Haematoporphyrin derivative (Photofrin II) photosensitization of isolated mitochondria: inhibition of ADP/ATP translocator

To gain further insight into the mechanism by which irradiation of mitochondria in the presence of haematoporphyrin derivative (Photofrin II) (PF II) causes impairment of mitochondrial oxidative phosphorylation, the rate of ADP/ATP exchange via the ADP/ATP translocator was measured fluorometrically is isolated rat liver mitochondria. In accord with noncompetitive inhibition, PF II photosensitization decreases the maximum rate of exchange Vmax (20.8 and 9.6 nmol ATP effluxed min-1 x mg protein in the control and after 2 min irradiation, respectively) without changing the ADP affinity for the carrier (Km = 5 microM in both cases). Comparison of the rate of oxygen uptake by mitochondria stimulated by either ADP or by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) confirms that the adenine nucleotide carrier is a major target of photodynamic action which causes oxidative phosphorylation impairment.     

Dual signal (color change and fluorescence ON-OFF) ensemble system based on bis(Dpa-Cu) complex for detection of PPi in water

We have developed simple dual signal (color change and fluorescence ON-OFF) ensemble systems based on a bis(Dpa-Cu^II) complex 1 for the detection of PPi in water. Dual signal takes place because of weak binding and fluorescence quenching effect of coordinatively unsaturated Cu^II complex for indicators and replacement of the indicators by more strongly binding PPi. As a consequence, these ensembles show a high selectivity and sensitivity for PPi over various anions including phosphate and its derivatives (AMP, ADP, and ATP) in water.     

Size reduction of CdSe/ZnS core-shell quantum dots photosensitized by benzophenone: where does the Cd(0) go?

The size of core-shell CdSe/ZnS quantum dots can be decreased by using the combined action of an n,π* aromatic ketone and UVA light. Energy-dispersive X-ray spectroscopy as well as X-ray photoelectron spectroscopy techniques gave information on the photosensitization mechanism and the eventual destiny of Cd(2+) and Se(2-) core ions. Our data support the electron transfer from the BP ketyl radical to Cd(2+), leading to Cd(0) and H(+), as well as to the recovery of benzophenone. Elemental Cd remains on the core and, eventually, can be oxidized to CdO. In addition, Se(2-) counterions disperse inside the solution mainly attached to protonated amine ligands. The Se(2-) combines with H(+), leading to SeH(2), which is finally oxidized to Se(0) by oxygen. Therefore, quantum dots illumination in the presence of benzophenones brings about a profound nanoparticle reconstruction which takes place after dark storage; this agrees with the drastic quenching of the quantum dot emission detected immediately after illumination as well as the slow recovery in the dark.     

ADENYLATE KINASE BOUND TO THE CHROMATOPHORE MEMBRANES OF Rhodobactor spheroides G1C

Transformation of adenylates (AMP, ADP and ATP) by washed chromatophore membranes of Rhodobactor spheroides G1C in the dark and in the light indicated the functions of ATPase (ADP + Pi in equilibrium ATP) and of an adenylate kinase (2ADP in equilibrium AMP + ATP). The activity of adenylate kinase of the chromatophores was not inhibited by AP5A, and persisted even after sonication in the presence of EDTA or CaCl2; the results suggested the presence of an adenylate kinase bound to the chromatophore membrane. In search of the enzyme, the supernatant after sonication of the chromatophores in the presence of EDTA was subjected to a molecular sieve and then to ion-exchange HPLC; a fraction with high specific adenylate kinase activity, containing a very sharp peak at 55 kDa, was isolated. Preliminary characterization indicated that it is different from the well-documented water-soluble 33 kDa adenylate kinase.     

ENERGY-REGULATED FUNCTIONAL TRANSITIONS OF CHLOROPLAST ATPASE

The functional transitions of the membrane-bound chloroplast ATPase (CF₁) as influenced by low ADP and uncoupler concentrations are investigated by measurements of initial and steady-state ATP hyrolysis and concomitant membrane energization. Following activation of latent ATP hydrolysis by light in the presence of dithioerythritol, the resulting steady-state ATP hydrolysis depends on the dark-period ( t _d) bteween light activation and ATP addition. ADP, added during t _d, inhibits this activity ( K _i about 2 μ M ) and induces a lag in the onset of ATP hydrolysis. The extent of membrane energization as monitored by an aminoacridine fluorescent probe is proportional to the ATPase activity.
An uncoupler amplifies the inhibitory effect of ADP if added during f _d, whereas it induces the normal stimulation of ATP hydrolysis in the absence of ADP. The ADP effect, which is different from product inhibition, is interpreted as a conformational interaction with CF₁ causing an increase of the energy threshold required for the inactive → active transition of the CF₁ molecules. These results are in harmony with currently proposed models of CF₁ regulation by adenine nucleotides based on binding studies.
The inactive → active transition of CF₁ conformation is investigated by analysis of the lag in the onset of ATP hydrolysis at different ADP concentrations and by means of varied light pulses and single-turnover flashes, using the electric potential indicating absorption change at 515 nm as a probe for the onset of ATP hydrolysis. The half-time of the process leading to fully (re)activated ATP hydrolysis is about 0.25 s. The ATP-dependent flash-induced inactive → active transition occurs within a few turnovers of electron flow.     

