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1.
Monolayers of porcine kidney cells (LLC-PK) were grown in a series of Nu-Serum-supplemented media containing different Mg(2+) concentrations (480, 250, 25, 6.3 or 2.6 microM) to study the effect of Mg(2+) depletion on cellular phospholipid changes and the consequent effect on the membrane permeability to Ca(2+). Cells grown on 6.3 or 2.6 microM Mg(2+) showed a decrease in PE, PS, Sph, PI and an increase of PC. These changes were attributed mainly to the decreased rate of Sph synthesis through the transfer of phosphocholine from PC to ceramide, or due to the increase of PE N-methylation as found in Mg(2+)-deficient cells. The (45)Ca uptake was increased in cells grown on 25.0 microM Mg(2+), while it was decreased in cells grown on 6.3 or 2.6 microM Mg(2+). These changes in Ca(2+) uptake were related to changes of cellular phospholipids and fatty acids which affect adenylate cyclase activity in the membrane, as well as the membrane fluidity.  相似文献   

2.
Fluorescence energy transfer has been used to study the interaction of various phospholipids with the erythrocyte (Ca2+ + Mg2+)-ATPase. The fluorescence energy transfer between tryptophan residues of the (Ca2+ + Mg2+)-ATPase purified from erythrocytes and pyrene-labelled analogues of phosphatidylcholine (Pyr-PC), phosphatidylinositol (Pyr-PI), phosphatidylinositol 4-phosphate (Pyr-PIP), phosphatidylinositol 4,5-bisphosphate (Pyr-PIP2), phosphatidylglycerol (Pyr-PG) and phosphatidic acid (Pyr-PA) was measured. A positive correlation was found between the number of negative charges on the phospholipids (PIP2 greater than PIP greater than PA greater than PI = PG greater than PC) and the potency of their pyrene-labelled analogues to act as quantum acceptors in fluorescence energy transfer from the tryptophan residues of the (Ca2+ + Mg2+)-ATPase. This is the first time that a physical interaction between PIP/PIP2 and an intrinsic membrane protein has been demonstrated. The dependence of the energy transfer on the number of negative charges of the phospholipids closely resembles the previously demonstrated charge dependence of the enzymatic activity of the (Ca2+ + Mg2+)-ATPase (Missiaen, L., Raeymaekers, L., Wuytack, F., Vrolix, M., Desmet, H. and Casteels, R. (1989) Biochem. J. 263, 687-694). It is concluded that the stimulation of the (Ca2+ + Mg2+)-ATPase activity by negatively charged phospholipids is based on a binding of these lipids to the (Ca2+ + Mg2+)-ATPase and that the negative charges are a major modulatory factor for this interaction.  相似文献   

3.
The C2A domain is one of two calcium ion (Ca(2+))- and membrane-binding domains within synaptotagmin I (Syt I), the identified Ca(2+) sensor for regulated exocytosis of neurotransmitter. We propose that the mechanistic basis for C2A's response to Ca(2+) and cellular function stems from marginal stability and ligand-induced redistributions of protein conformers. To test this hypothesis, we used a combination of calorimetric and fluorescence techniques. We measured free energies of stability by globally fitting differential scanning calorimetry and fluorescence lifetime spectroscopy denaturation data, and found that C2A is weakly stable. Additionally, using partition functions in a fluorescence resonance energy transfer approach, we found that the Ca(2+)- and membrane-binding sites of C2A exhibit weak cooperative linkage. Lastly, a dye-release assay revealed that the Ca(2+)- and membrane-bound conformer subset of C2A promote membrane disruption. We discuss how these phenomena may lead to both cooperative and functional responses of Syt I.  相似文献   

4.
A phycoerythrin (PE) and phycocyanin (PC) mixture was separated from allophycocyanin on calcium phosphate chromatography from completely dissociated phycobilisomes of the blue-green alga, Nostoc sp. After dialysis of the PE-PC mixture in 0.75 m potassium phosphate, pH 7, which allows reassociation of the dissociated pigment-proteins, complexes of PE and PC in a 2:1 m ratio (PE/PC complex) as well as complexes predominantly of PC (PC/PE complex) were then separated by sedimentation on linear sucrose gradients. These complexes resemble the rods of intact phycobilisomes and transfer energy efficiently from PE to PC. They contain the Group II colorless polypeptides described by Tandeau de Marsac and Cohen-Bazire (1977 Proc Natl Acad Sci USA 74: 1635 61639). Phycobilisomes can be reconstituted by combining the allophycocyanin pool with (a) the PE-PC mixture, (b) the PE/PC complex, or (c) the PC/PE complex. Successful reconstitution is measured by absorption, fluorescence, circular dichroism, and electron microscopy. The major requirement for reconstitution is the 29-kilodalton colorless polypeptide. In its absence, no phycobilisomes are formed. It is the only colorless polypeptide common to both the PE/PC complex and the PC/PE complex, and appears to be the polypeptide responsible for rod attachment to the allophycocyanin. In addition, high phosphate concentrations and 20 degrees C temperatures are needed for reconstitution.  相似文献   

