PROPERTIES OF IMMOBILIZED THYLAKOID MEMBRANES IN A PHOTOSYNTHETIC PHOTOELECTROCHEMICAL CELL

Abstract— Thylakoid membranes isolated from spinach were immobilized in a cross-linked albumin-glutaraldehyde matrix. Their properties in a single-compartment photoelectrochemical cell using platinum electrodes in potentiostatic mode were compared with the native material. The porous network of the immobilized tissue retained a relatively significant quantity of aqueous media which could be modified at will by aspiration of the proper media. In the presence of the acceptor potassium ferricyanide the photocurrent generation was enhanced up to a value of one hundred times higher than the maximum output of thylakoids entrapped in a polyvinyl alcohol film deposited on a Sn0₂ electrode. Immobilization in the albumin-glutaraldehyde matrix resulted in a better resistance to high pH, elevated temperatures and continuous exposure to high light intensity in potentiostatic mode (working conditions) in the electrochemical cell.     

ORGANIZATION OF PIGMENT-PROTEIN COMPLEXES INTO MACRODOMAINS IN THE THYLAKOID MEMBRANES OF WILD-TYPE and CHLOROPHYLL fo-LESS MUTANT OF BARLEY AS REVEALED BY CIRCULAR DICHROISM

The organization of pigment-protein complexes into large chiral macrodomains was investigated in wild-type and chlorophyll b-less mutant thylakoid membranes of barley. The variations in the anomalous circular dichroism bands and in the angular-dependence of circular intensity differential scattering showed that in wild-type chloroplasts, the formation of macrodomains was governed by interactions of the light-harvesting chlorophyll alb complexes (LHCII). Two external factors could be identified which regulate the parameters of the anomalous circular dichroism signal: (i) electrostatic screening by divalent cations under conditions that favor membrane stacking and (ii) the osmotic pressure of the medium, which is suggested to affect the lateral interactions between complexes and influence the packing-density of particles. These two factors governed preferentially the negative and the positive anomalous circular dichroism signals, respectively. In the chlorina f-2 mutant thylakoid membranes, deficient in most chlorophyll b binding proteins, the formation of macrodomains which gave rise to the anomalous circular dichroism signals was still regulated by these same external factors. However, in the absence of major LHCII polypeptides the formation of macrodomains was apparently mediated by other complexes having weaker interaction capabilities. As a consequence, the size of the macrodomains under comparable conditions appeared smaller in the mutant than in the wild-type thylakoid membranes. Circular dichroism is a valuable probe for examining the long-range interactions between pigment-protein complexes which participate in the formation and stabilization of membrane ultrastruc-ture. A functional role of macrodomains in long-range energy migration processes is proposed.     

CYCLIC VOLTAMMETRY MEASUREMENTS OF THE PHOTOELECTROGENIC REACTIONS OF THYLAKOID MEMBRANES

Abstract— In this paper, the technique of cyclic voltammetry has been used in a photoelectrochemical cell in order to follow the redox species formed in solution by the photo-induced electron transfer between the thylakoids and various acceptors and donors. The photoelectrochemical behavior of artificial electron acceptors (such as 2,5-dichlorobenzoquinone and methylviologen) and donors (such as sym -diphenylcarbazide and durohydroquinone) specific for either Photosystem I or Photosystem II has been investigated. The influence of inhibiting agents (such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea and Tris) on the cell photoresponse has also been characterized, together with the capability of donors to restore the photocurrent. Evidence for H₂O₂ formation by way of a Mehler-type reaction has been provided and an electrochemical model of its coupled photochemical and electrochemical reactions in solution is reported.     

MULTIFREQUENCY CROSS-CORRELATION PHASE FLUOROMETRY OF CHLOROPHYLL a FLUORESCENCE IN THYLAKOID AND PSII-ENRICHED MEMBRANES

The changes in plasma membrane polarity of polymorphonuclear leukocytes (PMN) during the activation of the respiratory burst were investigated by measuring the steady-state fluorescence emission spectra of 2-dimethylamino(6-lauroyl) naphthalene (Laurdan), which is known to be incorporated at the hydrophobic-hydro-philic interface of the bilayer, displaying spectral sensitivity to the polarity of its surroundings. Laurdan shows a marked steady-state emission blue shift in nonpolar solvents, with respect to polar solvents. Our results show a blue shift of the fluorescence emission spectra of Laurdan during activation of PMN with phorbol myristate acetate or AT-formyl-methionyl-leucyl-phenylalanine. These results suggest that the activation of the respiratory burst of PMN is accompanied by a decrease in polarity in the hydrophobic-hydrophilic interface of the plasma membrane.     

