Extrinsic Peptide Research Articles

Abstract P-573: EXTRINSIC CALCITONIN GENE-RELATED PEPTIDE INHIBITS HYPEROXIA-INDUCED ALVEOLAR EPITHELIAL TYPE-II CELLS APOPTOSIS, OXIDATIVE STRESS AND REACTIVE OXYGEN SPECIES PRODUCTION BY ENHANCING NOTCH 1 AND HERP EXPRESSION

Aims & Objectives: This study investigated the effects of calcitonin gene- related peptide (CGRP) on AEC II cells exposed to hyperoxia. Methods Neonatal rat AEC II cells were isolated and identified by detecting surfactant protein C (SP-C). Three small in- terfering RNAs targeting Notch 1 were synthesized and transfected into AEC II. A hyperoxia-exposed AEC II cell injury model was established and was divided into 8 groups. MDA levels and SOD activity were examined using lipid peroxidation assay kits. Apoptosis and reactive oxygen species (ROS) production were evaluated using flow cytometry. Notch 1 mRNA expression was examined using RT-PCR. Homocysteine-induced endoplasmic reticulum protein (HERP) was examined using Western blot analysis. Results CGRP treatment significantly enhanced MDA levels and decreased SOD activity compared to hyperoxia-treated AEC II cells (P<0.05). CGRP treatment significantly inhibited hyperoxia-induced AEC II cell apoptosis, and significantly suppressed hyperoxia-induced ROS production compared to hyperoxia-treated AEC II cells (P<0.05) either undergoing γ secretase inhibitor or Notch RNAinterference. CGRP significantly triggered Notch 1 mRNA expression and significantly enhanced HERP expression compared to hyperoxia-treated AEC II cells (P<0.05) either undergoing γ secretase inhibitor or Notch RNA interference. Conclusions In AEC II cells, extrinsic peptide CGRP suppressed hyperoxia-induced apoptosis, oxidative stress, and ROS production, which may be triggered by Notch 1 and HERP signaling pathway.

Extrinsic Calcitonin Gene-Related Peptide Inhibits Hyperoxia-Induced Alveolar Epithelial Type II Cells Apoptosis, Oxidative Stress, and Reactive Oxygen Species (ROS) Production by Enhancing Notch 1 and Homocysteine-Induced Endoplasmic Reticulum Protein (HERP) Expression.

BackgroundLung alveolar epithelial type II cells (AEC II) are the most important stem cells in lung tissues, which are critical for wound repair of bronchopulmonary dysplasia (BPD). This study investigated the effects of calcitonin gene-related peptide (CGRP) on AEC II cells exposed to hyperoxia.Material/MethodsNeonatal rat AEC II cells were isolated and identified by detecting surfactant protein C (SP-C). Three small interfering RNAs targeting Notch 1 were synthesized and transfected into AEC II. A hyperoxia-exposed AEC II cell injury model was established and was divided into 8 groups. MDA levels and SOD activity were examined using lipid peroxidation assay kits. Apoptosis and reactive oxygen species (ROS) production were evaluated using flow cytometry. Notch 1 mRNA expression was examined using RT-PCR. Homocysteine-induced endoplasmic reticulum protein (HERP) was examined using Western blot analysis.ResultsCGRP treatment significantly enhanced MDA levels and decreased SOD activity compared to hyperoxia-treated AEC II cells (P<0.05). CGRP treatment significantly inhibited hyperoxia-induced AEC II cell apoptosis, and significantly suppressed hyperoxia-induced ROS production compared to hyperoxia-treated AEC II cells (P<0.05) either undergoing γ secretase inhibitor or Notch RNA interference. CGRP significantly triggered Notch 1 mRNA expression and significantly enhanced HERP expression compared to hyperoxia-treated AEC II cells (P<0.05) either undergoing γ secretase inhibitor or Notch RNA interference.ConclusionsIn AEC II cells, extrinsic peptide CGRP suppressed hyperoxia-induced apoptosis, oxidative stress, and ROS production, which may be triggered by Notch 1 and HERP signaling pathway.