5.
We examined energy transfer dynamics in phycobilisomes (PBSs) of cyanobacteria in relation to the morphology and pigment compositions of PBSs. We used Gloeobacter violaceus PCC 7421 and measured time-resolved fluorescence spectra in three types of samples, i.e., intact cells, PBSs, and rod assemblies separated from cores. Fremyella diplosiphon, a cyanobacterial species well known for its complementary chromatic adaptation, was used for comparison after growing under red or green light. Spectral data were analyzed by the fluorescence decay-associated spectra with components common in lifetimes with a time resolution of 3 ps/channel and a spectral resolution of 2 nm/channel. This ensured a higher resolution of the energy transfer kinetics than those obtained by global analysis with fewer sampling intervals. We resolved four spectral components in phycoerythrin (PE), three in phycocyanin (PC), two in allophycocyanin, and two in photosystem II. The bundle-like PBSs of G. violaceus showed multiple energy transfer pathways; fast ( approximately 10 ps) and slow ( approximately 100 ps and approximately 500 ps) pathways were found in rods consisting of PE and PC. Energy transfer time from PE to PC was two times slower in G. violaceus than in F. diplosiphon grown under green light.  相似文献   

6.
7.
Rusakov DA  Fine A 《Neuron》2003,37(2):287-297
Synaptic activation is associated with rapid changes in intracellular Ca(2+), while the extracellular Ca(2+) level is generally assumed to be constant. Here, using a novel optical method to measure changes in extracellular Ca(2+) at high spatial and temporal resolution, we find that brief trains of synaptic transmission in hippocampal area CA1 induce transient depletion of extracellular Ca(2+). We show that this depletion, which depends on postsynaptic NMDA receptor activation, decreases the Ca(2+) available to enter individual presynaptic boutons of CA3 pyramidal cells. This in turn reduces the probability of consecutive synaptic releases at CA3-CA1 synapses and therefore contributes to short-term paired-pulse depression of minimal responses. This activity-dependent depletion of extracellular Ca(2+) represents a novel form of fast retrograde synaptic signaling that can modulate glutamatergic information transfer in the brain.  相似文献   

8.
Unicellular marine cyanobacteria are abundant in both coastal and oligotrophic environments, where they contribute substantially to primary production. The physiological effect of future increases in atmospheric CO2 concentrations on the marine picocyanobacteria is still poorly known. We studied the physiological changes in marine phycocyanin (PC)-rich and phycoerythrin (PE)-rich Synechococcus strains under different CO2 partial pressures (350, 600 and 800 ppm). The PE strain showed no significant change in growth rate over the experimental CO2 range. A significant increase (25.4%) in carbohydrate was observed at 800 ppm CO2, but no significant change in protein and RNA/DNA ratio was observed in any CO2 treatment. The PC strain showed a significant increase (36.7%) in growth rate at 800 ppm CO2, but no significant change in carbohydrate or protein content was observed over the entire CO2 range. The RNA/DNA ratio increased with increasing CO2 concentration and was positively correlated with growth rate. Cellular red fluorescence and orange fluorescence of the PE strain tended to decline in all CO2 treatments. However, no such decline was observed at higher CO2 treatments in the PC strain. Our results suggest that the PC strain would probably benefit more than the PE strain from future increases in atmospheric CO2 concentrations.  相似文献   

