Limiting steps in Photosystem II and water decomposition in Chlorella and spinach chloroplasts

1. 1. By varying the redox potential of a chloroplast suspension, we obtained new evidence for an equilibrium between states S₀ and S₁ in the model of Kok, B., Forbush, B. and McGloin, N. (1970, Photochem. Photobiol. 11, 457–475). The mid-point potential of the S₀ to S₁ couple is close to that for the pool of the electron acceptor of System II, A to A⁻.

2. 2. The limiting steps between two consecutive photoreactions of System II in Chlorella and spinach chloroplasts, have been studied.

2.1. (a) The limiting step from S₁ to S₂ (noted γ₁ (Δt)) is not exponential. Its temperature coefficient becomes greater as the reaction proceeds. The shape of the kinetics is an intrinsic property of each center. Chloroplasts fixed with 2% glutaraldehyde, show simple first order kinetics.

2.2. (b) The limiting step from S₀ to S₁ (γ₀ (Δt)) exhibits the same characteristics as γ₁ (Δt)).

2.3. (c) The limiting step from S₂ to S₃ (γ₂ (Δt)) shows sigmoidal kinetics; two reactions are involved. One of the reactions exhibits the same properties as γ₀ (Δt) and γ₁ (Δt).

2.4. (d) The limiting step from S₃ to S₀ (γ₃ (Δt)) is a first order reaction, two times slower than the other transitions. This reaction is interpretated in terms of oxygen release.

3. 3. We also studied the limiting steps in the presence of low concentrations (50 μM) of hydroxylamine. The results favor the binding of two molecules of hydroxylamine to every photochemical center.

Evidence for the role of a bound ferredoxin as the primary electron acceptor of Photosystem I in spinach chloroplasts

The presence of a bound electron transport component in spinach chloroplasts with an EPR spectrum characteristic of a ferredoxin has been confirmed. The ferredoxin is photoreduced at 77 °K or at room temperature, it is not reduced in the dark by Na₂S₂O₄. The distribution of the ferredoxin in subchloroplast particles has been investigated. The ferredoxin is enriched in Photosystem I particles and it is proposed that it functions as primary electron acceptor for Photosystem I.

The EPR spectra indicate the presence of two components which are photoreduced sequentially. It is proposed that they may represent two active centres of a single protein.     

The effect of salt concentration on the fluorescence parameters of isolated chloroplasts

The previously reported effect of salt concentration on the fluorescence and other photochemical activities of Photosystem II is interpreted in terms of a change in the radiationless transition and the trapping probabilities. This is confirmed by quantitative comparison of the fluorescence and the photochemical activity. As a by-product of this analysis a method is devised to estimate the background fluorescence.

We did not eliminate the possibility that the radiationless transition constant may include a contribution of energy transfer from Photosystem II to Photosystem I.     

Freezing the effect of eutectic crystallization on biological membranes

Thylakoids from isolated spinach chloroplasts were frozen in the presence of various concentrations of inorganic and organic salts, amino acids and sugars and the kinetics of inactivation of cyclic photophosphorylation with phenazine methosulfate and of electron transport reactions were measured as a function of temperature.During freezing of membranes in the presence of neutral nontoxic compounds membrane damage did not occur until the eutectic temperature was reached. Then photophosphorylation became rapidly inactivated. With weakly membrane-toxic compounds there was a slow inactivation during freezing followed by rapid inactivation at the eutectic temperature. Freezing in the presence of strongly membrane-toxic compounds led to inactivation of photophosphorylation before the eutectic temperature was reached. The temperature at which eutectic crystallization occurred was dependent on the nature of the solutes present. The ratio between solute and membranes was also important: the lower the initial concentration of solutes added to membrane suspensions the lower the temperature at which eutectic solidification occurred. Some compounds such as mannitol crystallized gradually during the decrease in temperature; in this case inactivation of photophosphorylation took place parallel to the crystallization process.In contrast to photophosphorylation, electron transport reactions were not decreased during eutectic freezing in the presence of neutral membrane-protective compounds. Rather a stimulation of electron transport was observed. However, in the presence of inorganic salts or of sodium succinate, electron transport reactions were also inactivated in addition to photophosphorylation during eutectic solidification. This inactivation seems to be a salt effect and may not directly be related to the crystallization process. Various soluble enzymes and the Ca²⁺-dependent ATPase of thylakoids were not affected by eutectic crystallization.The results demonstrate that eutectic crystallization which may take place during freezing is a factor in membrane damage and has to be considered as a possible cause of membrane alterations in in vitro studies on freezing resistance.     

