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211.
In flash-illuminated, oxygen-evolving spinach chloroplasts and green algae, a free radical transient has been observed with spectral parameters similar to those of Signal II (, ). However, in contrast with ESR Signal II, the transient radical does not readily saturate even at microwave power levels of 200 mW. This species is formed most efficiently with “red” illumination ( and occurs stoichiometrically in a 1 : 1 ratio with . The Photosystem II transient is formed in less than 100 μs and decays via first-order kinetics with a halftime of 400–900 μs. Additionally, the for radical decay is temperature independent between 20 and 4 °C; however, below 4 °C the transient signal exhibits Arrhenius behavior with an activation energy of approx. 10 kcal · mol?1. Inhibition of electron transport through Photosystem II by , 3-(3,4-dichlorophenyl)-1,1-dimethylurea or reduced suppresses the formation of the light-induced transient. At low concentrations (0.2 mM), partially inhibits the free radical formation, however, the decay kinetics are unaltered. High concentrations of (1–5 mM) restore both the transient signal and electron flow through Photosystem II. These findings suggest that this “quinoidal” type ESR transient functions as the physiological donor to the oxidized reaction center chlorophyll, . 相似文献
212.
PsaD is required for the stable binding of PsaC to the photosystem I core protein of Synechococcus sp. PCC 6301. 总被引:2,自引:0,他引:2
The psaC gene product from Synechococcus sp. PCC 7002 and the psaD gene product from Nostoc sp. PCC 8009 were synthesized in Escherichia coli and purified to homogeneity. Incubation of the PsaC apoprotein with the Synechoccus sp. PCC 6301 photosystem I core protein in the presence of FeCl3, Na2S, and beta-mercaptoethanol resulted in a time-dependent transition in the flash-induced absorption change from a 1.2-ms, P700+ FX- back-reaction to a long-lived, P700+ [FA/FB]- back-reaction. ESR studies showed that FB and FA were photoreduced about equally at 19 K, and while the resonances were shifted upfield, they remained as broad as in the free PsaC holoprotein. When the reconstituted complex was purified in a sucrose gradient containing 0.1% Triton X-100, most of the optical absorption transient reverted to that characteristic of the P700+ FX- back-reaction. Addition of purified PsaD to the incubation mixture led to a greater extent of recovery of electron flow to FA/FB for any given concentration of PsaC. ESR studies showed that FA, rather than FB, became the preferred electron acceptor at 19 K; moreover, the resonances moved upfield and sharpened to become nearly identical with those of a control photosystem I complex. When the sample was purified in a sucrose gradient containing 0.1% Triton X-100, the long-lived P700+ [FA/FB]- optical transient remained stable. Analysis by denaturing polyacrylamide gel electrophoresis showed that the PsaC and PsaD proteins had rebound to the photosystem I core. The data indicate that although PsaC can bind loosely, the presence of PsaD leads to a stable, isolatable photosystem I complex which is spectroscopically indistinguishable from the native complex. Since a PsaC1 fusion protein which contains an amino-terminal extension of five amino acids (MEHSM...) does not bind in the absence of PsaD [Zhao, J., et al. (1990) FEBS Lett. 276, 175-180], the N-terminus of the PsaC protein could provide a site of interaction with the photosystem I core. We propose that the binding of PsaC to the PsaA/PsaB heterodimer is potentiated by insertion of the FA/FB clusters into PsaC, and stabilized by the presence of PsaD. 相似文献
213.
The origin of the "S3" EPR signal from calcium-depleted photosystem 2 samples has been investigated. This signal is observed after freezing samples under illumination and has been assigned to an interaction between the manganese cluster and an oxidized histidine radical [Boussac et al. (1990) Nature 347; 303-306]. In calcium-depleted samples prepared by three different methods, we observed the trapping of the tyrosine radical YZ+ under conditions which also formed the "S3" signal. An "S3"-type signal and YZ+ were also formed in PS2 samples treated with the water analogue ammonia. Following illumination at 277 K, the "S3" and YZ+ signals decayed at the same rate at 273 K in the dark. Both the YZ+ and "S3" signals decayed on storage at 77 K and could be subsequently regenerated by illumination at 8-77 K. No evidence to support histidine oxidation was found. The effects of DCMU, chelators, and alkaline pH on the dark-stable multiline S2 and the "S3" signals from calcium-depleted samples were determined. Both signals required the presence of EGTA or citrate for maximum yield. The addition of DCMU caused a reduction in the yield of "S3" generated by freezing under illumination. Incubation at pH 7.5 resulted in the loss of both signals. We propose that a variety of treatments which affect calcium and chloride binding cause a stabilization of the S2 state and slow the reduction of YZ+. This allows the trapping of YZ+, the interaction with the manganese cluster (probably in the S2 state) resulting in the "S3" signal. The data allow the position of the manganese cluster to be estimated as within 10 A of tyrosine Z (D1-161). 相似文献
214.