9.
Blebbistatin, a myosin II inhibitor, interferes with myosin-actin interaction and microtubule assembly. By influencing cytoskeletal dynamics blebbistatin counteracts apoptosis of several types of nucleated cells. Even though lacking nuclei and mitochondria, erythrocytes may undergo suicidal cell death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include energy depletion and osmotic shock, which enhance cytosolic Ca(2+) activity with subsequent Ca(2+)-sensitive cell shrinkage and cell membrane scrambling. The present study explored the effect of blebbistatin on eryptosis. Cell membrane scrambling was estimated from binding of annexin V to phosphatidylserine at the erythrocyte surface, cell volume from forward scatter in fluorescence-activated cell sorting analysis and cytosolic Ca(2+) concentration from Fluo3 fluorescence. Exposure to blebbistatin on its own (1-50 μM) did not significantly modify cytosolic Ca(2+) concentration, forward scatter, or annexin V binding. Glucose depletion (48 h) was followed by a significant increase of Fluo3 fluorescence and annexin V binding, effects significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 μM, annexin V binding ≥ 10 μM). Osmotic shock (addition of 550 mM sucrose) again significantly increased Fluo3 fluorescence and annexin binding, effects again significantly blunted by blebbistatin (Fluo3 fluorescence ≥ 25 μM, annexin V binding ≥ 25 μM). The present observations disclose a novel effect of blebbistatin, i.e., an influence on Ca(2+) entry and suicidal erythrocyte death following energy depletion and osmotic shock.  相似文献   

10.
The Ca(2+) depletion of the endoplasmic reticulum (ER) activates the ubiquitous store-operated Ca(2+) entry (SOCE) pathway that sustains long-term Ca(2+) signals critical for cellular functions. ER Ca(2+) depletion initiates the oligomerization of stromal interaction molecules (STIM) that control SOCE activation, but whether ER Ca(2+) refilling controls STIM de-oligomerization and SOCE termination is not known. Here, we correlate the changes in free luminal ER Ca(2+) concentrations ([Ca(2+)](ER)) and in STIM1 oligomerization, using fluorescence resonance energy transfer (FRET) between CFP-STIM1 and YFP-STIM1. We observed that STIM1 de-oligomerized at much lower [Ca(2+)](ER) levels during store refilling than it oligomerized during store depletion. We then refilled ER stores without adding exogenous Ca(2+) using a membrane-permeable Ca(2+) chelator to provide a large reservoir of buffered Ca(2+). This procedure rapidly restored pre-stimulatory [Ca(2+)](ER) levels but did not trigger STIM1 de-oligomerization, the FRET signals remaining elevated as long as the external [Ca(2+)] remained low. STIM1 dissociation evoked by Ca(2+) readmission was prevented by SOC channel inhibition and was associated with cytosolic Ca(2+) elevations restricted to STIM1 puncta, indicating that Ca(2+) acts on a cytosolic target close to STIM1 clusters. These data indicate that the refilling of ER Ca(2+) stores is not sufficient to induce STIM1 de-oligomerization and that localized Ca(2+) elevations in the vicinity of assembled SOCE complexes are required for the termination of SOCE.  相似文献   

11.
The delipidated sarcoplasmic reticulum (SR) Ca(2+)-ATPase was reconstituted into proteoliposomes containing different phospholipids. The result demonstrated the necessity of phosphatidylcholine (PC) for optimal ATPase activity and phosphatidylethanolamine (PE) for the optimal calcium transport activity. Fluorescence intensity of Fluorescein 5-isothiocyanate (FITC)-labeled enzyme at Lys515 as well as the measurement of the distance between 5-((2-[(iodoacetyl) amino] ethyl) amino)naphthalene-1-sulphonic acid (IAEDANS) label sites (Cys674/670) and Pr3+ demonstrated a conformational change of cytoplasmic domain, consequently, leading to the variation of the enzyme function with the proteoliposomes composition. Both the intrinsic fluorescence of Trp and its dynamic quenching by HB decreased with increasing PE content, revealing the conformational change of transmembrane domain. Time-resolved fluorescence study characterized three classes of Trp residues, which showed distinctive variation with the change in phospholipid composition. The phospholipid headgroup size caused the conformational change of SR Ca(2+)-ATPase, subsequent the ATPase activity and Ca2+ uptake.  相似文献   