Free amino acids in rat ocular tissues during postnatal development

Amino acid changes in the retina, vitreous, lens, iris-ciliary body and cornea of the rat eye were determined during postnatal growth. The amino acid concentrations of the ocular tissues showed varying profiles at various developmental stages. These results suggest a different timetable for development of each ocular tissue or indicate a synthesis of specific proteins in the postnatal period. Adult amino acid levels appeared to be fully reached on the 30th day after birth at the latest. Quantitatively the greatest changes were observed in taurine concentrations, which increased in all five ocular tissues during maturation. GABA changes paralleled those of taurine in the retina, whereas in the other ocular tissues GABA changes were very low. The greatest decrease in glutamic acid and aspartic acid concentration during postnatal development was in the lens, where these amino acids probably are needed for the synthesis of the lenticular proteins, the alpha-, beta-, and gamma-crystallines.     

Effects of freezing on biological membranes in vivo and in vitro

Membrane inactivation by freezing has been investigated using intact spinach leaves and isolated thylakoid membranes from chloroplasts of leaf cells as test material. During freezing in vitro in solutions containing neutral solute and a slight excess of inorganic salts such as NaCl, electron transport is stimulated while photophosphorylation is lost. Under more drastic freezing conditions damage increases, affecting dichlorophenolindophenol reduction, the rise in variable fluorescence, ferricyanide reduction and electron transport through Photosystem I, in that order. Semipolar compounds such as phenylalanine or phenylpyruvate exhibit a much higher membrane toxicity during freezing than inorganic salts. The profile of damage caused by this class of compounds is different from that caused by salts. Damage to membranes isolated rapidly from frost-killed leaves is similar to that produced by semipolar compounds during freezing in vitro. A few sites of damage could be identified, among them the site responsible for oxidation of water during photosynthesis. The results support the view that the sensitivity of their membranes limits the ability of cells to withstand freezing and suggest that freezing sensitivity is due to the accumulation in the cells of potentially membrane-toxic organic and norganic cell constituents.     

Electron paramagnetic resonance spectra of mitochondrial and microsomal cytochrome P-450 from the rat adrenal

The electron paramagnetic resonance (EPR) spectra of rat adrenal zona fasciculata mitochondria showed peaks corresponding to low spin ferric cytochrome P-450 with apparent g values of 2.424, 2.248 and 1.917, and weak signals due to high spin ferric cytochrome P-450 with g_x values of 8.08 and 7.80. The former is attributed to cholesterol side chain cleavage cytochrome P-450, the latter to 11β-hydroxylase cytochrome P-450. On addition of deoxycorticosterone the g = 7.80 signal was elevated and there was an associated drop in the low spin signal. As the pH was reduced from 7.4 to 6.1, the g = 8.08 signal increased with again a drop in intensity of the low spin signal. Mitochondria from the zona glomerulosa showed similar spectral properties to those described above. Addition of succinate, isocitrate or pregnenolone caused a loss of the g = 8.08 signal. Addition of calcium increased the magnitude of the g = 8.08 signal, and caused a slight reduction in the magnitude of the low spin signal. Also, addition of deoxycorticosterone, pregnenolone, succinate or isocitrate caused slight shifts of the outer lines of the low spin spectrum. Interaction of mitochondrial cytochrome P-450 with metyrapone and aminoglutethimide modified the low spin parameters. Adrenal microsomal cytochrome P-450 had low spin ferric g values of 2.417, 2.244 and 1.919 and high spin ferric g_xy values of 7.90 and 3.85, distinct from the values obtained with mitochondria.     