Chemical neuroanatomy of the Drosophila central complex: Distribution of multiple neuropeptides in relation to neurotransmitters

The central complex of the insect brain is an integration center, receiving inputs from many parts of the brain. In Drosophila it has been associated with the control of both locomotor and visually correlated behaviors. The central complex can be divided into several substructures and is comprised of a large number of neuronal types. These neurons produce classical neurotransmitters, biogenic amines, and different neuropeptides. However, the distribution of neurotransmitters and neuromodulators in central-complex circuits of Drosophila is poorly known. By immunolabeling and GAL4-directed expression of marker proteins, we analyzed the distribution of acetylcholine, glutamate, GABA, monoamines, and eight different neuropeptides; Drosophila tachykinin, short neuropeptide F, myoinhibitory peptide, allatostatin A, proctolin, SIFamide, neuropeptide F, and FMRFamide. All eight neuropeptides were localized to the fan-shaped body, the largest substructure of the central complex, and were mapped to different layers within this structure. Several populations of peptide-immunoreactive tangential and columnar neurons were identified, of which some colocalized acetylcholine. Fewer peptides were found to be expressed in the other substructures: the ellipsoid body, the protocerebral bridge, and the noduli. The ellipsoid body and the protocerebral bridge were innervated by extrinsic peptide expressing neurons. Our findings reveal that numerous neuropeptides are expressed in the central complex and that each peptide has a distinct distribution pattern, suggesting important roles for neuropeptides as neuromediators and cotransmitters in this brain area.

Transgenic expression of HLA‐E single chain trimer protects porcine endothelial cells against human natural killer cell‐mediated cytotoxicity

The susceptibility of porcine endothelial cells (pEC) to human natural killer (NK) cells is related to the failure of human major histocompatibility complex (MHC)-specific killer inhibitory receptors to recognize porcine MHC class I molecules. The aims of this study were (i) to assess the protection of pEC against xenogeneic NK-mediated cytotoxicity afforded by the stable expression of HLA-E single chain trimers (SCT) composed of a canonical HLA-E binding peptide antigen, VMAPRTLIL, the mature human beta2-microglobulin, and the mature HLA-E heavy chain, and (ii) to test whether HLA-E expression on pEC and porcine lymphoblastoid cells affects the adhesion of human NK cells. Porcine EC lines expressing different levels of HLA-E SCT were generated by Ca(2)PO(4)-transfection followed by limiting dilution cloning. Surface expression of HLA-E was measured by flow cytometry. Susceptibility of transfected pEC lines against human NK cells was tested in (51)Cr-release cytotoxicity assays. Interactions between human NK cells and HLA-E positive pEC or porcine lymphoblastoid cells were further addressed in adhesion and conjugation assays. The level of protection of pEC from human NK-mediated cytotoxicity correlated with the intensity of surface HLA-E expression. Furthermore, the HLA-E SCT-mediated protection was specifically reversed by blocking the HLA-E specific NK inhibitory receptor CD94/NKG2A. HLA-E expression does neither affect the adhesion of human NK cells to pEC nor the heteroconjugate formation between human NK and porcine 13271.10 cells. Stable surface expression of HLA-E on pEC was achieved in the absence of extrinsic peptide pulsing and provided partial protection from human NK cytotoxicity. Though insufficient to inhibit xenogeneic NK cell reactivity completely, transgenic HLA-E expression on pig organs might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.

Binding of pediocin PA-1 with anionic lipid induces model membrane destabilization.

To obtain molecular insights into the action mode of antimicrobial activity of pediocin PA-1, the interactions between this bacteriocin and dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylglycerol (DMPG) model membranes have been investigated in D(2)O at pD 6 by Fourier transform infrared spectroscopy. The interactions were monitored with respect to alteration of the secondary structure of pediocin, as registered by the amide I' band, and phospholipid conformation, as revealed by the methylene nu(s)(CH(2)) and carbonyl nu(C;O) stretching vibrations. The results show that no interaction between pediocin and DMPC occurs. By contrast, pediocin undergoes a structural reorganization in the presence of DMPG. Upon heating, pediocin self-aggregates, which is not observed for this pD in aqueous solution. The gel-to-crystalline phase transition of DMPG shifts to higher temperatures with a concomitant dehydration of the interfacial region. Our results indicate that pediocin is an extrinsic peptide and that its action mechanism may lie in a destabilization of the cell membrane.

