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1.
Cytochrome bd is a tri-heme (b
558, b
595, d) respiratory oxygen reductase that is found in many bacteria including pathogenic species. It couples the electron transfer from quinol to O2 with generation of an electrochemical proton gradient. We examined photolysis and subsequent recombination of CO with isolated cytochrome bd from Escherichia coli in one-electron reduced (MV) and fully reduced (R) states by microsecond time-resolved absorption spectroscopy at 532-nm excitation. Both Soret and visible band regions were examined. CO photodissociation from MV enzyme possibly causes fast (τ<1.5 µs) electron transfer from heme d to heme b
595 in a small fraction of the protein, not reported earlier. Then the electron migrates to heme b
558 (τ∼16 µs). It returns from the b-hemes to heme d with τ∼180 µs. Unlike cytochrome bd in the R state, in MV enzyme the apparent contribution of absorbance changes associated with CO dissociation from heme d is small, if any. Photodissociation of CO from heme d in MV enzyme is suggested to be accompanied by the binding of an internal ligand (L) at the opposite side of the heme. CO recombines with heme d (τ∼16 µs) yielding a transient hexacoordinate state (CO-Fe2+-L). Then the ligand slowly (τ∼30 ms) dissociates from heme d. Recombination of CO with a reduced heme b in a fraction of the MV sample may also contribute to the 30-ms phase. In R enzyme, CO recombines to heme d (τ∼20 µs), some heme b
558 (τ∼0.2–3 ms), and finally migrates from heme d to heme b
595 (τ∼24 ms) in ∼5% of the enzyme population. Data are consistent with the recent nanosecond study of Rappaport et al. conducted on the membranes at 640-nm excitation but limited to the Soret band. The additional phases were revealed due to differences in excitation and other experimental conditions. 相似文献
2.
The effect of Zn2+ on the rates of electron transfer and of voltage generation in the cytochrome bc1 complex (bc1) was investigated under excitation of Rhodobacter capsulatus chromatophores with flashing light. When added, Zn2+ retarded the oxidation of cytochrome b and allowed to monitor (at 561-570 nm) the reduction of its high potential heme bh (in the absence of Zn2+ this reaction was masked by the fast re-oxidation of the heme). The effect was accompanied by the deceleration of both the cytochrome c1 reduction (as monitored at 552-570 nm) and the generation of transmembrane voltage (monitored by electrochromism at 522 nm). At Zn2+ <100 μM the reduction of heme bh remained 10 times faster than other reactions. The kinetic discrepancy was observed even after an attenuated flash, when bc1 turned over only once. These observations (1) raise doubt on the notion that the transmembrane electron transfer towards heme bh is the main electrogenic reaction in the cytochrome bc1 complex, (2) imply an allosteric link between the site of heme bh oxidation and the site of cytochrome c1 reduction at the opposite side of the membrane, and (3) indicate that the internal redistribution of protons might account for the voltage generation by the cytochrome bc1 complex. 相似文献
3.
Chun-Yen Lin Po-Li Wei Wei-Jen Chang Yung-Kai Huang Sheng-Wei Feng Che-Tong Lin Sheng-Yang Lee Haw-Ming Huang 《PloS one》2013,8(3)
The aim of this study was to assess the cryoprotective effect of static magnetic fields (SMFs) on human erythrocytes during the slow cooling procedure. Human erythrocytes suspended in 20% glycerol were slowly frozen with a 0.4-T or 0.8-T SMF and then moved to a −80°C freezer for 24 hr. The changes in survival rate, morphology, and metabolites of the thawed erythrocytes were examined. To understand possible cryoprotective mechanisms of SMF, membrane fluidity and dehydration stability of SMF-exposed erythrocytes were tested. For each test, sham-exposed erythrocytes were used as controls. Our results showed that freezing coupled with 0.4-T or 0.8-T SMFs significantly increased the relative survival ratios of the frozen-thawed erythrocytes by 10% and 20% (p<0.001), respectively. The SMFs had no effect on erythrocyte morphology and metabolite levels. However, membrane fluidity of the samples exposed to 0.8-T SMF decreased significantly (p<0.05) in the hydrophobic regions. For the dehydration stability experiments, the samples exposed to 0.8-T SMF exhibited significantly lower (p<0.05) hemolysis. These results demonstrate that a 0.8-T SMF decreases membrane fluidity and enhances erythrocyte membrane stability to resist dehydration damage caused by slow cooling procedures. 相似文献
4.