Transport of cationic and zwitterionic amino acids in preimplantation rat conceptuses

The ability of preimplantation rat conceptuses to take up several amino acids was examined under a variety of conditions, and the characteristics of uptake were compared to those determined previously for mouse conceptuses. Mediated leucine transport in two-cell rat conceptuses is Na(+)-independent and inhibited almost completely by 2-amino-endobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), so it resembles system L which predominates in two-cell mouse conceptuses. System L becomes less conspicuous than homoarginine-sensitive, Na(+)-independent leucine transport (provisionally designated system bo,+) by the time rat conceptuses develop into blastocysts, as is also the case for mouse conceptuses. In contrast to leucine transport, system bo,+ appears to be the most conspicuous transporter of cationic amino acids throughout preimplantation development of both species. A Na(+)-independent cation-preferring amino acid transport process also appears to be present in rat as well as in mouse conceptuses. Moreover, rat conceptuses resemble mouse conceptuses because Na(+)-dependent transport system Gly activity virtually disappears from them by the time they form blastocysts. Unlike mouse conceptuses, however, Na(+)-dependent system Bo,+ activity appears to be present throughout preimplantation development of rat conceptuses, whereas it has not been detected until at least the two-cell stage in the mouse. Although system Bo,+ becomes more conspicuous in mouse than in rat conceptuses by the time they form blastocysts, system Bo,+ activity appears to increase when blastocysts of both species are removed from the uterus just prior to implantation. The latter observation is consistent with the possibility that system Bo,+ activity is controlled, in part, by the uterus near the time of implantation, although further studies are needed to verify this possibility. Similarities as well as differences in the amino acid transport processes present in conceptuses of rats and mice may eventually be understood best in relation to the environments in which they develop in vitro and in situ.     

Reduced nicotinamide adenine dinucleotide dependent reduction of fumarate coupled to membrane energization in a cytochrome deficient mutant of Escherichia coli K12

Escherichia coli SASX76 does not form cytochromes unless supplemented with 5-aminolevulinic acid. It can grow anaerobically on glycerol and dl-glycerol 3-phosphate in the absence of 5-aminolevulinic acid with fumarate but not with nitrate as the terminal electron acceptor. Cytochrome-independent NADH oxidase, glycerol 3-phosphate- and NADH-fumarate oxidoreductase activities are induced by anaerobic growth on a glycerol-fumarate medium. The pathway of electrons from substrate to fumarate involves menaquinone. The NADH-fumarate oxidoreductase and cytochrome-independent NADH oxidase systems are inhibited by piericidin A, 2-heptyl-4-hydroxyquinoline N-oxide, and iron chelating agents. Both systems can energize the membrane particles as indicated by quenching of atebrin fluorescence.     

Transport systems for GABA and for other amino acids in incubated chick brain tissue during development

The transport of six amino acids (GABA(γ-aminobutyric acid), glycine, AIB (α-aminoisobutyric acid), leucine, d-glutamate, and lysine) was studied in brain slices from chick embryos and young chicks at different developmental stages.     

