Inner and outer immobilization of chloroplasts]

The effects of glutaric aldehyde on pea leave chloroplasts and their inactivation kinetics were studied. Optimization of the chloroplasts fixation by glutaric aldehyde resulted in a 5-fold increase of stability of the chloroplasts. Immobilization of the chloroplasts in agar-agar gels was performed; the ability of chloroplasts for photooxidation of H2O was thereby retained. Immobilization did not actually affect the stability of chloroplasts. The inactivation kinetics of fixed and immobilized chloroplasts are in good agreement with the previously described model for inactivation of native chloroplasts.     

Absorption and Transfer of Light and Photoreduction Activities of Spinach Chloroplasts under Calcium Deficiency: Promotion by Cerium

Chloroplasts were isolated from spinach cultured in calcium-deficient, cerium-chloride-administered calcium-present Hoagland’s media or that of calcium-deficient Hoagland’s media and demonstrated the effects of cerium on distribution of light energy between photosystems II and I and photochemical activities of spinach chloroplast grown in calcium-deficient media. It was observed that calcium deprivation significantly inhibited light absorption, energy transfer from LHCII to photosystemII, excitation energy distribution from PSI to PSII, and transformation from light energy to electron energy and oxygen evolution of chloroplasts. However, cerium treatment to calcium-deficient chloroplasts could obviously improve light absorption and excitation energy distribution from photosystem I to photosystem II and increase activity of whole chain electron transport, photosystems II and I DCPIP photoreduction, and oxygen evolution of chloroplasts. The results suggested that cerium under calcium deficiency condition could substitute for calcium in chloroplasts, maintain the stability of chloroplast membrane, and improve photosynthesis of spinach chloroplast, but the mechanisms still need further study.     

Second set method for determining the immunogenicity of heart valves

The "second set" method was used on inbred rats to study immunogenicity of the heart valves treated with a proteolytic enzyme and glutaric aldehyde and to compare it with immunogenicity of the valves treated with glutaric aldehyde alone according to Hancock's method. The valves treated by the enzyme and 0.2-0.5% glutaric aldehyde did not lead to the body sensitization in contrast to the valves exposed to 0.5% glutaric aldehyde alone. During transplantation of the latter ones, there were signs of the immunologic response on the part of the recipients and calcification of valvular tissue 70 days after subcutaneous implantation. It is assumed that pretreatment with the enzyme makes it possible to appreciably reduce immunogenicity of the heart valves.     

Effect of chemical modification on thermal stability of horseradish peroxidase]

Soluble preparations of horse radish peroxidase are obtained by means of its amino groups modification with glutaric aldehyde, maleic anhydride and inert proteins including albumin. The enzyme activity is found to decrease under the modification with glutaric aldehyde and to be unchanged at all other cases. Thermal stability of the enzyme preparations obtained is studied within the temperature range from 56 to 80 degrees C. Thermostability of glutaric aldehyde-modified peroxidase is approximately 2.5-fold decreased at 56 degrees C. Thermostability of other preparations exceeds the stability of native peroxidase in 25--90 times at 56 degrees C. Thermodynamic parameters of activation for the process of irreversible thermoinactivation of native and modified enzyme are calculated. A strong compensation effect between activation enthalpy and entropy values is observed, which were changed in 1.5--2 times, while the free activation energy is changed by 2--3 kcal/mol only. Possible mechanism of the change of the enzyme thermal stability under its chemical modification is discussed.     