Proteine der Photosynthese: Aus Licht wird Leben

Photosynthesis is based on a fascinating interplay between proteins, pigments and membranes. The cleavage of water molecules takes place in photosystem II which is excited by light. Recent structure elucidation of photosystem II helped in understanding the electron transfer reaction but the water splitting mechanism is still not solved. The light energy is stored in ATP, which is synthesized by the ATP Synthase, a molecular motor. Its membrane part, the c‐ring, mediates the transfer of protons. Structure and function of ATP Synthase are currently under intensive investigation.     

EFFECTS OF HEMATOPORPHYRIN DERIVATIVE ON THE CELLULAR ENERGY METABOLISM IN THE ABSENCE AND PRESENCE OF LIGHT

Effects of Photofrin II on energy metabolism and metabolic viability were studied in a mammalian transformed cell line (BHK-21) in dark and after photo-irradiation with visible light. Cells were allowed to accumulate Photofrin by incubating for 4 h in buffer containing Photofrin (5-60 micrograms/ml). The results show that Photofrin significantly affects the cellular energy metabolism even in the absence of light; activity of cytochrome c oxidase is decreased and glucose utilization and lactate production (glycolysis) are increased. Irradiation with light resulted in a significant decrease in the activity of cytochrome c oxidase, glycolysis, ATP content, energy charge, ratios of adenine nucleotides like ATP/ADP, ATP/AMP and cell viability (dye exclusion test). Presence of inhibitors of energy metabolism, potassium cyanide (respiration) and 2-deoxyglucose (glycolysis), further enhanced the cytotoxic effects induced by hematoporphyrin derivative and light.     

Design of a coupled bioluminescent assay for a recombinant pyruvate kinase from a thermophilic Geobacillus

A simple and rapid method using coupled bioluminescent assay was developed to determine level of ADP. ADP is involved in many biological reactions and ADP assay can be used for assaying some reactions universally by monitoring ADP formation or depletion. ADP analysis involves incubation of ADP or extracts containing ADP with pyruvate kinase (PK) and PEP. The ATP formed by this reaction is determined by measuring the intensity of the initial light flash produced when luciferin-luciferase preparation injected into the reaction mixture. In regard to the main role of the PK in this assay, the gene of PK from a Geobacillus species has been cloned in expression vector pET28a (+), sequenced and overexpressed in Escherichia coli. Recombinant protein was purified using Ni-NTA column and then the purified PK was used in a coupled bioluminescent assay for ADP measurement. Kinetic properties of PK are determined according to a bioluminescent assay using firefly luciferase.     

Influence of gas atmosphere and temperature on the conductivity and the photoconductivity of a TiO2 single crystal in the surface region

The electrical photoconductivity and conductivity at (and near) the surface of a TiO(2) single crystal (rutile) was studied in a range of temperatures between 300 and 573 K and under different ambient gases (oxygen and nitrogen) by means of impedance spectroscopy. The long times required (many hours) to reach steady state photoconductivity can be explained by the reduction of the material upon illumination. At about 475 K a maximum is observed in the equilibrium photoconductivity and a minimum in the rate constants of the rise and decay after switching on and off, respectively, the light. After switching off the light a fast decay takes place during the first milliseconds followed by a slow exponential decay. The first one is related to recombination through defects, while the latter is due to re-oxidation processes of the material. The results are correlated with measurements of photocatalytic activity.     

Colour change of co-doped yttrium aluminium borate crystals under illumination with different white light sources

A colour change of doped YAB crystals and microcrystalline powders takes place upon illumination with different white light sources. This paper presents extensive experimental data on the new materials Ho,Nd:YAB; Ho,Cr:YAB, Nd,Cr:YAB and Ho,Nd,Cr:YAB, resulting in a chart providing the observed crystal colours and chromaticity differences of the materials under illumination with seven different types of white light sources. The microcrystalline powders could be used as coatings in order to produce a special colour changing effect. Furthermore, the colour table presented enables the observer to discriminate the investigated light sources at one glance.      

