Light-induced ATP Formation from Acetyl Phosphate and ADP by Broken Spinach Chloroplasts

Spinach chloroplasts catalyzed ATP formation from acetyl phosphateand ADP when exposed to light. No ATP formation was detectablein the dark. In the absence of ADP, chloroplasts did not hydrolyzeacetyl phosphate in the light or dark. Neither high-energy phosphatessuch as creatine phosphate and phosphoenol pyruvate nor inhibitorsof photophosphorylation competitive with P_i, such as ß-naphthylmonophosphate, phenyl phosphate and pyridoxal 5-phosphate, couldsubstitute for acetyl phosphate as a P_i donor. The apparentK_m values for acetyl phosphate and P_i were 0.81 mM and 0.25mM, respectively. The maximal rate of ATP formation with acetylphosphate and P_i were 331 and 521 µmol ATP formed mg chl^–1hr^–1, respectively. The optimum pH value for acetyl phosphate-dependentATP formation was about 8.0. NH₄Cl, dicyclohexylcarbodiimideand triphenyltin chloride inhibited the acetyl phosphate-dependentATP formation. Acid-base transition also could induce subsequentATP formation from acetyl phosphate and ADP. These results suggestthat the acetyl phosphate-dependent ATP formation requires theformation and the utilization of a proton-motive force as ordinaryphotophosphorylation does. ¹ This work was supported in part by Grants-in-Aid for ScientificResearch from the Ministry of Education, Science and Culture,Japan to H. S. Part of this work was reported at the 1981 AnnualMeeting of the Japanese Society of Plant Physiologists (Sapporo,May 8, 1981). (Received August 25, 1981; Accepted November 1, 1981)     

Properties of a Membrane-bound Phosphatase from the Thylakoids of Spinach Chloroplasts

A 3-phosphoglycerate phosphatase activity of about 2 micromoles per minute per milligram chlorophyll is associated with the thylakoid membranes of spinach chloroplasts. The K_m for 3-phosphoglycerate is 3 millimolar. The enzyme can be solubilized from thylakoid membranes by treatment with 0.33 molar MgCl₂ or sodium deoxycholate. The activity is not stimulated by sulfhydryl reagents or the addition of 10 millimolar MgCl₂. The enzymic activity is insensitive to ethylenediaminetetraacetate. The pH optimum is broad, between 5.5 to 7.5. Although the substrate specificity is broad, 3-phosphoglycerate is the best substrate of those tested at neutral pH. However, p-nitrophenyl phosphate was a more effective substrate at pH 5.5. The enzyme exhibits the general characteristics of an acid phosphatase.     

菠菜叶片蔗糖磷酸合成酶的调节

电泳均一的菠菜叶片蔗糖磷酸合成酶的活力受G6P,Mg~(2 ),Mn~(2 )的调节;G6P对此酶的促进作用在F6P浓度较低时表现得比较明显;此酶对Mn~(2 )较对Mg~(2 )敏感,Mg~(2 ),Mn~(2 )对此酶的促进作用可被EDTA解除。底物F6P的饱和曲线为S型,底物UDPG的饱和曲线为双曲线型。NADP是此酶的负效应剂,NADP对F6P表现为混合型抑制,使V_m(F6P)降低和K_m(F6P)增大,3mmol/L NADP使F6P的K_m值从2.5mmol/L上升至3.8mmol/L,但不影响希尔系数,n=1.3。NADP对UDPG表现为K_m不变的非竞争性抑制,K_m(UDPG)=3.8mmol/L。     

菠菜叶片蔗糖磷酸合成酶的纯化

经硫酸铵分部沉淀,DEAE-纤维素(DE 52),Sepharose 6B和 AH—4B连续三次柱层析,得到纯化88倍电泳均一的菠菜叶片蔗糖磷酸合成酶。电泳分析该酶分子量为490 kD,是由八个分子量为60 kD的相同亚基组成的寡聚体,等电点为PI=4.l,其最适pH值为6.9。     