ALL-trans β-CAROTENE-5,6-EPOXIDE IN THYLAKOID MEMBRANES

All- trans β-carotene-5,6-epoxide has been found in the thylakoid membranes of spinach and of the cyanobacterium Synechococcus vulcanus Copeland. The epoxide was extracted from the thylakoid membranes with acetone, and was isolated by high-performance liquid chromatography (HPLC). The structure of the epoxide was identified by means of mass, Raman, and electronic absorption spectroscopy. Changes in the amount of the epoxide, as a result of epoxidation and (apparent) de-epoxidation reactions in the membranes, were traced by analysis of extracts on HPLC. In isolated thylakoid membranes, only the epoxidation reaction took place. The reaction was caused by irradiation or by the addition of ferricyanide, suggesting that electron transport reactions in the membranes are involved in the epoxidation. In intact spinach leaves, however, both epoxidation and de-epoxidation took place; the extent of epoxidation correlated with the intensity of light incident on the leaves. The epoxidation and de-epoxidation of all- trans β-carotene are contrasted with those of xanthophylls (in the violaxanthin cycle).     

SIMULTANEOUS SATURATION OF VARIABLE FLUORESCENCE YIELD AND PHOTOACOUSTICALLY MONITORED THERMAL EMISSION IN THYLAKOID MEMBRANES

Thermal and fluorescence emission from thylakoid membranes were monitored simultaneously in a photoacoustic cell. A close relationship existing between variable fluorescence and variable thermal emissions is demonstrated. The modulated measuring beam of the photoacoustic spectrophotometer was used to study the effect of light intensity on the energy storage yield and on the variable fluorescence yield. The half-saturation light intensities obtained for both parameters were 8.5 and 9.1 W m^?2, respectively. The similar sensitivity of energy storage and variable fluorescence yield to light intensity indicates that electron transfer with plastoquinone as last acceptor is probably responsible for the photoacoustically monitored energy storage.     

OXYGEN MEDIATED PHOTOSYSTEM I ACTIVITY IN THYLAKOID MEMBRANES MONITORED BY A PHOTOELECTROCHEMICAL CELL

Abstract— A photoelectrochemical cell has been used to monitor the effects of three enzymes on the photocurrent produced by isolated spinach thylakoids. The enzymes were glucose oxidase, superoxide dismutase and catalase. It is shown that all three inhibit the photocurrent to varying degrees. The results demonstrate that electron transport to the working electrode is mediated by oxygen. Further, the activity monitored originated from photosystem I with oxygen as the acceptor and photosystem II/plastoquinone as the donor. Thus, the photoelectrochemical cell constitutes a potential new approach for the monitoring of pseudocyclic electron transport.     

ANOXIC PHOTODAMAGE IN THE PRESENCE OF PORPHYRINS: EVIDENCE FOR THE LACK OF EFFECTS ON MITOCHONDRIAL MEMBRANES

Abstract When isolated mitochondria are irradiated in the presence of 50 μg/ml hematoporphyrin or hematoporphyrin derivative in the respiratory medium, the irradiation is at least twenty-fold less effective in impairing the Ca²⁺ pump in the absence of oxygen than in its presence. This result suggests that photosensitization by non oxygen-depending pathways has few or no effects on cell membranes, a result somewhat at variance with those observed with liposomes.     

ENERGY DISSIPATION AND PHOTOPROTECTION MECHANISMS DURING CHLOROPHYLL PHOTOBLEACHING IN THYLAKOID MEMBRANES

The kinetics of chlorophyll photobleaching were followed in whole thylakoid membranes as well as in photosystem I and photosystem II submembrane fractions. The onset of photobleaching was characterized by a slow rate which indicated the presence of energy traps implicated in the photoprotection of the bulk pigments. The pigments in photosystem I submembrane fractions bleached at a faster rate than those in photosystem II counterparts, the latter being more sensitive towards photoinhibition. An analysis of the pigment-protein complexes isolated from whole thylakoid membranes during the course of a photobleaching experiment has shown that the core-antenna complexes, including CP29, are more sensitive to illumination than the peripheral complexes. The absorption spectra of the CPI and CP29 complexes presented a blue shift of the red absorption maximum after partial photobleaching, indicative of a non-homogeneous bleaching of the holochromes in these complexes. An analysis of these data points towards the involvement of CP29 in a photoprotection mechanism at the level of photosystem II. The weaker resistance of photosystem I to photobleaching relative to photosystem II and its stronger resistance to photoinhibition is discussed in terms of an energy dissipation pathway in thylakoid membranes.     