5.
Dayue Tong Yi Jin Tianyu Xue Xiaoyan Ma Jinxiang Zhang Xueling Ou Jianding Cheng Hongyu Sun 《PloS one》2015,10(9)
To evaluate the identification method using the microRNA markers miR10b and miR135b to distinguish semen stains from menstrual blood, peripheral blood, vaginal fluid and so on body fluid stains. The expression levels of miR10b and miR35b in semen stains and menstrual blood and so on were detected utilizing a real-time quantitative PCR technique with a specific fluorescence-labeled TaqMan probe. RNU6b was used as the internal reference gene; the difference in their expression was analyzed, and the specificity, sensitivity, and detection capability of the techniques were evaluated. The expression of miR10b and miR135b in semen stains was significantly higher than that of other body fluid stains, with a mean value of ΔCт from-6 to-7. However, it ranged from-2 to-4 for other body fluid stains. The initial criteria for judging which semen stains can be identified were determined by analyzing the research results. When the threshold value was set to 0.04, the CT value could be detected in the target genes miR10b, miR135b and in the internal reference gene RNU6b, and CT values are<40, ΔCT[10b-U6]<-5.5, and ΔCT[135b-U6]<-6, respectively, and the semen stain could be identified. The expression levels of miR10b and miR135b are higher in semen with strong tissue specificity; thus, they can be used to differentiate semen stains from other body fluid stains in forensic science. 相似文献
6.
Jos M. Oliva-Lozano Daniel Rojas-Valverde Carlos D. Gmez-Carmona Víctor Fortes Jos Pino-Ortega 《Biology of sport / Institute of Sport》2020,37(4):429
This study aimed to describe the worst-case scenarios (WCS) of professional soccer players by playing position in different durations and analyse WCS considering different contextual variables (match half, match location and match outcome). A longitudinal study was conducted in a professional soccer team. Data were collected from different WCS durations in the total distance (TD), high-speed running distance (HSRD), and sprinting distance (SPD). A mixed analysis of variance was performed to compare different WCS durations between playing positions and contextual variables, making pairwise comparisons by Bonferroni post hoc test. Positional differences were found for TD (p < 0.01, ωp2 = 0.02), HSRD (p < 0.01, ωp2 = 0.01) and SPD (p < 0.01, ωp2 = 0.02). There was a significant interaction when comparing WCS by match half in TD (F = 6.1, p < 0.01, ωp2 = 0.07) but no significant differences in HSRD (p = 0.403, ωp2 = 0) or SPD (p = 0.376, ωp2 = 0). A significant interaction was identified when comparing WCS by match location in TD (F = 51.5, p < 0.01, ωp2 = 0.14), HSRD (F = 19.15, p < 0.01, ωp2 = 0.05) and SPD (F = 8.95, p < 0.01, ωp2 = 0.01) as well as WCS by match outcome in TD (F = 36.4, p < 0.01, ωp2 = 0.08), HSRD (F = 13.6, p < 0.01, ωp2 = 0.04) and SPD (F = 7.4, p < 0.01, ωp2 = 0.02). Positional differences exist in TD, HSRD, and SPD in match-play WCS, and contextual variables such as match half, match location and match outcome have a significant impact on the WCS of professional soccer players. 相似文献
7.