Photosynthetic carbon metabolism in isolated pea chloroplasts: metabolite levels and enzyme activities

We report here that enzyme activation precedes the rise in metabolite levels, which appear to limit photosynthetic CO2 fixation during induction in pea leaf chloroplasts. Therefore light activation may be required for the build-up of photosynthetic intermediates and hence for photosynthesis in isolated chloroplasts. Analysis of metabolite levels and the known kinetic properties of the chloroplast enzymes indicates that the reductive pentose phosphate cycle is subject to control which fluctuates between several points during induction and when CO2 fixation is maximal. The transketolase-aldolase-catalyzed reactions around sedoheptulose-biphosphatase appear to provide a simple and effective primary control for photosynthetic CO2 fixation. When substrate levels and enzyme active site concentrations are taken into account, there is insufficient glyceraldehyde 3-phosphate dehydrogenase, aldolase, and transketolase activity to support photosynthetic CO2 fixation at observed rates. These results suggest that there may be direct transfer of glyceraldehyde 3-phosphate among these enzymes in the pea chloroplast.     

The role of charge-transfer states in the spectral tuning of antenna complexes of purple bacteria

Local damage (mainly burning, heating, and mechanical wounding) induces propagation of electrical signals, namely, variation potentials, which are important signals during the life of plants that regulate different physiological processes, including photosynthesis. It is known that the variation potential decreases the rate of CO₂ assimilation by the Calvin–Benson cycle; however, its influence on light reactions has been poorly investigated. The aim of our work was to investigate the influence of the variation potential on the light energy flow that is absorbed, trapped and dissipated per active reaction centre in photosystem II and on the flow of electrons through the chloroplast electron transport chain. We analysed chlorophyll fluorescence in pea leaves using JIP-test and PAM-fluorometry; we also investigated delayed fluorescence. The electrical signals were registered using extracellular electrodes. We showed that the burning-induced variation potential stimulated a nonphotochemical loss of energy in photosystem II under dark conditions. It was also shown that the variation potential gradually increased the flow of light energy absorbed, trapped and dissipated by photosystem II. These changes were likely caused by an increase in the fraction of absorbed light distributed to photosystem II. In addition, the variation potential induced a transient increase in electron flow through the photosynthetic electron transport chain. Some probable mechanisms for the influence of the variation potential on the light reactions of photosynthesis (including the potential role of intracellular pH decrease) are discussed in the work.     

THE OSMOTIC EFFECTS OF ELECTRON MICROSCOPE FIXATIVES

The reflecting cells on the scales of sprat and herring contain ordered arrays of guanine crystals. The spacing of the crystals within these cells determines the wave bands of the light which they reflect, hence volume changes in the reflecting cells can be observed as color changes directly. This property of the scales is used to show that (a) fixation with osmium tetroxide solutions destroys osmotic activity; (b) fixation with aldehyde solutions does not destroy osmotic activity and does not cause volume changes if the aldehydes are made up in salt or sucrose solutions whose osmolarities, discounting the aldehyde, are about 60% of those to which the cells are in equilibrium in life, and (c) after aldehyde fixation the cells are osmotically active but come to a given volume in salt and sucrose solutions of concentrations only 60% of those which give their volume before fixation. Various possible mechanisms underlying the change of osmotic equilibrium caused by aldehyde fixation are discussed.     

Sedimentation behavior of chloroplast ribosomes from Chlamydomonas reinhardtii.

The identity of peaks generated by chloroplast ribosomes of Chlamydomonas reinhardtii were determined by zone velocity sedimentation on sucrose density gradients, and analysis of distribution of ribosomal RNAs in the gradients. The sedimentagion coefficient of the principal peak was 66-70 S (usually 69 S), in good agreement with previously reported values for chloroplast ribosomes of C. reinhardtii, and other organisms. The fast sedimenting side of the 69 S peak contained an excess of chloroplast large subunit. When ribosome dissociation was prevented by sedimentation at low velocity, by aldehyde fixation, or by the presence of nascent polypeptide chains, the principal peak had a sedimentation coefficient of about 75 S. Thus the 69 S peak was an artifact caused by dissociation during centrifugation. Peaks that contained chloroplast ribosomal RNAs were also observed at '60 S' and '45 S' when chloroplast ribosomes were centrifuged unfixed at high velocity. The amounts of '60 S' and '45 S' components were decreased by centrifugation at low speed, or fixation, but sedimentation coefficients remained unchanged. The '60 S', and '45 S' components were identified as large, and small subunits of chloroplast ribosomes, respectively. The artifacts produced by centrifugation of chloroplast ribosomes, are similar to the artifacts produced by centrifuging ribosomes of Escherichia coli. Similar explanations appear to apply to both. We concluded that the 69 S chloroplast ribosome peak occurs because of dissociation of 'tight' couples, and incomplete separation of subunits. Subunit peaks (60 S and 45 S) arise from free subunits, and/or from dissociation of 'loose' couples.     