GENETIC VARIATIONS AND LINOLENIC ACID INDUCED CHANGES IN THE ORIENTATION PATTERN OF CHLOROPHYLL a IN THYLAKOID MEMBRANES

Abstract Orientation pattern of the Q_y absorption and emission dipoles of chlorophyll a were studied in wild type Scenedesmus obliquus and in mutants deficient in chlorophyll b and carotenoids. Fluorescence polarization ratio at –140°C and linear dichroism at 25°C were measured in whole cells and thylakoids aligned in polyacrylamide gel. Unlike normal thylakoids, mutants displayed fluorescence polarization ratios significantly lower than 1.0 and showed a negative LD signal around 672 nm, indicating the tendency of the Q_y dipoles to tilt out from the membrane plane. Such an orientation pattern can also be artificially induced by treating normal thylakoids with linolenic acid.     

EVIDENCE FOR A MAGNESIUM DEPENDENT ATPase IN BOVINE ROD OUTER SEGMENT DISK MEMBRANES*†

Abstract—In the presence of Mg²⁺ and adenosine triphosphate (ATP), a rapid. light-induced, light-scattering transient is observed from bovine rod outer segments (ROS). This light-scattering transient we have labelled 'A'. Ca²⁺ cannot replace Mg²⁺. nor can guanosine triphosphate (GTP) replace ATP. 'A' is observed at ATP concentrations as low as a few μM.
The half-time of 'A', 60 ms at 20° and 20 ms at 37°, is consistent with a process possibly involved in visual transduction.
'A' has the action spectrum of rhodopsin bleaching and its amplitude is strictly proportional to the fraction of rhodopsin bleached per flash. 'A' can be regenerated by 11- cis retinal.
Inhibition studics with ATP analogues, which cannot be hydrolysed and fail to evoke an 'A' response, reveal that an ATP hydrolysis process has to precede illumination in order for 'A' to occur.
On the basis of the above findings. it is proposed that there is a Mg²⁺ dependent ATPase in ROS that allows the disk membrane to assume a new membrane state which, upon illumination, is altered. giving rise to the structural phenomenon monitored as light-scattering transient 'A'.     

MODE OF ACTION OF α-TERTHIENYL ON ESCHERICHIA COLI: EVIDENCE FOR A PHOTODYNAMIC EFFECT ON MEMBRANES

The photodynamic effects of α-terthienyl (αT) in near-UV light (UV-A) on Escherichia coli showed close agreement with the light absorption of αT at different wavelengths suggesting that αT is the primary absorbing molecule responsible for the photosensitized reaction. Studies with DNA repair deficient mutants of E. coli indicated that the bactericidal action of αT/UV-A was not mediated by DNA damage, in direct contrast to the well-known photosensitizer, 8-methoxypsoralen. By using a closed borosilicate glass reaction vessel and various gas mixtures, it was demonstrated that photosensitization of both E. coli and a more resistant bacterium, Pseudomonas aeruginosa , was absolutely dependent on the presence of oxygen. The rate of killing by αT/UV-A showed a rather small dependence on preincubation temperatures, with quite rapid killing at 5°C, suggesting that the movement of αT across the cytoplasmic membrane of E. coli is not the rate limiting step in killing and perhaps is not even necessary for killing. Sodium dodecyl sulphate-polyacrylamide gels of cell membrane proteins after 15 and 30min of treatment with αT/UV-A showed substantial random crosslinking of these proteins. The results taken overall suggest that αT is a photodynamic photosensitizer which exerts its primary effect at the level of the cytoplasmic membrane.     