The vital parameter data for 62 stocks, covering 38 species, collected from the literature, including parameters of age, growth, and reproduction, were log-transformed and analyzed using multivariate analyses. Three groups were identified and empirical equations were developed for each to describe the relationships between the predicted finite rates of population increase (λ’) and the vital parameters, maximum age (Tmax), age at maturity (Tm), annual fecundity (f/Rc)), size at birth (Lb), size at maturity (Lm), and asymptotic length (L∞). Group (1) included species with slow growth rates (0.034 yr-1 < k < 0.103 yr-1) and extended longevity (26 yr < Tmax < 81 yr), e.g., shortfin mako Isurus oxyrinchus, dusky shark Carcharhinus obscurus, etc.; Group (2) included species with fast growth rates (0.103 yr-1 < k < 0.358 yr-1) and short longevity (9 yr < Tmax < 26 yr), e.g., starspotted smoothhound Mustelus manazo, gray smoothhound M. californicus, etc.; Group (3) included late maturing species (Lm/L∞ ≧ 0.75) with moderate longevity (Tmax < 29 yr), e.g., pelagic thresher Alopias pelagicus, sevengill shark Notorynchus cepedianus. The empirical equation for all data pooled was also developed. The λ’ values estimated by these empirical equations showed good agreement with those calculated using conventional demographic analysis. The predictability was further validated by an independent data set of three species. The empirical equations developed in this study not only reduce the uncertainties in estimation but also account for the difference in life history among groups. This method therefore provides an efficient and effective approach to the implementation of precautionary shark management measures. 相似文献
8.
Reinhard Schweitzer-Stenner Michael Bosenbeck Wolfgang Dreybrodt 《Biophysical journal》1993,64(4):1194-1209
The depolarization ratios of heme protein Raman lines arising from vibrations of the heme group exhibit significant dependence on the excitation wavelength. From the analysis of this depolarization ratio dispersion, one obtains information about symmetry-lowering distortions δQΓ of the heme group that can be classified in terms of the symmetry races Γ = A1g, B1g, B2g, and A2g in D4h symmetry. The heme-protein interaction can be changed by the protonation of distinct amino acid side chains (i.e., for instance the Bohr groups in hemoglobin derivates), which gives rise to specific static heme distortions for each protonation state. From the Raman dispersion data, it is possible to obtain parameters by fitting to a theoretical expression of the Raman tensor, which provide information on these static distortions and also about the pK values of the involved titrable side chains. We have applied this method to the ν4 (1,355 cm-1) and ν10 (1,620 cm-1) lines of deoxygenated hemoglobin of the fourth component of trout and have measured their depolarization ratio dispersion as a function of pH between 6 and 9. From the pH dependence of the thus derived parameters, we obtain pK values identical to those of the Bohr groups, which were earlier derived from the corresponding O2-binding isotherms. These are pKα1 = pKα2 = 8.5 for the α and pKβ1 = 7.5, pKβ2 = 7.4 for the β chains. We also obtain the specific distortion parameters for each protonation state. As shown in earlier studies, the ν4 mode mainly probes distortions from interactions between the proximal histidine and atoms of the heme core (i.e., the nitrogens and the Cα atoms of the pyrroles). Group theoretical argumentation allows us to relate specific changes of the imidazole geometry as determined by its tilt and azimuthal angle and the iron-out-of-plane displacement to distinct variations of the normal distortions δQΓ derived from the Raman dispersion data. Thus, we found that the pH dependence of the heme distortions δQA1g (totally symmetric) and δQB1g (asymmetric) is caused by variations of the azimuthal rather than the tilt angle of the Fe-His (F8) bond. In contrast to this, the ν10 line mainly monitors changes resulting from the interaction between peripheral substituents of the porphyrin macrocycle (vinyl). From the pH dependence of the parameters, it is possible to separately identify distortions δQΓ affecting the hemes in the α and β chains, respectively. From this, we find that in the α subunit structural changes induced on protonation of the corresponding Bohr groups are mainly transferred via the Fe—Nε bond and give rise to changes in the azimuthal angle. In the β subunit, however, in addition, structural changes of the heme pocket arise, which most probably result from protonation of the imidazole of the COOH-terminal His (HC3 β). This rearranges the net of H bonds between His HC3 β, Ser (F9 β), and Glu (F7 β). 相似文献
9.