Effects of Green Light on the Chloroplasts of Spinach Leaf Discs

The development and photosynthetic capacity of chloroplastsformed in green light in cultured spinach leaf discs has beenstudied. At intensities of 4 to 6 mW cm^–2 green lightstimulates chloroplast replication to about the same extentas white, blue, and red light. However, practically no chloroplastreplication occurs in discs grown in low intensity green orwhite light but considerable chloroplast growth takes place.Ultrastructural studies have shown that these chloroplasts,which can be two to five times the area of control plastids(high intensity white light), have an essentially normal thylakoidsystem. Double isotope labelling experiments have establishedthat the synthesis of chloroplast ribosomal-RNA is similar incontrols and in discs grown in low intensity green or whitelight. On a per unit chlorophyll basis the CO₂ fixation rateof spinach discs grown in low intensity green (or white) lightsaturates with increasing light intensity or increasing CO₂concentration at values well below control discs. In this respecttheir photosynthetic characteristics bear a similarity to thoseof shade plants.     

Subunit Movements in Single Membrane-bound H+-ATP Synthases from Chloroplasts during ATP Synthesis

Subunit movements within the H⁺-ATP synthase from chloroplasts (CF₀F₁) are investigated during ATP synthesis. The γ-subunit (γCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5′-(β,γ-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one α-subunit. The labeled CF₀F₁ is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the γ-subunit relative to the α-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each αβ-pair. Without catalysis the central stalk interacts with only one specific αβ-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP.     

On the Mechanism of Activation by Light of the NADP-dependent Malate Dehydrogenase in Spinach Chloroplasts

With intact spinach (Spinacia oleracea L. cv. Vital R) chloroplasts, the activity of the NADP-dependent malate dehydrogenase after activation by light was 30 micromoles of malate formed per milligram of chlorophyll per hour; an identical rate of O₂ evolution was obtained upon oxaloacetate reduction by the intact plastids. However, when the activity of NADP-dependent malate dehydrogenase was measured subsequently to maximal activation of the enzyme by dithiothreitol (DTT) an average rate of 113 micromoles per milligram of chlorophyll per hour was obtained. When membranes and stroma were separated after osmotic disruption of the chloroplasts, 28% of NADP-dependent malate dehydrogenase activity inducible by DTT was found with the membranes and 72% was found in the stromal fraction. The membrane-associated portion of the enzyme corresponds well with the activity achieved after activation by light. About 64% of an activator system was found to be associated also with the membrane fraction. Washing the membranes with buffer removed more activator than enzyme. However, both were removed almost completely by ethylenediaminetetraacetate. It was concluded that both a portion of the enzyme and the total activator system are associated with the chloroplast membranes in vivo and that the activator is more loosely bound than the enzyme. A model describing the partial activation of chloroplastic NADP-dependent malate dehydrogenase by light and the total activation by DTT is presented.     

菠菜叶绿体的光抑制部位

有氧条件下,叶绿体的光抑制部位不是专一的。强光可使PSⅡ氧化侧、PSⅡ反应中心、PSⅡ还原侧,PSⅡ及类囊体膜透性都有不同程度的破坏。这种非专一性可能与类囊体膜蛋白在强光下的降解有关。无氧条件下,叶绿体的光抑制部位只是在PSⅡ反应中心及Q_B蛋白上。     

Effects of Nanoanatase TiO2 on Photosynthesis of Spinach Chloroplasts Under Different Light Illumination

With a photocatalyzed characteristic, nanoanatase TiO₂ under light could cause an oxidation–reduction reaction. Our studies had proved that nano-TiO₂ could promote photosynthesis and greatly improve spinach growth. However, the mechanism of nano-TiO₂ on promoting conversion from light energy to electron energy and from electron energy to active chemistry energy remains largely unclear. In this study, we report that the electron transfer, oxygen evolution, and photophosphorylation of chloroplast (Chl) from nanoanatase-TiO₂-treated spinach were greatly increased under visible light and ultraviolet light illumination. It was demonstrated that nanoanatase TiO₂ could greatly improve whole chain electron transport, photoreduction activity of photosystem II, O₂-evolving and photophosphorylation activity of spinach Chl not only under visible light, but also energy-enriched electron from nanoanatase TiO₂, which entered Chl under ultraviolet light and was transferred in photosynthetic electron transport chain and made NADP⁺ be reduced into NADPH, and coupled to photophosphorylation and made electron energy be transformed to ATP. Moreover, nanoanatase h⁺, which photogenerated electron holes, captured an electron from water, which accelerated water photolysis and O₂ evolution.     