AQUEOUS AGGREGATES OF BACTERIOCHLOROPHYLL c AS A MODEL FOR PIGMENT ORGANIZATION IN CHLOROSOMES

Chlorosomes isolated from Chloroflexus aurantiacus were extracted with chloroform/methanol. The extract contained bacteriochlorophylls c and a and lipids, but was devoid of proteins. This crude extract spontaneously formed aggregates when a methanol solution was dispersed in aqueous buffer. The aggregates could be sedimented by ultracentrifugation and appeared in electron micrographs as stain-excluding bodies with diameters between 70 and 170 nm. The absorption spectrum is remarkably similar to that of intact chlorosomes with an absorption maximum of bacteriochlorophyll c at around 740 nm. The circular dichroism spectrum of the aggregate is also very similar to that of intact chlorosomes. A conservative (±) band centered at 740 nm confirms the highly aggregated state of bacteriochlorophyll c in both systems. Steady-state fluorescence studies showed that in the aggregate energy-transfer from bacteriochlorophyll c to a component emitting at 830 nm took place. When the aggregate was suspended in buffer saturated with 1-hexanol the 740 nm form of bacteriochlorophyll c was reversibly converted to a form with spectral properties resembling the monomer absorbing at 670 nm but still in an aggregated state. This form of bacteriochlorophyll c showed no circular dichroism signal.     

认可机制下实验室人员培训工作的组织与开展

针对部分实验室在人员培训工作中存在的培训模式不清、培训机制不健全、培训质量较低的问题,系统地分析了计量实验室人员培训所涉及的主要类别、内容及要求等。对培训工作的流程逐个进行详尽地描述,分内部培训和外部培训两个方面对培训中应注意的问题进行重点说明,为广大计量实验室管理人员开展人员培训工作提供借鉴。     

USE OF THE FLUORESCENT LANTHANIDE Tb3+ AS A PROBE FOR CATION-BINDING SITES ASSOCIATED WITH ISOLATED CHLOROPLAST THYLAKOID MEMBRANES

Abstract— The fluorescent properties of the rare-earth ion, Tb³⁺ have been utilized to probe the nature of cation-binding sites associated with thylakoid membranes. At low concentrations (< 100μ M ), Tb³⁺ was observed to inhibit the increase in chlorophyll a fluorescence normally seen on adding 5 m M MgCl₂ (or 100 m M NaCl) to isolated, broken chloroplasts. We also observed under these conditions, the appearance of a new band around 280 nm in the excitation spectrum of Tb³⁺ ion fluorescence. However, similar changes in Tb³⁺ fluorescence could be observed in the presence of a membrane-free preparation of chloroplast coupling factor protein (CF₁). From this and other results it is concluded that changes in Tb³⁺ fluorescence reflect an association of the ion with CF₁ followed by intermolecular transfer of excitation energy from protein ligands (possibly un-ionized tyrosine residues) to the lanthanide. The interaction of Tb³⁺ with sites which control chlorophyll a fluorescence does not seem to modify Tb³⁺ fluorescence, suggesting that in this case, simple membrane-bound ligands such as carboxyl or phosphate groups are involved.     

EVIDENCE FOR THE EXISTENCE OF MEMBRANE-ASSOCIATED PHYTOCHROME IN THE CELL

Abstract Comparative fluorescence and photochemical studies of phytochrome in etiolated seedlings of maize and in soluble and membrane-containing fractions isolated from them were camed out. The membrane fractions prepared in the absence of Mg²⁺ from etiolated coleoptiles contained 13% of total photoreversible phytochrome, which was readily solubilized by mild detergents. Its molecular size was indistinguishable from soluble phytochrome and equal to nondegraded maize phytochrome. Low-temperature fluorescence studies with intact tissue found that the position of the emission maximum at 85 K (λ_max) and the extent of the phototransformation of the red-absorbing form (Pr) into the first stable photoproduct, lumi-R, at 85 K (γ₁), varied in different parts of etiolated seedlings: λ_max and γ₁ reached their maximum values in the tips of coleoptiles and roots, 686 nm and 0.30–0.40, whereas the lowest values, 682 nm and ca 0.05, were observed in the root base. These parameters correlated well with those obtained for the pigment in the soluble and membrane-containing fractions: 684 and 680 nm, and 0.33 and 0.06, respectively. The extent of the Pr phototransformation into the far red-absorbing form (Pfr) (γ₂) did not differ much: values of 0.80–0.85 and 0.70–0.75 correlated with the high and low values of γ₁. These variations of the parameters were interpreted in agreement with our previous observations in terms of two phytochrome A species whose relative concentrations vary depending on the experimental conditions—the longer wavelength bulk light-labile species with high γ₁ (Pr″), and the shorter wavelength minor light-stable species with low γ₁ (Pr″). Close similarity between Pr’and the soluble phytochrome and between Pr″ and the membrane-bound phytochrome points to the possible origin of the native Pr’and PrPrime; species, thus providing evidence for the existence of membrane-bound pigment in the cell.     