高效液相色谱法测定细胞内三磷酸腺苷及其代谢物的含量（英文）

研究了三磷酸腺苷(ATP)及其代谢物在细胞内的含量以及2-叔丁基-1,4-苯醌(TBBQ)对ATP及其代谢产物在细胞内含量的影响。建立了一种高效液相色谱法(HPLC)用于快速分离、检测细胞内ATP及其代谢产物(二磷酸腺苷(ADP)和一磷酸腺苷(AMP))的含量。使用岛津高效液相系统及艾杰尔Venusil MP C18柱,采用等度洗脱的方式。流动相A相为50 mmol/L磷酸氢二钠和15 mmol/L三甲胺(TEA),用醋酸(HAc)调节pH至7.88;流动相B相为甲醇。采用建立的高效液相色谱法得到了3种代谢物的工作曲线,相关系数高(R~2≥0.999 6),MRC-5细胞中3种代谢物的含量均在线性范围(0.1~100μmol/L)内。该方法检出限低。采用预冷的80%(体积分数)甲醇水溶液提取细胞内的代谢物。该研究建立的方法成功地应用于检测MRC-5细胞中的ATP、ADP和AMP的含量,结果表明,TBBQ会对ATP、ADP、AMP在细胞内的含量产生影响,但TBBQ浓度和ATP、ADP以及AMP在MRC-5细胞内浓度的关系比较复杂。     

CONTROL OF CHLOROPHYLL b BIOSYNTHESIS BY PHYTOCHROME

In the mustard seedling cotyledons, chlorophyll b appears from the very beginning in white light provided that a red light pulse pretreatment was given 12 h prior to the onset of white light. The red light pulses act through phytochrome. Without pretreatment no chlorophyll b is detectable at least during the first 60 min after the onset of white light (25°C). Biogenesis of chlorophyll b specifically depends on the action of phytochrome during the pre-steady state period as well as during the steady state period of chlorophyll accumulation. In light pulse experiments, it was found that formation of chlorophyll b takes place stoichiometrically at the cost of chlorophyll(ide) a.     

Light-induced transformations of hematoporphyrin diacetate and hematoporphyrin

Illumination of hematoporphyrin diacetate (HP-Diac) and hematoporphyrin (HP) in phosphate-buffered solutions causes the formation of a stable photoproduct absorbing at 636 nm. Simultaneously the photo-oxidation of HP-Diac and HP takes place. These phototransformations depend on the illumination dose and the concentration of the HP-Diac or HP solution, and cause qualitatively the same spectral changes independently of the light source used.     

INTRACELLULAR DAMAGE IN YEAST CELLS CAUSED BY PHOTODYNAMIC TREATMENT WITH TOLUIDINE BLUE

Abstract— The positively charged photosensitizer toluidine blue (TB) can induce loss of clonogenicity in Kluyveromyces marxianus. Previous studies have revealed that, as a consequence of the localization of this dye at the cell surface, photodynamic action results in extensive damage at the level of the plasma membrane. In this paper, a study is reported on the effect of photodynamic treatment with TB on intracellular enzymes. It is shown that treatment with TB and light resulted in the inhibition of alcohol dehydrogenase, cytochrome c oxidase, glyceraldehyde-3-phosphate dehydrogenase and hexokinase. Photodynamic treatment also lowered the ATP levels. The ATP levels could be partially restored in the presence of glucose but not with ethanol. Toluidine blue binding experiments revealed that photodynamic treatment caused a rapid increase in the amount of cell-associated dye. Moreover, it also appeared that this treatment decreased the binding of TB to the cell surface. It is concluded that TB enters the cell during the first minutes of illumination, whereafter intracellular enzymes are inactivated. The data indicate that photodynamic damage of intracellular sites contributes to the loss of viability.     

SENSITIZED PHOTOOXYGENATION ACCORDING TO TYPE I MECHANISM (RADICAL MECHANISM) — PART III. EXPERIMENTS WITH CONTINUOUS ILLUMINATION

Abstract— The photochemical reaction in the system thionine (sensitizer), allylthiourea (ATU, acceptor), and oxygen was studied with continuous illumination. In oxygen-free aqueous solution thionine is photoreduced to leucothionine. With oxygen, however, a photooxygenation of the acceptor takes place. At the same time the quantum yield of the bleaching reaction of thionine decreases markedly in comparison with that of the oxygen-free solution. At about 100 sec after the beginning of illumination, the overall quantum yield of the bleaching reaction diminishes further because the leucothionine formed during the reaction now becomes transformed into thionine. The quantum yields do not change significantly over the range of oxygen concentrations studied (initial concentration 1 × 10^-5 to 5 × 10^-5 M ). In addition they are independent of the light intensity. The influence of the pH and the acceptor concentration were also investigated.
The sensitizer is not only bleached reductively, but is also partly destroyed by oxidation. The results are in agreement with the reaction scheme elucidated by flash photolysis measurements.
In accordance with this reaction scheme, the primary reaction (a) of the reductive bleaching of the sensitizer, (b) of the photooxygenation of the acceptor and (c) of the oxidative destruction of the sensitizer, is identical in all cases. This process is the redox reaction between the sensitizer triplet and the acceptor, where a semithionine and an ATU-radical are formed. The reaction represents an example of a Type I photooxygenation according to the notation of Gollnick.     