Light Modulation of Enzyme Activity in Chloroplasts: Generation of Membrane-bound Vicinal-Dithiol Groups by Photosynthetic Electron Transport

Inhibitor experiments indicate that photosynthetic electron transport is required for light activation of the pea (Pisum sativum) leaf chloroplast enzymes NADP-linked glyceraldehyde-3-phosphate dehydrogenase, NADP-linked malic dehydrogenase, ribulose-5-phosphate kinase and sedoheptulose-1,7-diphosphate phosphatase, and for inactivation of glucose-6-phosphate dehydrogenase. Modulation of the activity of the dehydrogenases and kinase apparently involves a component preceding ferredoxin in the photosynthetic electron transport chain; activation of the phosphatase involves an electron transport component at the level of ferredoxin. Modulation of enzyme activity can be obtained in a broken chloroplast system consisting of membrane fragments and stromal extract. The capacity for light regulation in this system is reduced or eliminated when the membrane fraction is exposed to arsenite in the light or to sulfite in light or dark. Light-generated vicinal-dithiols seem therefore to be involved in modulation of the activity of the enzymes included in this study.     

Induction of Hexose-Phosphate Translocator Activity in Spinach Chloroplasts

Many environmental and experimental conditions lead to accumulation of carbohydrates in photosynthetic tissues. This situation is typically associated with major changes in the mRNA and protein complement of the cell, including metabolic repression of photosynthetic gene expression, which can be induced by feeding carbohydrates directly to leaves. In this study we examined the carbohydrate transport properties of chloroplasts isolated from spinach (Spinacia oleracea L.) leaves fed with glucose for several days. These chloroplasts contain large quantities of starch, can perform photosynthetic 3-phosphoglycerate reduction, and surprisingly also have the ability to perform starch synthesis from exogenous glucose-6-phosphate (Glc-6-P) both in the light and in darkness, similarly to heterotrophic plastids. Glucose-1-phosphate does not act as an exogenous precursor for starch synthesis. Light, ATP, and 3-phosphoglyceric acid stimulate Glc-6-P-dependent starch synthesis. Short-term uptake experiments indicate that a novel Glc-6-P-translocator capacity is present in the envelope membrane, exhibiting an apparent Km of 0.54 mM and a Vmax of 2.9 [mu]mol Glc-6-P mg-1 chlorophyll h-1. Similar results were obtained with chloroplasts isolated from glucose-fed potato leaves and from water-stressed spinach leaves. The generally held view that sugar phosphates transported by chloroplasts are confined to triose phosphates is not supported by these results. A physiological role for a Glc-6-P translocator in green plastids is presented with reference to the source/sink function of the leaf.     

Two Forms of Division Profile in Spinach Chloroplasts

CHLOROPLAST division is generally thought to take place by constriction (fission)^1–7. Division may also occasionally occur by the growth of a central baffle before separation^8–11. Usually only one type has been observed for a given tissue although searches have been made for the alternative form. The present communication, forming part of a study on the effect of light and dark treatments on cultured leaf disks, describes the ultrastructure of spinach chloroplasts which exhibit both dumb-bell and central baffle profiles suggestive of division.     

Ferricyanide Reduction in Photosystem II of Spinach Chloroplasts

Ferricyanide can be reduced in Photosystem II of spinach chloroplasts at 2 separate sites, both of which are sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but only one of which is sensitive to dibromothymoquinone. Data presented in this paper emphasize ferricyanide site II of Photosystem II, which is sensitive to thiol inhibition and may reflect a cyclic pathway around Photosystem II. Ferricyanide reduction sites 1 and 2 also differ from each other in fractions isolated from discontinuous sucrose gradients, from fragmented chloroplasts, and upon trypsin treatment. Sucrose density gradient centrifugation shows that ferricyanide reduction site 1 activity at pH 6 decreases from 30 to 50% in various isolated fractions, while the dibromothymoquinone-insensitive activity at pH 8 (site 2) is stimulated from 15 to 35%.     

