从二溴百里香醌的抑制效应分析融合膜电子传递的途径

甲基紫精(MV)系统中,在对类囊体膜的光合磷酸化(PSP)活力近于完全抑制的二溴百里香醌(DBMIB)浓度下,由类囊体残缺膜与线粒体嵴膜组成的融合膜PSP活力不仅不被抑制,反而受到不同程度的促进。在铁氰化钾(FeCy)系统中,DBMIB对类囊体膜的PSP活力不能完全抑制,同样浓度的DBMIB对融合膜的PSP活力有抑制效应。检测了不同膜在不同系统中,光下耗氧、放氧、FeCy还原和融合效应的关系等,论证了融合膜中电子传递的途径。     

Isolation of a brain peptide identical to the intestinal PHI (peptide HI)

The isolation of a brain peptide identical to the intestinal peptide PHI (peptide HI) is described. The peptide was isolated from porcine brain extract using a chemical assay method based on its C-terminal isoleucine amide structure. The complete amino acid sequence of the peptide was found to be: His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Phe-Ser-Arg-Leu-Leu-Gly-Gln-Leu-Ser-Ala- Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Ile-NH2. This sequence is identical to the intestinal peptide thus demonstrating PHI to be a brain-gut peptide. The role of PHI in the central nervous system as a neurotransmitter or neuromodulator is discussed.     

Interaction of exogenous benzoquinone with Photosystem II in chloroplasts. The semiquinone form acts as a dichlorophenyldimethylurea-insensitive secondary acceptor

Chloroplasts were submitted to a sequence of saturating short flashes and then rapidly mixed with dichlorophenyldimethylurea (DCMU). The amount of singly reduced secondary acceptor (B^?) present was estimated from the DCMU-induced increase in fluorescence in the dark caused by the reaction: QB^?

Q^?B. By varying the time interval between the preillumination and the mixing, the time course of B^? reoxidation by externally added benzoquinone was investigated. It was found that benzoquinone oxidizes B^? in a bimolecular reaction, and does not interact directly with Q^?. When a sufficient delay after the preillumination was allowed in order to let benzoquinone reoxidize B^? before the injection of DCMU, the fluorescence increase caused by one subsequent flash fired in the presence of DCMU was followed by a fast decay phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 100 μ}\text{s}$). The amplitude of this phase was proportional to the amount of B^? produced by the preillumination. This fast decay was observed only after the first flash in the presence of DCMU. These results are interpreted by assuming a binding of the singly reduced benzoquinone to Photosystem II where it acts as an efficient, DCMU-insensitive, secondary (exogenous) acceptor.     

Membrane phosphorylation leads to the partial detachment of the chlorophyll a/b protein from Photosystem II

The hypothesis that the chlorophyll fluorescence decline due to membrane phosphorylation is caused principally by the detachment and removal of LHCP from the LHCP-PS II matrix is examined. It is demonstrated that when membranes are phosphorylated in the dark (a) the fluorescence decline is greater when excited by light enriched in wavelengths absorbed mainly by LHCP (475 nm) than when excited by light absorbed to a large extent also by the PS II complex (435 nm), (b) titration with different artificial quenchers of chlorophyll fluorescence is unchanged after the phosphorylation-induced fluorescence decline, and (c) the F_v/F_m ratio does not change after the phosphorylation-induced fluorescence decline. These data indicate that it is indeed principally LHCP that interacts with the quencher (PS I presumably). This interaction involves a small fraction of the total PS II-coupled LHCP, which becomes functionally detached from the LHCP-PS II matrix.     

Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme

Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes. We could identify a significant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled Photosystem II complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.     

Alterations in Electron Transport Characteristics during Senescence of Cucumis Cotyledonary Leaves. Analysis of the Effects of Inhibitors

Cotyledonary leaves of Cucumis sativus cv. Poinsette exhibited senescence-induced losses in chlorophyll (Chl) and protein contents within three weeks since germination. Chl and protein concentrations in cotyledonary leaves approached maximum on 6th d after germination and they declined to 50 and 41 %, respectively, by the 20th day of growth. Activities of both photosystem (PS) 2 and PS1 decreased by 33 and 31 %, respectively, on the 20th day, compared to the control 6th day. Changes in sensitivity of PS2 to inhibitors like atrazine and dibromothymoquinone and sensitivity of PS1 to KCN accompanied the changes in PS2 and PS1 activities. Hence both the acceptor side of PS2 and the donor side of PS1 are affected by senescence-induced changes in cucumber cotyledonary leaves.     

Lack of site-specificity of spinach chloroplast coupling factor 1

The irreversible inhibition of chloroplast phosphorylation by either sulfate anions, or N-ethylmaleimide, is energy dependent. Chloroplasts must first be illuminated in the presence of the inhibitors and a mediator of electron flow, for the subsequent phosphorylation to show any inhibition. Both inhibitors affect the chloroplast coupling factor 1.Electron transport only through Photosystem I can be used to activate either of these inhibitions. The subsequent inhibition in a second light reaction is the same whether ATP synthesis is supported by Photosystem I, or by Photosystem II electron transport. The reverse experiment, activating inhibition by electron transport only through Photosystem II, is possible in the case of sulfate. Again, the inhibition is expressed whether Photosystem II or Photosystem I electron flow supports ATP synthesis. We conclude that the two electron transport regions probably generate the same high energy state which is able to activate all members of a functionally uniform coupling factor population. These enzyme molecules must catalyze phosphorylation coupled to electron transport through either region of the chain. The results tend to discredit models requiring a separate group of coupling factor molecules unique to each part of the chain.     

Three Substrate Binding Sites on Spinach Ferredoxin:NADP+ Oxidoreductase. Studies with Selectively Acting Inhibitors

The effects of phenylmercuric acetate (PMA) and apoferredoxin (apoFd) on the diaphorase activity of spinach ferredoxin:NADP⁺ oxidoreductase (FNR) in the presence of dibromothymoquinone (DBMIB) or cytochrome c (Cyt c) were studied. PMA inhibited effectively (I₅₀ = < 5 M) ferredoxin-dependent Cyt c reduction but did not affect evidently the enzyme activity in the presence of DBMIB as an electron acceptor. ApoFd caused also inhibition of Cyt c reduction but slightly stimulated, like ferredoxin, DBMIB reduction. We confirm a hypothesis according to which three binding sites for substrates [NADP(H), Fd-Cyt c, quinone/dichlorophenol indophenol] occur within the molecule of isolated FNR.     

Effect of dibromothymoquinone on Chlorophyll <Emphasis Type="Italic">a</Emphasis> Fluorescence in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> cells incubated in complete or sulfur-depleted medium

The effect of dibromothymoquinone on chlorophyll fluorescence was studied in Chlamydomonas reinhardtii cells using PAM and PEA fluorometers. Dibromothymoquinone was shown to affect differently control cells incubated in complete medium and S-starved cells. The fluorescence yield in the control suspension considerably increased in the presence of the inhibitor. Presumably, this can be due to inactivation of protein kinase, as a result of which part of light-harvesting complex II that could have diffused from the stacking zone of the membrane into the lamellar zone towards photosystem I remains close to photosystem II. In S-starved cells, whose photosynthetic apparatus is in state 2, the fluorescence level declines in the presence of dibromothymoquinone. The JIP testing of induction curves (O-J-I-P fluorescence transient) suggests that dibromothymoquinone inhibits both light-harvesting complex II kinase and photosynthetic electron transport when added to the control, while in the starved cells it acts predominantly as an electron acceptor.