamp-loader from this complex occurred after guiding the polymerase onto the correct face of the clamp bound to DNA. The final holoenzyme complex consists of the clamp, DNA, and the polymerase. Although this sequential assembly mechanism can be generally applied to most other replication systems studied to date, the specifics of ATP utilization seem to vary across replication systems.     

Homebuilt single-molecule scanning confocal fluorescence microscope studies of single DNA/protein interactions

Many technical improvements in fluorescence microscopy over the years have focused on decreasing background and increasing the signal to noise ratio (SNR). The scanning confocal fluorescence microscope (SCFM) represented a major improvement in these efforts. The SCFM acquires signal from a thin layer of a thick sample, rejecting light whose origin is not in the focal plane thereby dramatically decreasing the background signal. A second major innovation was the advent of high quantum-yield, low noise, single-photon counting detectors. The superior background rejection of SCFM combined with low-noise, high-yield detectors makes it possible to detect the fluorescence from single-dye molecules. By labeling a DNA molecule or a DNA/protein complex with a donor/acceptor dye pair, fluorescence resonance energy transfer (FRET) can be used to track conformational changes in the molecule/complex itself, on a single molecule/complex basis. In this methods paper, we describe the core concepts of SCFM in the context of a study that uses FRET to reveal conformational fluctuations in individual Holliday junction DNA molecules and nucleosomal particles. We also discuss data processing methods for SCFM.     

Regulation of photosynthesis in isolated spinach chloroplasts during orthophosphate limitation

The stromal concentration of orthophosphate in intact spinach chloroplasts (prepared in the absence of orthophosphate or pyrophosphate but supplied with both in the reaction medium) fell from a value of approx. 20 mM in the dark to a steady-state concentration of approx. 8 mM in the light. Chloroplasts illuminated in the absence of orthophosphate or pyrophosphate showed a similar trend. However, in this situation the stromal inorganic phosphate (P_i) concentration rapidly decreased from approx. 10 mM in the dark to a constant steady-state concentration of between 1.5 and 2.5 mM in the light. This P_i concentration was not further diminished (even though CO₂-dependent O₂ evolution had ceased) and was therefore considered to be stromal orthophosphate not freely available to metabolism. In the P_i-deficient chloroplasts the rate of photosynthesis declined rapidly after 1–2 min in the light such that CO₂-dependent O₂ evolution ceased with 5 min of the onset of illumination. The decline in O₂ evolution was accompanied by an increase in the transthylakoid ΔpH (as measured by 9-aminoacridine fluorescence quenching) and in the high-energy state, non-photochemical component of chlorophyll fluorescence quenching (q_E). Measurements of stromal metabolite concentrations showed that the ATP/ADP ratio was decreased in the P_i-deficient chloroplasts relative to chloroplasts illuminated in the presence of P_i. The stromal concentration of glycerate 3-phosphate was comparable in the P_i-deficient chloroplasts and those to which P_i had been supplied. Chloroplasts which were illuminated in P_i-free media showed a large accumulation of ribulose-1,5-bisphosphate relative to those supplied with P_i, suggesting inhibition of ribulose-1,5-bisphosphate carboxylase under these conditions. When P_i was added to chloroplasts illuminated in the absence of P_i, both non-photochemical quenching (q_E), photochemical quenching (q_Q) and ΔpH increased. This suggests that electron transport was not limited by inability to discharge transthylakoid ΔpH. These observation are consistent with the hypothesis that P_i limitation results in decreased ATP production by the thylakoid ATP synthase. The data presented here show that there are multiple sites of flux control exerted by low stromal P_i in the chloroplast. At least three factors contribute to the inhibition of photosynthesis under phosphate limitation: (1) there appears to be a direct effect of P_i on the energy-transducing system; (2) there is direct inhibition of the Calvin cycle decreasing the ability of the pathway to act as a sink for ATP and NADPH; and (3) feedback inhibition of primary processes occurs either via ΔpH or the redox state of electron carriers. However, ΔpH does not appear to be a limiting factor, but rather an inability to regenerate NADP as electron acceptor is suggested. The addition of DCMU to chloroplasts during illumination in the absence of P_i for periods of up to 10 min showed that there was very little loss of variable fluorescence despite a 60% reduction in the capacity for O₂ evolution. This would suggest that photoinhibitory damage to Photosystem II was not the major cause of the inhibition of photosynthesis observed with low P_i.     