Enzymatic Evidence for a Complete Oxidative Pentose Phosphate Pathway in Chloroplasts and an Incomplete Pathway in the Cytosol of Spinach Leaves

The intracellular localization of transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase was reexamined in spinach (Spinacia oleracea L.) leaves. We found highly predominant if not exclusive localization of these enzyme activities in chloroplasts isolated by isopyknic centrifugation in sucrose gradients. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose phosphate isomerase, and triose phosphate isomerase activity was present in the chloroplast fraction but showed additional activity in the cytosol (supernatant) fraction attributable to the cytosol-specific isoforms known to exist for these enzymes. Anion-exchange chromatography of proteins of crude extracts on diethylaminoethyl-Fractogel revealed only a single enzyme each for transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase. The data indicate that chloroplasts of spinach leaf cells possess the complete complement of enzymes of the oxidative pentose phosphate path-way (OPPP), whereas the cytosol contains only the first two reactions, contrary to the widely held view that plants generally possess a cytosolic OPPP capable of cyclic function. The chloroplast enzymes transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase appear to be amphibolic for the Calvin cycle and OPPP.     

Effects of Azide on Photophosphorylation in Isolated Spinach Chloroplasts

The influence of sodium azide on open-chain and flavine mononucleotide mediated cyclic photophosphorylation in isolated spinach chloroplasts was investigated under anaerobic conditions. Open chain phosphorylation was completely inhibited with DCMU both in the presence and absence of sodium azide in the experimental medium. Flavine mononucleotide mediated photophosphorylation was only slightly inhibited by DCMU in the absence of sodium azide but inhibited in two steps by increasing amounts of DCMU when sodium azide was present in the medium. The first step can be explained as being mainly an effect of DCMU on an open chain electron transport, with water and H₂O₂ as electron donors and with flavine mononucleotide — kept in an oxidized state by sodium azide — as the electron acceptor. The second step, as well as the comparatively insensitivity to DCMU in the absence of sodium azide, depends on cyclic photophosphorylation mediated by flavine mononucleotide.     

A Calcium-Selective Site in Photosystem II of Spinach Chloroplasts

After acid-treatment of spinach (Spinacia oleracea) chloroplasts, various partial electron transport reactions are inactivated from 25 to 75%. Divalent cations in concentrations from 10 to 50 millimolar can partially restore electron transport rates. Two cation-specific sites have been found in photosystem II: one on the 3-(3,4-dichlorophenyl)-1, 1-dimethylurea-insensitive silicomolybdate pathway, which responds better to restoration by Mg²⁺ than by Ca²⁺ ions, the other on the forward pathway to photosystem I, located on the 2,5-dimethylbenzoquinone pathway. This site is selectively restored by Ca²⁺ ions. When protonated chloroplasts are treated with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aziridine, a carboxyl group modifying reagent, presumed to react with glutamic and aspartic acid residues of proteins, restoration of electron transport at the Ca²⁺-selective site on the 2,5-dimethylbenzoquinone pathway is impaired, while no difference in restoration is seen at the Mg²⁺ site on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive silicomolybdate pathway.

Trypsin treatment of chloroplasts modifies the light-harvesting pigment-protein complex, destroys the dibromothymoquinone-insensitive 2,5-dimethyl-benzoquinone reduction, but does not interfere with the partial restoration of activity of this pathway by Ca²⁺ ions, implying that the selective Ca²⁺ effect on photosystem II (selective Ca²⁺ site) is different from its effects as a divalent cation on the light-harvesting pigment-protein complex involved in the excitation energy distribution between the two photosystems.

     

菠菜叶中存在两种谷氨酰胺合成酶同工酶

运用非变性聚丙稀酰胺凝胶电泳结合活性染色的方法,在菠菜(Spinacia oleracea L．)生长发育过程中,观察到叶片中至少存在2种谷氨酰胺合成酶(GS),其中一种GS的活性随发育进程而逐渐升高,而另一种GS的活性逐渐降低。在不同来源的成熟的菠菜叶片中同样观察到2种GS的存在。     

Action of Mercury on the Photosynthetic Apparatus of Spinach Chloroplasts

In chloroplasts of Spinacea oleracea L., Hg2+ ions interact with some sites in the photosynthetic electron transport chain: (l) with the intermediates Z+/D+ situated in the D1 and D2 proteins and with the manganese cluster in the oxygen evolving complex which are located on the donor side of photosystem (PS) 2, (2) with the chlorophyll a dimer in the core of PS1 (P700). P700 is oxidized in the dark by HgCl2. The Hg2+ ions form organometallic complexes with amino acids contained in chloroplast proteins.