PHOTOBLEACHING OF A CYANINE DYE IN SOLUTION AND IN MEMBRANES

Abstract— N,N'-bis(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC), a lipophilic dye with a delocalized positive charge, photosensitizes cells to visible irradiation. In phosphate-buffered saline (PBS), EDKC absorbs maximally at 700 nm (ε= 1.2 × 10⁵ M⁻¹ cm⁻¹) and in methanol, the absorption maximum is at 706 nm (ε= 2.3 × 10⁵ M⁻¹ cm⁻¹). EDKC partitions from PBS into small unilamellar liposomes prepared from saturated phospholipids and into membranes prepared from red blood cells (RBC) and binds to human serum albumin (HSA). The EDKC fluorescence maximum red shifts from 713 nm in PBS to 720–725 nm in liposomes and RBC membranes and the fluorescence intensity is enhanced by factors of 14–35 compared to PBS (φ= 0.0046). EDKC is thermally unstable in PBS (T_1/2= 2 h at 1.3 × 10⁻⁵ M EDKC), but stable in methanol. In liposomes and RBC membranes, EDKC is 10 times more stable than in PBS, indicating that it is only partially exposed to the aqueous phase. Quenching of EDKC fluorescence in liposomes and RBC membranes by trinitrobenzene sulfonate also indicates that EDKC is not buried within the membranes. Photodecomposition of EDKC was oxygen-dependent and occurred with a low quantum yield (6.4 × 10⁻⁴ in PBS). Singlet oxygen was not detected upon irradiation of EDKC in membranes or with HSA since the self-sensitized oxidation of EDKC occurred at the same rate in D₂O as in H₂O and was not quenched by sodium azide or histidine.     

EVIDENCE FOR INVOLVEMENT OF PHYTOCHROME REGULATION IN MALE STERILITY OF A MUTANT OF Oryza sativa L.

Abstract— A rice mutant ( Oryza sativa L. Nongken 58S) "Hubei photoperiod-sensitive genie male-sterile rice" ( ms mutant) has been found to be male sterile under long day cycles (LD) and fertile in short day cycles (SD). After formation of the secondary rachis-branch primodia the mutant plants under SD were interrupted in the middle of the long night phase (night break) for 10 days with 5 min pulses of red light (R) or far-red light (FR). Rates of normal pollen and seed setting of the mutant treated by R or R → FR → R declined significantly, while the rates after FR or R → FR treatments were similar to those under SD alone. The result of these induction reversion experiments is consistent with the operational criteria for the involvement of photochrome. Wild-type rice ( O. sativa L. Nongken 58) under the same treatment showed no change in fertility. Experiments on the effect of different dark intervals (20 s to 15 min) between R and FR on male sterility of the ms mutant showed that the longer the dark interval, the greater the escape of R induction from FR reversibility. Treatment with SD or LD after formation of pollen mother cells had no influence on fertility of the ms mutant plants treated previously with R or FR night breaks.     

PHOTOREACTIVATION IN A phrB MUTANT OF Escherichia coli K-12: EVIDENCE FOR THE ROLE OF A SECOND PROTEIN IN PHOTOREPAIR

Abstract In Escherichia coli , the light-dependent repair of pyrimidine dimers in UV-irradiated DNA is now accepted as being due to enzymatic photoreactivation (PR) by a 50 kDa enzyme, photolyase (EC 4.1.99.3). The gene for this enzyme has been mapped at 16.2 min and designated phr . This gene was earlier described as phr B, another locus phr A having been proposed in association with PR. The relevance of the putative phr A gene has now been placed in doubt. The recent report of the discovery of a photoreactivating enzyme in Drosphila melanogaster . which specifically repairs pyrimidine (6–4) pyrimidone photoproducts ([6–4] photoproducts), and that E. coli does possess a protein with specific affinity for the (6–4) photoproduct, has cast new light on the prospective role of phr A in PR. We have determined the nucleotide sequence of the putative phr A gene, which suggests it codes for a protein of 38 kDa. When the putative phr A gene was cloned into an expression vector and transformed into a phr A phr B mutant of E. coli , a level of photorepair was observed, which could correspond to repair of (6–4) photoproducts.