An investigation into the ATP requirement for phosphorylation of thylakoid proteins and for the ATP-induced decrease in the yield of chlorophyll fluorescence in chloroplasts at different stages of development

The phosphorylation of thylakoid membrane polypeptides has been investigated in chloroplasts prepared from peas that had been grown under intermittent light and then exposed to between 4 and 48 h of continuous light. At 4 h, when the ratio of the total amount of labelling of a 9 kDa-polypeptide relative to light-harvesting chlorophyll protein (LHCP) polypeptides was much greater than 1, the affinity for ATP was found to be the same (S_0.5, approx. 100 μM) for both polypeptides. In contrast, in fully greened chloroplasts, when labelling of LHCP was much greater than that of the 9 kDa-polypeptide, the S_0.5 for ATP was 40 μM for LHCP and 500 μM for the 9 kDa-polypeptide. A correlation was observed during development between the affinity for ATP of the 9 kDa-species and its abundance relative to LHCP. It is suggested that these polypeptides compete for phosphorylation by the same protein kinase. Simultaneous assay of the ATP-induced fluorescence decrease at different ATP concentrations revealed a close correlation with LHCP labelling but not with labelling of the 9 kDa-polypeptide. This correlation held irrespective of which polypeptide was the major phosphoprotein.     

Synthesis and hydrolysis of ATP by intact chloroplasts under flash illumination and in darkness

ATP concentrations were measured in isolated intact spinach chloroplasts under various light and dark conditions. The following results were obtained: (1) Even in darkened chloroplasts and in the absence of exogenous substrates, ATP levels in the chloroplast stroma were significant. They decreased on addition of glycerate, phosphoglycerate or dihydroxyacetone phosphate. When dihydroxyacetone phosphate and oxaloacetate were added together, ATP levels increased in darkened chloroplasts owing to substrate level phosphorylation. (2) Under illumination with saturating single turnover flashes, oxygen evolution in the presence of phosphoglycerate, whose reduction requires ATP, was no lower on a unit flash basis at the low flash frequency of 2 Hz than at higher frequencies. Quenching of 9-aminoacridine fluorescence, which indicates the formation of a proton gradient in intact chloroplasts, decreased with decreasing flash frequencies, until there was no significant fluorescence quenching at a flash frequency of about 2 Hz. In contrast to intact chloroplasts, broken chloroplasts did not phosphorylate much ADP at the low flash frequency of 2 Hz. (3) Flashing at extremely low frequencies (0.2 Hz) caused ATP hydrolysis rather than ATP synthesis in intact chloroplasts. At higher flash frequencies, synthesis replaced hydrolysis. Still, even at high frequencies (10 Hz), the first flashes of a series of flashes given after a long dark time always decreased chloroplast ATP levels.From these results, it is concluded that the enzyme, which mediates ATP synthesis in the light, is inactive in darkened intact chloroplasts. Its light activation can be separated from the formation of the high energy condition, which results in ATP synthesis. After its activation, the enzyme catalyzes a reversible reaction.     

Synthesis and hydrolysis of ATP by intact chloroplasts under flash illumination and in darkness.

ATP concentrations were measured in isolated intact spinach chloroplasts under various light and dark conditions. The following results were obtained: (1) Even in darkened chloroplasts and in the absence of exogenous substrates, ATP levels in the chloroplast stroma were significant. They decreased on addition of glycerate, phosphoglycerate or dihydroxyacetone phosphate. When dihydroxyacetone phosphate and oxaloacetate were added together, ATP levels increased in darkened chloroplasts owing to substrate level phosphorylation. (2) Under illumination with saturating single turnover flashes, oxygen evolution in the presence of phosphoglycerate, whose reduction requires ATP, was no lower on a unit flash basis at the low flash frequency of 2 Hz than at higher frequencies. Quenching of 9-aminoacridine fluorescence, which indicates the formation of a proton gradient in intact chloroplasts, decreased with decreasing flash frequencies, until there was no significant fluorescence quenching at a flash frequency of about 2 Hz. In contrast to intact chloroplasts, broken chloroplasts did not phosphorylate much ADP at the low flash frequency of 2 Hz. (3) Flashing at extremely low frequencies (0.2 Hz) caused ATP hydrolysis rather than ATP synthesis in intact chloroplasts. At higher flash frequencies, synthesis replaced hydrolysis. Still, even at high frequencies (10 Hz), the first flashes of a series of flashes given after a long dark time always decreased chloroplast ATP levels. From these results, it is concluded that the enzyme, which mediates ATP synthesis in the light, is inactive in darkened intact chloroplasts. Its light activation can be separated from the formation of the high energy condition, which results in ATP synthesis. After its activation, the enzyme catalyzes a reversible reaction.