Camila Nassif Aline Regina Gomes Gustavo H. C. Peixoto Mauro Heleno Chagas Danusa Dias Soares Emerson Silami-Garcia Eric J. Drinkwater Jack Cannon Frank E. Marino 《PloS one》2014,9(8)
This study evaluated double blind ingestions of placebo (PLA) versus 6% carbohydrate (CHO) either as capsules (c) or beverage (b) during 60 km self-paced cycling in the heat (32°C and 50% relative humidity). Ten well-trained males (mean ± SD: 26±3 years; 64.5±7.7 kg and 70.7±8.8 ml.kg−1.min−1 maximal oxygen consumption) completed four separate 60 km time trials (TT) punctuated by 1 km sprints (14, 29, 44, 59 km) whilst ingesting either PLAb or PLAc or CHOb or CHOc. The TT was not different among treatments (PLAb 130.2±11.2 min, CHOb 140.5±18.1 min, PLAc 143.1±29.2 min, CHOc 137.3±20.1 min; P>0.05). Effect size (Cohen’s d) for time was only moderate when comparing CHOb – PLAb (d = 0.68) and PLAb – PLAc (d = 0.57) whereas all other ES were ‘trivial’ to ‘small’. Mean speed throughout the trial was significantly higher for PLAb only (P<0.05). Power output was only different (P<0.05) between the sprints and low intensity efforts within and across conditions. Core and mean skin temperatures were similar among trials. We conclude that CHO ingestion is of little or no benefit as a beverage compared with placebo during 60 km TT in the heat. 相似文献
10.
Enika Nagababu Joy G. Mohanty Surya Bhamidipaty Graciela R. Ostera Joseph M. Rifkind 《Life sciences》2010,86(3-4):133-138
AimsRed blood cells (RBCs) have an extensive antioxidant system designed to eliminate the formation of reactive oxygen species (ROS). Nevertheless, RBC oxidant stress has been demonstrated by the formation of a fluorescent heme degradation product (excitation (ex) 321 nm, emission (em) 465 nm) both in vitro and in vivo. We investigated the possibility that the observed heme degradation results from ROS generated on the membrane surface that are relatively inaccessible to the cellular antioxidants.Main methodsMembrane and cytosol were separated by centrifugation and the fluorescence intensity and emission maximum were measured. The effect on the maximum emission of adding oxidized and reduced hemoglobin to the fluorescent product formed when hemin is degraded by hydrogen peroxide (H2O2) was studied.Key findings90% of the fluorescent heme degradation products in hemolysates are found on the membrane. Furthermore, these products are not transferred from the cytosol to the membrane and must, therefore, be formed on the membrane. We also showed that the elevated level of heme degradation in HbCC cells that is attributed to increased oxidative stress was found on the membrane.SignificanceThese results suggest that, although ROS generated in the cytosol are neutralized by antioxidant enzymes, H2O2 generated by the membrane bound hemoglobin is not accessible to the cytosolic antioxidants and reacts to generate fluorescent heme degradation products. The formation of H2O2 on the membrane surface can explain the release of ROS from the RBC to other tissues and ROS damage to the membrane that can alter red cell function and lead to the removal of RBCs from circulation by macrophages. 相似文献
11.
Peter Lachmann Yafei Huang Joachim Reimann Ulrika Flock Pia ?delroth 《The Journal of biological chemistry》2010,285(33):25531-25537
Nitric -oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N2O) (2NO + 2H+ + 2e− →N2O + H2O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b3, and a non-heme iron FeB. Here, we have studied the reaction between fully reduced NOR and NO using the “flow-flash” technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b3 occurs with a time constant of ∼1 μs at 1.5 mm NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to FeB before ligation to heme b3. Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s−1 and 3 s−1 at 1.5 mm NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s−1 and 12 s−1 at 75 μm NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed. 相似文献
12.