Kinky microtubules: bending and breaking induced by fixation in vitro with glutaraldehyde and formaldehyde.

We have employed video-enhanced light microscopy to study alterations of the overall shape of microtubules that are produced by the aldehyde fixation methods commonly employed to study them in vitro. Changes brought about by these methods include deformation and breakage. The severity of the effects depends on the fixative employed and increases with its concentration, and with the time of fixation. The changes are observed under a variety of conditions, such as brief exposure to 3.7% formaldehyde, or somewhat longer exposure to glutaraldehyde at concentrations as low as 0.05%. The observed distortion explains why microtubules usually appear curved or sinuous in electron micrographs while appearing relatively rigid and linear in video-enhanced light microscopy. The observed breakage implies that caution must be used in inferring length distributions from measurements of aldehyde-fixed microtubules.     

Some physicochemical properties of modified trypsin]

Physico-chemical properties of trypsin covalently bound with human serum albumin by glutaric aldehyde have been studied. The modification of the enzyme practically caused no changes in the pH optimum of trypsin. The inhibition of modified trypsin by inhibitors from soy beans and human blood serum has been also studied. The apparent inhibition constants have been calculated. The modification has been shown to result in a deceleration of autolytic degradation. The autolysis rate constants have been calculated at 50 degrees C.     

The Relationships between Nitrogen Fixation and Several Kinds of Energy Metabolism by Gloeocapsa sp

Gloeocapsa sp., a species of anicellular blue-green alga, fixes dinitrogen mostly under light. The energy (ATP and reductant) needed for nitrogen fixation may be provided by photoreaction and aerobic catabolism. The nitrogenase activity (acetylene reduction) in vivo was decreased under the conditions of dark and inhibition of photo-phosphorylation or oxidative phosphorylation in the light. When photosystem Ⅱ was inhibited by the presence of DCMU, nitrogenase activities in both reactions of acetylene reduction and hydrogen evolution may be muchenhanced probably due to eliminating of the damage caused by the oxygen produced in the photolysis of water. The effects of the oxygen present in the atmosphere of the reaction systemand produced by the cells are different. It is shown that some trace oxygen seems to be required for nitrogen fixation by the energy supply of aerobic actabolism and oxidative phosphorylation. While the fixation of dinitrogen was inhibited by CO or no any reducible substrate was present, 70-100% of the energy accepted by nitrogenase was evolved as hydrogen. The algal cells also showed hydrogen uptake reaction, but no enhancement of nitrogen fixation by the hydrogen uptake was found.     

The relevance of dynamic thylakoid organisation to photosynthetic regulation

The higher plant chloroplast thylakoid membrane system performs the light-dependent reactions of photosynthesis. These provide the ATP and NADPH required for the fixation of CO₂ into biomass by the Calvin-Benson cycle and a range of other metabolic reactions in the stroma. Land plants are frequently challenged by fluctuations in their environment, such as light, nutrient and water availability, which can create a mismatch between the amounts of ATP and NADPH produced and the amounts required by the downstream metabolism. Left unchecked, such imbalances can lead to the production of reactive oxygen species that damage the plant and harm productivity. Fortunately, plants have evolved a complex range of regulatory processes to avoid or minimize such deleterious effects by controlling the efficiency of light harvesting and electron transfer in the thylakoid membrane. Generally the regulation of the light reactions has been studied and conceptualised at the microscopic level of protein-protein and protein-ligand interactions, however in recent years dynamic changes in the thylakoid macrostructure itself have been recognised to play a significant role in regulating light harvesting and electron transfer. Here we review the evidence for the involvement of macrostructural changes in photosynthetic regulation and review the techniques that brought this evidence to light.     