Phase II study of cetrorelix, a luteinizing hormone-releasing hormone antagonist in patients with platinum-resistant ovarian cancer

Objective The goal of this work was to study the anticancer activity of cetrorelix, a decapeptide with LHRH receptor antagonist properties in patients with platinum-resistant ovarian cancer. About 80% of primary ovarian cancers and cell lines bear LHRH receptors. Cetrorelix has anticancer activity in in vitro and in vivo ovarian cancer models. Methods Eligible patients with ovarian or mullerian carcinoma resistant to platinum chemotherapy received cetrorelix 10 mg subcutaneously every day. Eligibility criteria included age ≥ 18, PS ≤ 2, measurable disease, chemistries and blood counts in normal range, no estrogen replacement for at least 2 weeks, and no known allergic reactions to extrinsic peptide. In patients volunteering for a biopsy, tissue was taken to perform a LHRH receptor assay. Results Seventeen patients were treated. Median age was 58 years. Median performance status was 0. Median number of prior chemotherapies was 3. Three patients had partial remissions lasting 9, 16, and 17 weeks. Toxicities effects included grade 4 anaphylactoid reaction (one patient) controlled by cortisol and cimetidine, grade 2 histamine reaction (two patients), grade 2 arthralgia (one patient) 20% cholesterol increase (two patients, who did not require specific treatment), minor hot flushes, headache, and local skin reaction at the injection site. Six of seven samples were LHRH receptor positive for mRNA and/or ligand assay. Two responding patients were LHRH receptor positive. The patient who had no receptor did not respond. Conclusion Cetrorelix has activity against ovarian cancer in this refractory population, and has minimal toxicity, except for potential anaphylactoid reactions. Activity may be mediated through the LHRH receptor.

Cooperative binding of oxygen to the water-splitting enzyme in the filamentous cyanobacterium Oscillatoria chalybea

In the filamentous cyanobacterium Oscillatoria chalybea photolysis of water does not take place in the complete absence of oxygen. A catalytic oxygen partial pressure of 15×10 −6 Torr has to be present for effective water splitting to occur. By means of mass spectrometry we measured the photosynthetic oxygen evolution in the presence of H 2 18O in dependence on the oxygen partial pressure of the atmosphere and analysed the liberations of 16O 2, 16O 18O and 18O 2 simultaneously. The observed dependences of the light-induced oxygen evolution on bound oxygen yield sigmoidal curves. Hill coefficient values of 3.0, 3.1 and 3.2, respectively, suggest that the binding is cooperative and that four molecules of oxygen have to be bound per chain to the oxygen evolving complex. Oxygen seems to prime the water-splitting reaction by redox steering of the S-state system, putting it in the dark into the condition from which water splitting can start. It appears that in O. chalybea an interaction of oxygen with S 0 and S 1 leads to S 2 and S 3, thus yielding the typical oxygen evolution pattern in which even after extensive dark adaptation substantial amounts of Y 1 and Y 2 are found. The interacting oxygen is apparently reduced to hydrogen peroxide. Mass spectrometry permits to distinguish this highly specific oxygen requirement from the interaction of bulk atmospheric oxygen with the oxygen evolving complex of the cyanobacterium. This interaction leads to the formation H 2O 2 which is decomposed under O 2 evolution in the light. The dependence on oxygen-partial pressure and temperature is analysed. Structural peculiarities of the cyanobacterial reaction centre of photosystem II referring to the extrinsic peptides might play a role.

Heat Resistance in Photosynthesis of Sorrel Vine(Cayratia japonica(Thunb.)Gagn.) Leaf, Chloroplasts and Photosystem II Particles.