Alpha-fetoprotein is dynamically expressed in rat pancreas during development

To identify proteins involved in pancreatic development, we used a differential proteomics approach by comparing pancreatic extracts from four biologically significant stages of development: embryonic day (E) 15.5, E18.5, postnatal (P) days 0 and adult. By two-dimensional gel electrophoresis (2D-E) and MALDI-TOF MS (Matrix Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) following database searching and protein annotation, 15 proteins were identified as being differently expressed in the pancreas between the four phases. The expression pattern and the localization of alpha-fetoprotein (AFP), one of significant changed proteins observed, were further determined. Four isoforms of AFP (72 kDa, 60 kDa, 48 kDa and 37 kDa) were found by Western blotting in the pancreas tested, most of them showed a stronger signal in E18.5 followed by a steady decrease and only a 60-kDa isoform was detected in the adult pancreas. Immunolocalization for AFP revealed that a positive reactivity was detectable at E15.5 pancreas, became stronger in the cytoplasm of mesenchyme cells at E18.5, and declined after birth to a nearly undetectable level in adults. The dynamic expression of AFP in rat pancreas from different stages indicates that AFP might be involved in some aspects of pancreatic development.     

Free amino acids in Phycomyces blakesleeanus during development

In Phycomyces blakesleeanus the total level of free amino acids and their relative distributions change strikingly during the development of sporangiophores. Very high levels of serine, glutamate and histidine are found. The variety of amino acids becomes more restricted in sporangiophores grown on nitrogen-rich media; levels of arginine increase dramatically. The distribution differs in mycelia. No unusual amino acids or oligopeptides were found.     

Free amino acids in rat blood serum during A-hypovitaminosis]

A pool of free aminoacids of the blood serum has been studied in growing rats with a decreased retinol content in the liver tissue (to 5 g/g). The study shows a reliable drop in the concentrations of all essential aminoacids, except for phenylalanine. It is determined that the total amount of essential aminoacids decreases by 31%. At the same time concentrations of replaceable aminoacids increase, namely: glutamic acid and arginine--by 15.7%, ornithine and histidine--by 24%-45%, though proline concentration decreases abruptly by 31%. A disturbance in ultrastructural organization of microvilli in the apical membrane cells absorptive epithelium of the small intestine has been found. The results of the study confirm changes in a free aminoacid pool of the blood serum in growing rats and these changes occur first of all due to the disturbance of the absorption processes in the small intestine.     

Transport of branched-chain amino acids in Corynebacterium glutamicum

The transport of branched-chain amino acids was characterized in intact cells of Corynebacterium glutamicum ATCC 13032. Uptake and accumulation of these amino acids occur via a common specific carrier with slightly different affiniteis for each substrate (K _m[Ile]=5.4 M, K _m[Leu]=9.0 M, K _m[Val]=9.5 M). The maximal uptake rates for all three substrates were very similar (0.94–1.30 nmol/mg dw · min). The optimum of amino acid uptake was at pH 8.5 and the activation energy was determined to be 80 kJ/mol. The transport activity showed a marked dependence on the presence of Na⁺ ions and on the membrane potential, but was independent of an existing proton gradient. It is concluded, that uptake of branched-chain amino acid transport proceeds via a secondary active Na⁺-coupled symport mechanism.Abbreviations CCCP Carboxyl cyanide m-chlorophenylhydrazone - dw dry weight - MES 2[N-morpholino]ethanesulfonic acid - mon monensin - nig nigericin - TPP tetraphenylphosphonium bromide - Tris tris[hydroxymethyl]aminomethane - val valinomycin     

Transport of basic amino acids in Candida albicans

In Candida albicans, ATCC 46977, transport of basic amino acids is mediated by two systems (S1 and S2). Kinetic data and competitive inhibition studies of the different systems showed that transport of L-lysine, L-arginine and L-histidine have distinct specificities. System S1 of L-lysine and L-arginine was highly specific for the respective single basic amino acid. However, S2 of L-lysine and S1 of L-histidine were shown to be specific systems for most of basic amino acids. S2 of L-arginine was different from S2 of L-lysine and S1 of L-histidine. The effect of a thiol reagent, N-ethylmalemide, revealed that S2 of L-lysine and S1 of L-histidine were sensitive to this reagent, while all other systems were insensitive. The transport activity of different systems of L-lysine, L-arginine and L-histidine was followed during the growth of C. albicans. It was observed that different basic amino-acid systems have maximum activity during different stages of C. albicans growth.