12.
The time-course of acclimation (0-5h) of the red alga Porphyridium purpureum with respect to total proteins, phycoerythrin (PE) and phycobilisomes (PBS) has been studied at different N availability and different light regimes. After a high N input, acclimation takes place in two phases. The first one, which is photoindependent is characterized by simultaneous increase of proteins and PE. At low N input, this first phase is not detected. In the second phase the PE content increases only under low light together with an increase of the PBS size, followed probably by an increase in the number of PBS. The effectiveness of the energy transfer increases under these conditions. A rapid decrease in the PBS size correlated with a decrease of the energy transfer is observed at high irradiance. Free PE plays an important role in the organization-disorganization of the PBS at low N concentration (inverse correlation between free PE and PE attached to PBS). Free PE is not accumulated in the cell after a high N input at high irradiance. Independently of photoacclimation, two species of PBS appear with different PE content and different capacities to aggregate with other compounds. A clear correlation appears between the level of coupling of the PBS and the fluorescence ‘in vivo’ of the whole cells. The comparison between dissociated and undissociated PBS as well as between PBS obtained after the different acclimation processes allows the determination of the presence of two linker polypeptides probably associated with B-PE (37 and 32–5 kDa) and two associated with PC and APC (27 and 25 kDa). That suggests that acclimation of PBS requires a parallel stoichiometric response of biliproteins and the linker polypeptides involved in the efficiency of the energy transfer.  相似文献   

13.
In various cell types, depletion of intracellular Ca(2+)-stores results in store-operated Ca(2+)-entry (SOCE) across the cellular membrane. However, the effects of SOCE on neuronal membrane excitability and mitochondrial functions in central neurons are not well defined. We investigated such cellular downstream effects in pyramidal neurons of rat organotypic hippocampal slice cultures by applying electrophysiological and fluorescence imaging techniques. We report that SOCE is associated with (i) elevations of Ca(2+)-concentration in individual neuronal mitochondria ([Ca(2+)](m)). In addition, SOCE can result in (ii) hyperpolarizing neuronal membrane currents, (iii) increase in extracellular K(+)-concentration ([K(+)](o)), (iv) mitochondrial membrane depolarization, and (v) changes in intracellular redox state (NAD(P)H and FAD fluorescence), the latter reflecting responses of energy metabolism. These additional downstream effects of SOCE required concomitant muscarinic receptor activation by carbachol or acetylcholine, and were suppressed by agonist washout or application of antagonist, atropine. We conclude that muscarinic receptor activation determines the downstream effects of SOCE on neuronal membrane excitability and energy metabolism. This mechanism might have significant impact on information processing and neurometabolic coupling in central neurons.  相似文献   

14.
Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Fo?rster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at micromolar concentrations, for FRET to occur this complex must have an affinity in the micromolar range. We present here a fluorescence dynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytia gregaria, the photoprotein clytin in its Ca(2+)-discharged form that is highly fluorescent (λ(max) = 506 nm) and its GFP (cgreGFP; λ(max) = 500 nm). Ca(2+)-discharged clytin shows a predominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product, coelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 °C), consistent with tight binding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant and molecular brightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolar concentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolarization via FRET with a transfer correlation time of 0.5 ns. The 34 ns time of cgreGFP showed no change upon addition of a 1000-fold excess of Ca(2+)-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRET complex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond) intermediate in the clytin reaction pathway.  相似文献   

15.
Satoh K  Fork DC 《Plant physiology》1983,71(3):673-676
In the red alga Porphyra perforata, the level of chlorophyll fluorescence in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased during illumination of the thallus. The results showed that: (a) this decay was related to the photooxidative activity of photosystem I; (b) Q, the primary electron acceptor of photosystem II, became oxidized during the decay of the fluorescence; (c) reagents which inhibit the back reaction of photosystem II inhibited the decay.

From these results, it is suggested that, when conditions in the chloroplasts of this red alga become too oxidative, excess light energy can be converted to heat as a result of an accelerated back reaction of photosystem II. This may be one of the mechanisms by which this alga can cope with the high salt and high light conditions that can occur in its natural habitat.

  相似文献   

16.
Bioluminescence recording of Ca(2+) signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca(2+) in various cell compartments. In addition, they would also serve to monitor Ca(2+) in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R(0)) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca(2+) oscillations in single HeLa cells expressing tdTA. Ca(2+) rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca(2+) activity of HeLa cells injected subcutaneously into mice, and Ca(2+) signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca(2+) sensor reported to date and is, therefore, a promising probe to study Ca(2+) dynamics in whole organisms or tissues expressing the transgene.  相似文献   

17.
Kazuhiko Satoh  David C. Fork 《BBA》1983,722(1):190-196
Time courses of chlorophyll fluorescence and fluorescence spectra at 77 K after various light treatments were measured in the red alga, Porphyra perforata. Photosystem (PS) I or II light (light 1 or 2) induced differences in the fluorescence spectra at 77 K. Light 2 decreased the two PS II fluorescence bands (F-685 and F-695) in parallel, while light 1 preferentially increased F-695. Light 1 and 2 also produced different effects on the activities of PS I and II. Preillumination with light 1 increased PS II activity and decreased PS I activity. However, preillumination with light 2 decreased PS II activity with no effect on PS I activity. These results show that there are at least two mechanisms that can alter the transfer of light energy in P. perforata. The dark state in this alga was found to be State 2 and light 1 induced a State 2-State 1 transition which retarded the transfer of light energy from PS II to PS I. Light 2 induced another change (which we have called a State 2-State 3 transition) that was accompanied by a change only in PS II activity.  相似文献   