The sorrel vine (Cayratia japonica (Thunb.) Gagn.), a winder plant (liana) in the Vitaceae family, was heat resistant in photosynthetic oxygen (O2) evolution. The O2 evolution of leaf, chloroplasts and photosystemII particles of sorrel vine was little affected by heating at 45°C for 5min, while the O2 evolution of spinach and many other plants was inhibited at 45°C. Inhibition of the O2 evolving center of a heated leaf was monitored with greater depression of chlorophyll-fluorescence induction, reflecting the depression of plastoquinone photoreduction by photosystem II (PS II). The leaf fluorescence of spinach, pumpkin, maize, sorrel (Rumes japonicus) and arrowroot (Pueraria lobata Ohwi) was depressed and modified in a rising pattern, while the fluorescence rise of sorrel vine leaf was little affected by heating at 45°C; this indicated that sorrel vine leaf was more heat resistant than others tested. Heating of spinach and sorrel leaf, and their chloroplasts and/or PS II particles to 45°C destroyed the O2 evolving center so that most of the essential Mn and the 18-, 24- and 33-kDa extrinsic peptides of the center were lost, while the center of the sorrel vine was protected from the destruction at 45°C.

Structural characterization of free and membrane-bound nisin by infrared spectroscopy

This study reports two new trends about nisin affinity for lipid membranes. First, there is a very strong dependence of nisin binding on the membrane surface charge. As illustrated in this work, the binding of nisin is much greater for phosphatidylglycerol (PG) than for phosphatidylcholine (PC) membranes. This can be rationalized by electrostatic attraction between the positively charged peptide and the negatively charged PG. Second, the affinity of nisin shows a very weak dependence on the lipid phase, the binding to fluid or gel phase membranes being nearly equivalent. Therefore, our results suggest that nisin behaves as an extrinsic peptide. This work also presents the first piece of information relative to the structure of membrane-bound nisin. The Amide I band of the peptide is different for free nisin in water and for membrane-bound nisin. By analyzing this region using self-deconvolution and band fitting, and by comparing with results obtained from nisin dissolved in various H 2O/trifluoroethanol mixtures, it can be inferred that the binding of nisin to phospholipid membranes leads to an increased proportion of β-turns.

Influence of CO2 and SO2 on Growth and Structure of Photosystem II of the Chinese Tung-Oil Tree Aleurites montana

Three months old plants of the Chinese tung-oil tree Aleurites montana were cultivated for 4 months in air containing an increased amount of 700 ppm CO2. During the exposure to 700 ppm CO2 the plants exhibited a considerably stronger growth (30-40% ) in comparison to the control plants (grown in normal air). In these CO2-plants during the entire analyzing period the amount of soluble proteins, of soluble sugars and the chlorophyll content were lower than in control plants. The protein content, referred to leaf area, increased during this time in both plant types by approx. 50% but with a different time course. The increase is faster in CO2-plants compared to control plants, and ends up with similar values in both plants after 4 months. No difference is seen between sun and shade leaves. The chlorophyll content in both sun and shade leaves is 20% lower in CO2-plants. Whereas the chlorophyll content in sun leaves stays constant during developm ent, it has increased in shade leaves by 20% at the end of the 4 months period. The content of soluble sugars is lower in CO2-plants compared to control plants. The difference is bigger in sun leaves than in shade leaves. The ribulose 1.5-bisphosphate carboxylase/oxygenase content almost doubles within the experimentation period, but seems to be subject to large variations. CO2-plants contain in general less ribulose 1.5-bisphosphate carboxylase/oxygenase than control plants. The content of coupling factor of photophosphorylation is 20% lower in CO2-plants when compared to control plants and remains during development more constant in CO2-plants. The molecular structure of the photosystem II-complex undergoes under the influence of the increased CO2-content a quantitative modification. The light harvesting complex (LHCP) and the extrinsic peptide with the molecular mass of 33 kDa increase in CO2-plants. Gassing with SO2 (0.3 ppm in air) leads to a strong damage of the plants. The damaging influence is already seen after 6 days and leads to a partial leaf-shedding of the tree. In the visually still intact remaining leaves the chlorophyll content referred to unit leaf area decreases by 63%, that of soluble sugars by 65%, the content of soluble proteins and that of Rubisco decrease by 26% and 36% respectively. The light harvesting complex and the chlorophyll- binding peptides (43 and 47 kDa) increase whereas the extrinsic peptides decrease. It looks as if the simultaneous application of SO2 (0.3 ppm) and increased CO2 (700 ppm) releaves the damaging effect of SO2. Plant growth does not exhibit a difference in comparison to control plants. Soluble proteins and chlorophyll increase by 27% and 33% and the ribulose 1.5-bisphosphate carboxylase/oxygenase content as well as that of soluble sugars increases by 18 respectively 14%. The peptide composition of photosystem II shows a quantitative modification. The LHCP increases and the chlorophyll-binding peptides and the peptides with a molecular mass smaller than 24 kDa are reduced. The quantity of extrinsic peptides appears unchanged. Ribulose 1,5-bisphosphate carboxylase/oxygenase and the CF1-complex of Aleurites are immunochemically only partially identical to the corresponding enzymes of Nicotiana tabacum as demonstrated by tandem-cross-immune electrophoresis.