Effects of Lead on Activities of Photochemical Reaction and Key Enzymes of Carbon Assimilation in Spinach Chloroplast

Photosynthesis is one of the most sensitive processes to lead, but the effects of lead on the transformation of light energy of plants are still not clearly understood. In the present paper, spinach was cultivated in the experimental fields and was sprayed with various concentrations of PbCl2 solution. We investigated the effects of lead on the activities of photochemical reaction and the key enzymes of carbon assimilation in spinach chloroplast. The results showed that Pb2+ treatment could significantly inhibit the Hill reaction activity of spinach chloroplast and photophosphorylation, and it had a more conspicuous effect on cyclic photophosphorylation than non-cyclic photophosphorylation. The activities of ATPase on the thylakoid membrane were severely inhibited under Pb2+-treated condition, and Ca2+ ATPase activity was affected more obviously than Mg2+ ATPase activity. Meanwhile, the activities of the key enzymes of carbon assimilation were also significantly reduced by Pb2+, especially Rubisco activase. The reduction of dry weight of spinach caused by Pb2+ was more significant than that of fresh weight. It implied that Pb2+ could disturb light energy transformation of chloroplast.     

Betaine aldehyde oxidation by spinach chloroplasts

Chenopods synthesize betaine by a two-step oxidation of choline: choline → betaine aldehyde → betaine. Both oxidation reactions are carried out by isolated spinach (Spinacia oleracea L.) chloroplasts in darkness and are promoted by light. The mechanism of betaine aldehyde oxidation was investigated with subcellular fractions from spinach leaf protoplasts. The chloroplast stromal fraction contained a specific pyridine nucleotide-dependent betaine aldehyde dehydrogenase (about 150 to 250 nanomoles per milligram chlorophyll per hour) which migrated as one isozyme on native polyacrylamide gels stained for enzyme activity. The cytosol fraction contained a minor isozyme of betaine aldehyde dehydrogenase. Leaves of pea (Pisum sativum L.), a species that lacks betaine, had no betaine aldehyde dehydrogenase isozymes. The specific activity of betaine aldehyde dehydrogenase rose three-fold in spinach plants grown at 300 millimolar NaCl; both isozymes contributed to the increase. Stimulation of betaine aldehyde oxidation in illuminated spinach chloroplasts was due to a thylakoid activity which was sensitive to catalase; this activity occurred in pea as well as spinach, and so appears to be artifactual. We conclude that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H₂O₂-mediated reaction cannot be ruled out.     

The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 °C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit.2. CO₂ fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3. Phosphoglycerate reduction, which is a partial reaction of CO₂ fixation, shows very little pH dependency.3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO₂ fixation without affecting phosophoglycerate reduction. This inhibition of CO₂ fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP.4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO₂ fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO₂ fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport.5. It is shown that intact chloroplasts are able to fix CO₂ in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma.6. Dark CO₂ fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO₂ fixation in the light. This demonstrates that CO₂ fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8.7. It is shown that pH changes occurring in the stroma after illumination are sufficient to switch CO₂ fixation from zero to maximal activity. This offers a mechanism for light control of CO₂ fixation, avoiding wasteful CO₂ fixation in the dark.     