Molecular recognition of ADP over ATP in aqueous solution by a polyammonium receptor containing a pyrimidine residue

The synergistic action of the different binding groups of the polyfunctional HL receptor leads to the recognition of ADP over ATP in water, with a selectivity coefficient of up to 116, a phenomenon which is unprecedented in the context of synthetic receptors in water. The recognition is mostly due to the good matching between H(3)L(2+) and the protonated forms of ADP.     

Testing for violations of microscopic reversibility in ATP-sensitive potassium channel gating

In pancreatic beta cells, insulin secretion is tightly controlled by the cells' metabolic state via the ATP-sensitive potassium (KATP) channel. ATP is a key mediator in this signaling process, where its role as an inhibitor of KATP channels has been extensively studied. Since the channel contains an ATPase as an accessory subunit, the possibility that ATP hydrolysis mediates KATP channel opening has also been proposed. However, a rigorous test of coupling between ATP hydrolysis and channel gating has not previously been performed. In the present work, we examine whether KATP channel gating obeys detailed balance in order to determine whether ATP hydrolysis is strongly coupled to the gating of the KATP channel. Single-channel records were obtained from inside-out patches of transiently transfected HEK-293 cells. Channel activity in membrane patches with exactly one channel shows no violations of microscopic reversibility. Although KATP channel gating shows long closed times on the time scale where ATP hydrolysis takes place, the time symmetry of channel gating indicates that it is not tightly coupled to ATP hydrolysis. This lack of coupling suggests that channel gating operates close to equilibrium; although detailed balance is not expected to hold for ATP hydrolysis, it still does so in channel gating. On the basis of these results, the function of the ATPase active site in channel gating may be to sense nucleotides by differential binding of ATP and ADP, rather than to drive a thermodynamically unfavorable conformational change.     

Chloroplast Biogenesis 81: Transient Formation of Divinyl Chlorophyll a Following a 2.5 ms Light Flash Treatment of Etiolated Cucumber Cotyledons

Abstract— The possible conversion of nascent divinyl (DV) chloro-phyllide a (Chlide a ) to DV chlorophyll a (Chi a during the early stages of greening in a dark divinyl-light divinyl-light/dark divinyl (DDV-LDV-LDDV) plant species was investigated. Etiolated cucumber cotyledons ( Cucu-mis sativus L .) were subjected to a 2.5 ms light flash followed by darkness. The DV and monovinyl (MV) components of the protochlorophyllide a (Pchlide a ), Chlide a , Pchlide a ester and Chi a pools were monitored quantitatively by high-resolution spectrofluorometry, immediately following the light treatments and after various periods in darkness. The light treatment photoconverted DV and MV Pchlide a to DV and MV Chlide a . Some photoconversion of MV Pchlide a ester to MV Chi a also appeared to take place. A sharp rise in the level of DV Chi a following the light treatment could not be accounted for by photoconversion of DV Pchlide a ester. It must have arisen by rapid esterification of nascent DV Chlide a. After illumination, the level of DV Chi a rose for 5 s and then declined. The implications of the transient rise and fall of DV Chi a content following illumination to the Chi a biosynthetic heterogeneity is discussed.     

PHOTOREACTIVATION ACTION SPECTRUM OF TOBACCO MOSAIC VIRUS-RIBONUCLEIC ACID INACTIVATED BY U.V. RADIATION*

Abstract— Initially photoreactivation of irradiated (2537 Å) nucleic acid on pinto bean increases linearly with time of illumination with white light of 250 ft-c. Maximum amounts of photo-reactivation depend on the quality of light used. The action spectrum shows a peak in the ‘black light’ region, where the greater amount of photoreactivation is found, and a shoulder in the blue light region. Maximum repair is obtained with ‘black light.’ Photoreactivation does not occur at wavelengths above 550 nm. Photoprotection by illumination of leaves prior to inoculation by irradiated RNA was not found. The action spectrum for photoreactivation does not resemble the action spectrum for photosynthesis.     

Photoionization and recombination delayed fluorescence in anthracene single crystals

The delayed fluorescence (DF), the action DF spectrum, the incident light intensity dependence of the DF and the DF decay of an anthracene single crystal at 77°K are analyzed. It is proposed that the delayed fluorescence originates from electron-hole recombinations after photogeneration of charge carriers by the near UV light illumination.