18.
Ahn T  Oh DB  Lee BC  Yun CH 《Biochemistry》2000,39(33):10147-10153
The effect of phosphatidylethanolamine (PE) on the binding of apocytochrome c to model membranes was examined. When 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) of the standard vesicles composed of 80% of this lipid and 20% of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) was gradually replaced with upward of 50% of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), the binding increased appreciably. Ca(2+), causing the phase separation of PS, also brought about increased binding of apocytochrome c in the PC/PS system, underlining the importance of PS properties in membranes for the protein binding. The resonance energy transfer between Trp-59 in apocytochrome c and pyrene-PS incorporated into bilayers showed that the replacement of PC with PE increased the extent of apocytochrome c penetration into membranes by a PE concentration-dependent manner. However, in the absence of PS, PE had no apparent effect on these functions of apocytochrome c, suggesting that PE-induced change(s) of acidic membrane properties is important to the association of apocytochrome c with vesicles. From the observations that the excimer to monomer fluorescence ratio of pyrene-PS increased and the fluorescence of NBD-PS was quenched with increasing concentration of PE, it was deduced that PE caused PS-enriched domains in PC/PE/PS membranes. The colocalization of pyrene-PS with BODIPY-PS by PE further supported the possibility. We suggest that PE-induced formation of PS-enriched domains acts as binding sites for apocytochrome c in membranes.  相似文献   

19.
There is evidence that membranes of rod outer segment (ROS) disks are a high-affinity Ca(2+) binding site. We were interested to see if the high occurrence of sixfold unsaturated docosahexaenoic acid in ROS lipids influences Ca(2+)-membrane interaction. Ca(2+) binding to polyunsaturated model membranes that mimic the lipid composition of ROS was studied by microelectrophoresis and (2)H NMR. Ca(2+) association constants of polyunsaturated membranes were found to be a factor of approximately 2 smaller than constants of monounsaturated membranes. Furthermore, strength of Ca(2+) binding to monounsaturated membranes increased with the addition of cholesterol, while binding to polyunsaturated lipids was unaffected. The data suggest that the lipid phosphate groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in PC/PE/PS (4:4:1, mol/mol) are primary targets for Ca(2+). Negatively charged serine in PS controls Ca (2+) binding by lowering the electric surface potential and elevating cation concentration at the membrane/water interface. The influence of hydrocarbon chain unsaturation on Ca(2+) binding is secondary compared to membrane PS content. Order parameter analysis of individual lipids in the mixture revealed that Ca(2+) ions did not trigger lateral phase separation of lipid species as long as all lipids remained liquid-crystalline. However, depending on temperature and hydrocarbon chain unsaturation, the lipid with the highest chain melting temperature converted to the gel state, as observed for the monounsaturated phosphatidylethanolamine (PE) in PC/PE/PS (4:4:1, mol/mol) at 25 degrees C.  相似文献   

20.
《BBA》2020,1861(2):148139
An aerial green alga, Prasiola crispa (Lightf.) Menegh, which is known to form large colonies in Antarctic habitats, is subject to severe environmental stresses due to low temperature, draught and strong sunlight in summer. A considerable light-absorption by long-wavelength chlorophylls (LWC) at around 710 nm, which seem to consist of chlorophyll a, was detected in thallus of P. crispa harvested at a terrestrial environment in Antarctica. Absorption level at 710 nm against that at 680 nm was correlated with fluorescence emission intensity at 713 nm at room temperature and the 77 K fluorescence emission band from LWC was found to be emitted at 735 nm. We demonstrated that the LWC efficiently transfer excitation energy to photosystem II (PSII) reaction center from measurements of action spectra of photosynthetic oxygen evolution and P700 photo-oxidation. The global quantum yield of PSII excitation in thallus by far-red light was shown to be as high as by orange light, and the excitation balance between PSII and PSI was almost same in the two light sources. It is thus proposed that the LWC increase the photosynthetic productivity in the lower parts of overlapping thalli and contribute to the predominance of alga in the severe environment.  相似文献   

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