13.
Sebastian Pintscher Patryk Kuleta Ewelina Cieluch Arkadiusz Borek Marcin Sarewicz Artur Osyczka 《The Journal of biological chemistry》2016,291(13):6872-6881
In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemes b. The hemes display a large difference in redox midpoint potentials (ΔEm_b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on heme b ligand mutants of cytochrome bc1 in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔEm_b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔEm_b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functional in vivo. This reveals that cytochrome bc1 can accommodate large changes in ΔEm_b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product. We propose that hemes b in this cytochrome and in other membranous cytochromes b act as electronic connectors for the catalytic sites with no fine tuning in ΔEm_b required for efficient cross-membrane electron transfer. We link this concept with a natural flexibility in occurrence of several thermodynamic configurations of the direction of electron flow and the direction of the gradient of potential in relation to the vector of the electric membrane potential. 相似文献
14.
The Ion Permeability Induced in Thin Lipid Membranes by the Polyene Antibiotics Nystatin and Amphotericin B 总被引:23,自引:4,他引:19
Characteristics of nystatin and amphotericin B action on thin (<100 A) lipid membranes are: (a) micromolar amounts increase membrane conductance from 10-8 to over 10-2 Ω-1 cm-2; (b) such membranes are (non-ideally) anion selective and discriminate among anions on the basis of size; (c) membrane sterol is required for action; (d) antibiotic presence on both sides of membrane strongly favors action; (e) conductance is proportional to a large power of antibiotic concentration; (f) conductance decreases ~104 times for a 10°C temperature rise; (g) kinetics of antibiotic action are also very temperature sensitive; (h) ion selectivity is pH independent between 3 and 10, but (i) activity is reversibly lost at high pH; (j) methyl ester derivatives are fully active; N-acetyl and N-succinyl derivatives are inactive; (k) current-voltage characteristic is nonlinear when membrane separates nonidentical salt solutions. These characteristics are contrasted with those of valinomycin. Observations (a)–(g) suggest that aggregates of polyene and sterol from opposite sides of the membrane interact to create aqueous pores; these pores are not static, but break up (melt) and reform continuously. Mechanism of anion selectivity is obscure. Observations (h)–(j) suggest—NH3+ is important for activity; it is probably not responsible for selectivity, particularly since four polyene antibiotics, each containing two—NH3+ groups, induce ideal cation selectivity. Possibly the many hydroxyl groups in nystatin and amphotericin B are responsible for anion selectivity. The effects of polyene antibiotics on thin lipid membranes are consistent with their action on biological membranes. 相似文献
15.
Jeffrey A. Mayfield Neal D. Hammer Richard C. Kurker Thomas K. Chen Sunil Ojha Eric P. Skaar Jennifer L. DuBois 《The Journal of biological chemistry》2013,288(32):23488-23504
The chlorite dismutases (C-family proteins) are a widespread family of heme-binding proteins for which chemical and biological roles remain unclear. An association of the gene with heme biosynthesis in Gram-positive bacteria was previously demonstrated by experiments involving introduction of genes from two Gram-positive species into heme biosynthesis mutant strains of Escherichia coli, leading to the gene being renamed hemQ. To assess the gene product''s biological role more directly, a Staphylococcus aureus strain with an inactivated hemQ gene was generated and shown to be a slow growing small colony variant under aerobic but not anaerobic conditions. The small colony variant phenotype is rescued by the addition of exogenous heme despite an otherwise wild type heme biosynthetic pathway. The ΔhemQ mutant accumulates coproporphyrin specifically under aerobic conditions. Although its sequence is highly similar to functional chlorite dismutases, the HemQ protein has no steady state reactivity with chlorite, very modest reactivity with H2O2 or peracetic acid, and no observable transient intermediates. HemQ''s equilibrium affinity for heme is in the low micromolar range. Holo-HemQ reconstituted with heme exhibits heme lysis after <50 turnovers with peroxide and <10 turnovers with chlorite. The heme-free apoprotein aggregates or unfolds over time. IsdG-like proteins and antibiotic biosynthesis monooxygenases are close sequence and structural relatives of HemQ that use heme or porphyrin-like organic molecules as substrates. The genetic and biochemical data suggest a similar substrate role for heme or porphyrin, with possible sensor-regulator functions for the protein. HemQ heme could serve as the means by which S. aureus reversibly adopts an SCV phenotype in response to redox stress. 相似文献
16.