Isolation and Partial Characterization of a Manganese Requiring ʟ-Arginine Metabolizing Enzyme Being Present in Photosystem II Complexes of Spinach and Tobacco

Abstract A low ʟ-arginine metabolizing enzyme (L-AME) activity leading to ornithine, urea and additional products not identified so far could be detected in photosystem II (PS II) membranes of spinach and of the chlorophyll deficient tobacco mutant Su/su. The detectable L-AME activity was very low in untreated PS II membranes, but increased significantly (about 10 fold) when the extrinsic peptides (psbO, P and Q gene products) were removed - suggesting that the L-AME is exposed at the lumen side of PS II. It was possible to isolate the detergent-solubilized protein from CaCl2-washed PS II membranes of spinach by a combination of anion and cation exchange columns. On the basis of SDS PAGE the protein was homogenous and had an apparent molecular mass of 7 kDa. N-terminal sequencing of the polypeptide gave a contiguous sequence of 20 amino acids showing no homologies to PS II polypeptides as yet sequenced. After chromatography of the L-AME on an anion exchange column at pH 9.5 (last purification step) a completely inactive enzyme was obtained. Maximal reactivation was achieved by dialyzing the protein against Hepes-NaOH buffer in the pH range of 6.5 to 7.5 containing 100 mᴍ chloride or sulfate (being the most effective anions). The L-AME activity was totally dependent on manganese added to the reaction mixture. Moreover, there were indications of a second cation binding site being more sequestered and requiring bound Ca2+ or Mn2+ for activity (Sr2+ was less effective and Mg2+ was ineffective). There are indications that the protein contains a redox active group - possibly an aminoacid- derived quinonoid (based on a redox cycling assay with glycine and nitroblue tetrazolium). The capability of this PS II associated protein to bind the cofactors of water oxidation and having a redox active group (preliminary results) suggests that this protein might be functional in photosynthetic water oxidation. This is further supported by the fact that the isolated L-AME has a low catalase activity

Europium- and Dysprosium-Ions as Probes for the Study of Calcium Binding Sites in Photosystem II

Abstract Trivalent lanthanide cations are suitable probes for Ca2+-binding sites in photosystem II (PS II). PS II membranes prepared from Nicoticinci tabacum, intact and depleted of the extrinsic polypeptides were exposed to lanthanide ions (Dy3+ and Eu3+). Small concentrations of dysprosium and europium ions enhance oxygen evolution under short saturating flashes. Higher concentrations of the rare earth cations cause the release of the three extrinsic peptides (17, 23 and 33 kDa) and reduce O2 yield. The reactivation of the PS II membranes, thus depleted of the 33 kDa subunit, by Ca2+ ions is not possible. Comparing Eu3+ with Dy3+ in this effect shows that Eu3+ is more effective than Dy3+, because a lower Eu3+-concentration in comparison to that of Dy3+ inactivates O2-evolution. The differences between europium and dysprosium can be explained by their different ionic radius. Our results suggest the existence of two Ca-binding regions: one with a low affinity for calcium would be located on the contact surface of the 23 and 33 kDa proteins and the second one with a high affinity, should be located close to the Mn-cluster and to tyrosine-161 (Z ). The more tightly-bound calcium would be responsible for the activity of the PS II system.