The impact of photo-induced molecular changes of dairy proteins on their ACE-inhibitory peptides and activity

Among all dietary proteins, dairy proteins are the most important source of bio-active peptides which can, however, be affected by modifications upon processing and storage. Since it is still unknown to which extent the biological activity of dairy proteins is altered by chemical reactions, this study focuses on the effect of photo-induced molecular changes on the angiotensin I converting enzyme (ACE) inhibitory activity. Milk proteins were dissolved in phosphate buffer containing riboflavin and stored under light at 4 °C for one month during which the molecular changes and the ACE-inhibitory activity were analysed. An increase in the total protein carbonyls and the N-formylkynurenine content was observed, besides a decrease in the free thiol, tryptophan, tyrosine and histidine content. These changes were more severe in caseins compared with whey proteins and resulted moreover in the aggregation of caseins. Due to these photo-induced molecular changes, a significant loss of the ACE-inhibitory activity was observed for casein peptides. A peptide analysis moreover illustrated that the decreased activity was not attributed to a reduced digestibility but to losses of specific ACE-inhibitory peptides. The observed molecular changes, more specifically the degradation of specific amino acids and the casein aggregation, could be assigned as the cause of the altered peptide pattern and as such of the loss in ACE-inhibitory activity.     

Distribution of Metabolites between Chloroplast and Cytoplasm during the Induction Phase of Photosynthesis in Leaf Protoplasts

A method for rapid separation of the chloroplast and cytoplasmic fractions from isolated leaf protoplasts of wheat and spinach has been used to determine the distribution of ¹⁴C-labeled products during photosynthesis. In the dark, CO₂ fixation was only 1 to 2% of that in the light and the products were mainly in the cytoplasmic fraction suggesting fixation by phosphoenolpyruvate carboxylase. Label appeared rapidly in the chloroplast fraction following illumination but the amount leveled off after 4 to 5 minutes reflecting the buildup of intermediates to steady state levels. There was only a slight lag before label appeared in the cytoplasmic fraction and it continued to increase at a constant rate reflecting synthesis of neutral products. In the light, the percentage of label in the chloroplast fraction decreased rapidly in the first minute of illumination and was only 10 to 20% in the steady-state. It is suggested that the chloroplast phosphate transporter promotes a rapid transfer of sugar phosphates from the chloroplast to the cytoplasm, even during the induction phase of photosynthesis.     

Action Spectrum for Interaction between Visible and Far-Red Light on Face Chloroplast Orientation in Mougeotia

The orientation of chloroplasts from profile to face position in Mougeotia can be controlled in two ways: by a typical phytochrome-mediated system or by continuous, simultaneous irradiation with far-red and visible light. In experiments with dichromatic irradiation of Mougeotia, the light conditions applied prevented the formation of a far-red-absorbing form of phytochrome gradient in the cell. An unpolarized background of far-red light and linearly polarized monochromatic light of different wavelengths and vibrating parallel to the cell axis, if given by themselves, were completely ineffective in producing any changes in chloroplast orientation. Given together, however, changes in chloroplast orientation were induced. The action spectrum for this interaction between constant far-red and variable visible light was maximal at 620 nanometers. The chloroplast response in these dichromatic light conditions required a prolonged duration of exposure to simultaneous continuous irradiation of high fluence energy. The vibrating plane of linearly polarized 620 nanometer light had no significant influence on interaction with far-red light in chloroplast movement. The results obtained are different from the typical low energy phytochrome-mediated chloroplast orientation. This new type of chloroplast photoresponse might be mediated by an unknown sensory pigment.     

Influence of fixation, exciting light and section thickness on the primary fluorescence of samples for microfluorometric analysis

The primary fluorescence (autofluorescence) of some cell and tissue components depends on the fixative and fixing time, as well as on the thickness of paraffin sections and the wavelength of exciting light. The highest autofluorescence emission (pale green) was observed by using violet-blue excitation. After aldehyde fixation, the autofluorescence of some tissue structures was higher than after methanol or ethanolacetic acid. These features must be taken into account when fluorescence microscopy is applied to the study of cell smears and paraffin embedded tissues after flurochroming or immunofluorescence reactions.