Inhibition of hemozoin formation in Plasmodium falciparum trophozoite extracts by heme analogs: possible implication in the resistance to malaria conferred by the beta-thalassemia trait.
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BACKGROUND: Human falciparum malaria, caused by the intracellular protozoa Plasmodium falciparum, results in 1-2 million deaths per year. P. falciparum digests host erythrocyte hemoglobin within its food vacuole, resulting in the release of potentially toxic free heme. A parasite-specific heme polymerization activity detoxifies the free heme by cross-linking the heme monomers to form hemozoin or malaria pigment. This biochemical process is the target of the widely successful antimalarial drug chloroquine, which is rapidly losing its effectiveness due to the spread of chloroquine resistance. We have shown that chloroquine resistance is not due to changes in the overall catalytic activity of heme polymerization or its chloroquine sensitivity. Therefore, the heme polymerization activity remains a potential target for novel antimalarials. In this study, we investigated the ability of heme analogs to inhibit heme polymerization and parasite growth in erythrocytes. MATERIALS AND METHODS: Incorporation of radioactive hemin substrate into an insoluble hemozoin pellet was used to determine heme polymerization. Incorporation of radioactive hypoxanthine into the nucleic acid of dividing parasites was used to determine the effects of heme analogs on parasite growth. Microscopic and biochemical measurements were made to determine the extent of heme analog entry into infected erythrocytes. RESULTS: The heme analogs tin protoporphyrin IX (SnPP), zinc protoporphyrin IX (ZnPP), and zinc deuteroporphyrin IX, 2,4 bisglycol (ZnBG) inhibited polymerization at micromolar concentrations (ZnPP << SnPP < ZnBG). However, they did not inhibit parasite growth since they failed to gain access to the site of polymerization, the parasite's food vacuole. Finally, we observed high ZnPP levels in erythrocytes from two patients with beta-thalassemia trait, which may inhibit heme polymerization. CONCLUSIONS: The heme analogs tested were able to inhibit hemozoin formation in Plasmodium falciparum trophozite extracts. The increased ZnPP levels found in thalassemic erythrocytes suggest that these may contribute, at least in part, to the observed antimalarial protection conferred by the beta-thalassemia trait. This finding may lead to the development of new forms of antimalarial therapy. 相似文献
17.
The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h–1 to 0.01 h–1 when decreasing the pH from 7 to 6.8.Abbreviations D
dilution rate (h–1)
- kb
specific trypan-blue dead cells appearance rate (h–1)
- kL
specific lysis rate of viable cells (h–1)
- kd
specific death rate (h-1)
- LDH0
lactate dehydrogenase activity in the feed culture medium (IU.l–1)
- LDH
lactate dehydrogenase activity in the outlet culture medium (IU.l–1)
- LDHi
intracellular lactate dehydrogenase activity of viable cells (IU.10–9 cells)
- rLDH
total rate of LDH release (IU.h–1.L–1)
- rb
transformation rate of viable cells into blue dead cells (109 cells.h–1.L–1)
- xv
viable cell concentration (109 cells.l–1)
- xb
trypan-blue dead cell concentration (109 cells.l–1) 相似文献
18.
19.