Binding of Lipids onto Polypeptides of the Thylakoid Membrane I. Galactolipids and Sulpholipid as Prosthetic Groups of Core Peptides of the Photosystem II Complex

Photosystem II complexes were prepared from chloroplasts of wild type tobacco Nicotiana tabacum var. John William’s Broadleaf and from two chlorophyll mutants derived from it, namely N. tabacum Su/su and N. tabacum Su/su var. Aurea. The hydrophobic peptides of these complexes were analyzed for bound lipid molecules by means of monospecific lipid antisera. A comparison of the peptide composition of the complexes of the three chloroplast types by means of polyacrylamide gel electrophoresis showed that the peptide composition was qualitatively identical. A major quantitative difference referred to a 66 kDa peptide which appeared to be much stronger in gels of photosystem II peptides originating from the yellowgreen and the yellow tobacco variety. Furthermore, we were able to show that different SDS polyacrylamide gel electrophoresis runs of the same PS II preparation yielded differences in the band strength of this peptide. Comparative densitometric measurements showed that an increase in this 66 kDa peptide was always correlated with a decrease in the D1 and D2 peptides. Obviously, the 66 kDa peptide is the heterodimer of D1 and D2. Differences in the peptide composition of photosystem II preparations from the 3 tobacco species refer above all to peptides of the light-harvesting complex with molecular masses of 28 and 26 kDa. After the transfer of the peptides from the polyacrylamide gel to nitrocellulose membranes, they were incubated with monospecific antisera to monogalactolipid, digalactolipid or sulfolipid. These experiments showed that the 66 kDa peptide reacted with antibodies to digalactolipid and with those to sulfolipid. The 66 kDa peptide reacts in the Western blot procedure also with an antiserum to a 66 kDa peptide prepared and characterized earlier and which was shown to inhibit electron transport reactions in the region of the reaction center of photosystem II. The monospecific antiserum to monogalactolipid reacts with the D1 and D2 peptide as well as with the chlorophyll-binding polypeptides of the masses 42 and 48 kDa, and also with the 26 and 28 kDa peptides of the light-harvesting complex as well as with the extrinsic peptides exhibiting the molecular masses, 33 ,21 -23 and 18 kDa. Whereas lipase treatment apparently destroys the lipids as antigenic determinants of the peptides on the nitrocellulose membrane, periodate treatment or treatment of the photosystem II preparations with organic solvents do not prevent the reaction of the 66 kDa peptide with the sulfolipid antiserum. These experiments show as the 66 kDa peptide appears to be the heterodimer of D1 and D2, that the galactolipids mono- and digalactosyldiglyceride as well as the sulfolipid are bound, much like prosthetic groups, onto the core peptides.

Effect of Cyanide on the S2- and S3-State in the Filamentous Cyanobacterium Oscillatoria chalybea

Abstract Low concentrations of cyanide affect the flash-induced oxygen evolution pattern in thylakoid preparations of the filamentous cyanobacterium Oscillatoria chalybea. At a cyanide concentration of 2 x 10-6 ᴍ the effect lies exclusively on the S2- and S3-state whereas the subsequent flash yields and the steady state are barely affected, showing that the reaction center is not touched by the reagent. The influence of the chemical is characterized by the fact that at basic pH (7.5 -8.5) the deactivation mode of S2 and S3 is changed, showing that the cyanide-ion is the reactant. Whereas in the control the deactivation of S2 yields as usual S1 and S-1, the deactivation pattern in the presence of cyanide shows that S2 deactivates - without giving S1 - , to a more reduced state, probably S0. In the flash pattern the two signal amplitudes of the first two flashes are lowered to zero in the presence of 2 x 10-6 ᴍ cyanide and become uptake signals at higher cyanide concentrations. It is seen that in the presence of cyanide S2 and to a lesser extent S3 in the Oscillatoria system apparently react with oxygen. In tobacco thylakoids no effect of cyanide on the flash pattern itself is observed, even at higher concentrations (e.g. 10-4 ᴍ), although the signal amplitudes of the entire pattern are affected at such concentrations. Photosystem II of cyanobacteria is characterized by the absence of the two extrinsic 16 kDa and 23 kDa peptides. As shown by mass spectrometry inside-out vesicles from tobacco thylakoids, in which these two extrinsic peptides have been removed by NaCl-washing, exhibit simultaneously flash-induced O2-evolution and an O2-uptake which both require the presence of CL- and Ca2+ ions. Both the evolution signal and the uptake signal are DCMU sensitive and are inhibited by KCN. An amperometrically measured flash pattern of inside-out tobacco vesicles shows, if the assay is supplemented with CL- ions, the usual flash pattern known from the literature. The experiments seem to indicate that even in the washed inside-out condition the S-state system of higher plants is much less sensitive to ambient oxygen than in the phylogenetically older system of the filamentous cyanobacterium Oscillatoria chalybea.