A new photosystem I core has been isolated that is devoid of the bound iron-sulfur clusters but preserves electron flow from P700 to the intermediate electron acceptor A1. The particle is prepared by incubation of a Synechococcus sp. PCC 6301 photosystem I core protein (which contains electron acceptors A0, A1, and FX) with 3 M urea and 5 mM K3Fe(CN)6 to oxidatively denature the FX iron-sulfur cluster to the level of zero-valence sulfur. In this apo-FX preparation, over 90% of the flash-induced absorption change at 820 nm decays with a 10-microseconds half-time characteristic of the decay of the P700 triplet state formed from the backreaction of P700+ with an acceptor earlier than FX. Chemical reduction at high pH values with aminoiminomethanesulfinic acid results in kinetics identical with those seen in the P700 chlorophyll a protein prepared with sodium dodecyl sulfate (SDS-CP1, which contains only electron acceptor A0); the flash-induced absorption change decays primarily with a 25-ns half-time characteristic of the backreaction between P700+ and A0-, and the magnitude of the total absorption change is larger than can be accounted for by the P700 content alone. Addition of oxygen results in a reversion to the 10-microseconds kinetic decay component attributed to the decay of the P700 triplet state. At 77 K, the optical transient in the apo-FX preparation decays with a 200-microseconds half-time characteristic of the backreaction between P700+ and A1-.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
215.
A polymorphism affecting apolipoprotein A-II translational efficiency determines high density lipoprotein size and composition 总被引:13,自引:0,他引:13
M H Doolittle R C LeBoeuf C H Warden L M Bee A J Lusis 《The Journal of biological chemistry》1990,265(27):16380-16388
High density lipoproteins (HDL) are heterogeneous particles consisting of about equal amounts of lipid and protein that are thought to mediate the transport of cholesterol from peripheral tissues to liver. We show that a previously identified polymorphism affecting HDL electrophoretic mobility in mice is due to a monogenic variation controlling HDL size and apolipoprotein composition. Thus, the HDL particles of various inbred strains of mice exhibit a striking difference in the ratio fo the two major apolipoproteins of HDL, apoA-I and apoA-II. HDL particles in all strains examined contain an average of about five apoA-I molecules; however, whereas the strains with small HDL contain two to three apoA-II molecules per particle, the strains with large HDL contain about five apoA-II molecules per particle. This increase in the protein content of the large HDL is also accompanied by increased lipid content. The HDL size polymorphism and apoA-II levels cosegregate with the apoA-II structural gene on mouse chromosome 1, indicating that a mutation of the apoA-II gene locus is responsible. The rates of synthesis of apoA-II are increased in the strains with large HDL and high apoA-II levels as compared to the strains with small HDL and low apoA-II levels. On the other hand, the fractional catabolic rates of both apoA-I and apoA-II among the strains are very similar, confirming that apoA-II concentrations are controlled at the level of synthesis. Despite the difference in rates of apoA-II synthesis between strains, the apoA-II mRNA levels in the strains are not discernibly different, suggesting that a mutation of the apoA-II structural gene controls apoA-II translational efficiency. This was confirmed by translating apoA-II mRNA in vitro using a rabbit reticulocyte lysate system. Sequencing of apoA-II cDNA from the strains revealed a number of nucleotide substitutions, which may affect translational efficiency. We conclude that the assembly of apoA-II into HDL does not have a set stoichiometry but, rather, is controlled by the production of apoA-II. As apoA-II levels increase, the HDL particles become larger and acquire more lipid, but apoA-I content per particle remains unchanged. These studies with mice provide a model for the metabolic relationships between apoA-I, apoA-II, and HDL lipid in humans. 相似文献
216.
217.
BD Pascal MJ Chalmers SA Busby CC Mader MR Southern NF Tsinoremas PR Griffin 《BMC bioinformatics》2007,8(1):156
Background
The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange) experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues. 相似文献218.
Leticia Labriola Maria G Peters Karin Krogh Iván Stigliano Letícia F Terra Cecilia Buchanan Marcel CC Machado Elisa Bal de Kier Joffé Lydia Puricelli Mari C Sogayar 《BMC cell biology》2009,10(1):49-16
Background
The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. 相似文献219.
A stable T-2 hydrazide gel is prepared by activating T-2 toxin with tresyl chloride followed by coupling to agarose-adipic acid hydrazide. Utilized as an affinity chromatography column, this T-2 hydrazide gel purifies a monoclonal antibody for T-2 in high yield directly from ascites fluid. Specific antibody trapped on the column is eluted either with excess T-2 or at pH 11.6. Much less successful are two other T-2 affinity columns that were prepared and evaluated: T-2 bovine serum albumin Affi-Gel 15 and T-2 hexylamine Sepharose. 相似文献
220.