Yuan-Gang Qu Fei Zhou Linda Yu Chang-An Yu 《The Journal of biological chemistry》2013,288(2):1047-1054
Proton transfer involving internal water molecules that provide hydrogen bonds and facilitate proton diffusion has been identified in some membrane proteins. Arg-94 in cytochrome b of the Rhodobacter sphaeroides bc1 complex is fully conserved and is hydrogen-bonded to the heme propionate and a chain of water molecules. To further elucidate the role of Arg-94, we generated the mutations R94A, R94D, and R94N. The wild-type and mutant bc1 complexes were purified and then characterized. The results show that substitution of Arg-94 decreased electron transfer activity and proton pumping capability and increased O2˙̄ production, suggesting the importance of Arg-94 in the catalytic mechanism of the bc1 complex in R. sphaeroides. This also suggests that the transport of H+, O2, and O2˙̄ in the bc1 complex may occur by the same pathway. 相似文献
20.
Extracellular Calcium Regulates HeLa Cell Morphology during
Adhesion to Gelatin: Role of Translocation and Phosphorylation of
Cytosolic Phospholipase A2 总被引:1,自引:1,他引:0
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Attachment of HeLa cells to gelatin induces the release of arachidonic acid (AA), which is essential for cell spreading. HeLa cells spreading in the presence of extracellular Ca2+ released more AA and formed more distinctive lamellipodia and filopodia than cells spreading in the absence of Ca2+. Addition of exogenous AA to cells spreading in the absence of extracellular Ca2+ restored the formation of lamellipodia and filopodia. To investigate the role of cytosolic phospholipase A2 (cPLA2) in regulating the differential release of AA and subsequent formation of lamellipodia and filopodia during HeLa cell adhesion, cPLA2 phosphorylation and translocation from the cytosol to the membrane were evaluated. During HeLa cell attachment and spreading in the presence of Ca2+, all cPLA2 became phosphorylated within 2 min, which is the earliest time cell attachment could be measured. In the absence of extracellular Ca2+, the time for complete cPLA2 phosphorylation was lengthened to <4 min. Maximal translocation of cPLA2 from cytosol to membrane during adhesion of cells to gelatin was similar in the presence or absence of extracellular Ca2+ and remained membrane associated throughout the duration of cell spreading. The amount of total cellular cPLA2 translocated to the membrane in the presence of extracellular Ca2+ went from <20% for unspread cells to >95% for spread cells. In the absence of Ca2+ only 55–65% of the total cPLA2 was translocated to the membrane during cell spreading. The decrease in the amount translocated could account for the comparable decrease in the amount of AA released by cells during spreading without extracellular Ca2+. Although translocation of cPLA2 from cytosol to membrane was Ca2+ dependent, phosphorylation of cPLA2 was attachment dependent and could occur both on the membrane and in the cytosol. To elucidate potential activators of cPLA2, the extracellular signal-related protein kinase 2 (ERK2) and protein kinase C (PKC) were investigated. ERK2 underwent a rapid phosphorylation upon early attachment followed by a dephosphorylation. Both rates were enhanced during cell spreading in the presence of extracellular Ca2+. Treatment of cells with the ERK kinase inhibitor PD98059 completely inhibited the attachment-dependent ERK2 phosphorylation but did not inhibit cell spreading, cPLA2 phosphorylation, translocation, or AA release. Activation of PKC by phorbol ester (12-O-tetradecanoylphorbol-13-acetate) induced and attachment-dependent phosphorylation of both cPLA2 and ERK2 in suspension cells. However, in cells treated with the PKC inhibitor Calphostin C before attachment, ERK2 phosphorylation was inhibited, whereas cPLA2 translocation and phosphorylation remained unaffected. In conclusion, although cPLA2-mediated release of AA during HeLa cell attachment to a gelatin substrate was essential for cell spreading, neither ERK2 nor PKC appeared to be responsible for the attachment-induced cPLA2 phosphorylation and the release of AA. 相似文献