Quantitative Determination of the Distribution between Two Photosystem II-Mediated Oxidation Reactions in Oscillatoria chalybea: A Comparison between Water Oxidation and Hydrogen Peroxide Decomposition

Abstract Mass spectromctric analysis reveals that oxygen evolution measured as the consequence of short saturating light flashes in thylakoid preparations of the filamentous cyanobacterium Oscillatoria chalybea consists of two portions, one coming from photosynthetic water splitting and one coming from H2O2 decomposition. This H2O2 decomposition is photosystem II-mediated and it is the S-state system which oxidizes H2O2 to give protons and oxygen. Water is neither intermediate nor seems it to be the origin of the reaction. At the high oxygen partial pressure of normal air H2O2 production and its decomposition exceeds manyfold the H2O splitting reaction. H2O2 production seems to come from photosystem II but is not necessarily produced on the acceptor side of photosystem II in the sense of a Mehler type reaction. From the reaction rate and the observed labeling density, it is inferred that production and decomposition must take place within the same reaction site which might be for both, the production and decomposition, the S-state system. In this sense. H2O2 might be the product of the S-state system and seems somehow associated with S2 or S3. Thus, if oxygen evolution is measured as the consequence of short saturating light flashes in an ambient atmosphere of 21% oxygen, mass spectrometry reveals a flash pattern which bears the oxygen label of the ambient atmosphere and which has not much in common with an usual Kok sequence. Such a pattern might start in the first three flashes more or less as a Kok pattern would, but is then in the pattern portion usually characteristic for steady-state oxygen evolution characterized by a periodicity of two indicating that H2O2 decomposition requires only two light quanta. At high oxygen partial pressure (e.g. in 21% ,16O2) both reaction portions can be quantitatively deter- mined by labeling the assay with H2 18O and measuring the evolved (16O::18O)/18O2 ratio which is representative for water splitting. The measured evolution of 16O2 (mass 32) represents H2O2 decomposition, if the 16O2 portion calculated from the measured mass 34/36 ratio is subtracted. At low oxygen partial pressure the H2O2 forming and decomposing reaction is largely sup- pressed and oxygen evolution from water-splitting prevails. As a hypothesis, H2O2 production and its decomposition might be a defective performance of photosystem II at high ambient oxygen partial pressure in these cyanobacteria, perhaps due to the principal absence of two of the extrinsic peptides from photosystem II.

Stability of shark and human lens fiber cell membranes

1. 1. Scanning electron microscopy of minced dogfish, shark, and human lens fiber cell preparations showed that neither 8 M urea or 1% SDS was capable of totally disrupting the fiber cells, but that their membranes were totally disrupted by 1% SDS containing 50 mM dithiothreitol. 2. 2. Fiber cells were purified by sucrose centrifugation and extracted with Tris-buffer, 8 M urea, 1% SDS and 50 mM dithiothreitol plus 1% SDS. 3. 3. A residual 10% of the protein was capable of maintaining membrane integrity. 4. 4. DTT + SDS totally dissolved the membranes, implying that disulfide bonds are involved in maintaining structure. 5. 5. Non-tryptophan fluorescence was nearly totally extracted prior to the SDS-DTT step, indicating that the fluorescence associated with the membrane protein was not serving as crosslinks between extrinsic and intrinsic proteins. 6. 6. Polyacrylamide slab gel electrophoresis revealed that after exhaustive e extractions of lens fiber cells with Tris-buffer, 8 M urea and 1% SDS, the addition of DTT released major heat stable 27,000 and 24,000 dalton peptides. 7. 7. The data support the concept that fiber cell membranes contain high levels of extrinsic peptides and intrinsic peptides whose stability depends strongly on oxidized crosslinks, probably disulfide bonds.