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91.
Large-scale preparation of highly purified tonoplast from cucumber (Cucumis sativus L.) roots was obtained after centrifugation of microsome pellet (10,000 – 80,000 g) on discontinuous sucrose density gradient
(20, 28, 32 and 42 %). Lack of PEP carboxylase (cytosol marker) and cytochrome c oxidase (mitochondrial marker) together with
a slight activity of VO4-ATPase (plasma membrane marker) and NADH-cytochrome c reductase (ER marker) in tonoplast preparation confirmed its high purity.
Using latency of nitrate-inhibited ATPase and H+ pumping as criteria it was established that the majority of tonoplast vesicles were sealed and oriented right(cytoplasmic)-side-out.
Strong acidification of the interior of vesicles observed at the presence of both, ATP and PPi, confirmed that obtained tonoplast contains two classes of proton pumps: V-ATPase and H+PPiase.
To examine and characterise of proton-transport systems in tonoplast, the effect of various inhibitors on H+ pumping and hydrolytic activities of ATPase and PPiase were measured. ATP-dependent activities (H+ flux and ATP hydrolysis) were specifically decreased by nitrate and bafilomycin A1, whereas the PPiase activities were reduced in the presence of fluoride and Na+ ions. Both enzymes showed a similar sensitivity to DCCD and DES. The results of experiments with KCl and NaCl suggested that
the vacuolar ATPase was stimulated by Cl−, whereas the vacuolar Ppiase requires K+ ions for its activity. 相似文献
92.
Gilbert Berben 《Antonie van Leeuwenhoek》1996,69(1):305-315
Cytochrome c oxidase (EC 1.9.3.1) is one of the components of the electron transport chain by which Nitrobacter, a facultative lithoautotrophic bacterium, recovers energy from nitrite oxidation. The genes encoding the two catalytic core subunits of the enzyme were isolated from a Nitrobacter winogradskyi gene library. Sequencing of one of the 14 cloned DNA segments revealed that the subunit genes are side by side in an operon-like cluster. Remarkably the cluster appears to be present in at least two copies per genome. It extends over a 5–6 kb length including, besides the catalytic core subunit genes, other cytochrome oxidase related genes, especially a heme O synthase gene. Noteworthy is the new kind of gene order identified within the cluster. Deduced sequences for the cytochrome oxidase subunits and for the heme O synthase look closest to their counterparts in other -subdivision Proteobacteria, particularly the Rhizobiaceae. This confirms the phylogenetic relationships established only upon 16S rRNA data. Furthermore, interesting similarities exist between N. winogradskyi and mitochondrial cytochrome oxidase subunits while the heme O synthase sequence gives some new insights about the other similar published -subdivision proteobacterial sequences.Abbreviations COI
cytochrome oxidase subunit I
- COII
cytochrome oxidase subunit II
- COIII
cytochrome oxidase subunit III
- HOS
Heme O synthase
- ORF
open reading frame
- SDS
sodium dodecyl sulfate 相似文献
93.
Experimental observations reveal a number of characteristics of the redox-linked proton ejection from cytochrome c oxidase vesicles, which apparently cannot be explained by a proton pumping activity of the oxidase. These observations seem, on the other hand, to provide useful elements for alternative explanation(s) of the proton ejection. It is proposed here that the process is scalar and not vectorial and can derive from redox-linked rupture of protonated salt-bridges in the oxidase-lipid complex. 相似文献
94.
Glutamate transport in blood platelets resembles that in brain nerve terminals because platelets contain neuronal Na+-dependent glutamate transporters, glutamate receptors in the plasma membrane, vesicular glutamate transporters in secretory granules, which use the proton gradient as a driving force, and can release glutamate during aggregation/activation. The acidification of secretory granules and glutamate transport were assessed during acute treatment of isolated platelets with cholesterol-depleting agent methyl-β-cyclodextrin (MβCD). Confocal imaging with the cholesterol-sensitive fluorescent dye filipin showed a quick reduction of cholesterol level in platelets. Using pH-sensitive fluorescent dye acridine orange, we demonstrated that the acidification of secretory granules of human and rabbit platelets was decreased by ∼15% and 51% after the addition of 5 and 15 mM MβCD, respectively. The enrichment of platelet plasma membrane with cholesterol by the application of complex MβCD-cholesterol (1:0.2) led to the additional accumulation of acridine orange in secretory granules indicating an increase in the proton pumping activity of vesicular H+-ATPase. MβCD did not evoke release of glutamate from platelets that was measured with glutamate dehydrogenase assay. Flow cytometric analysis did not reveal alterations in platelet size and granularity in the presence of MβCD. These data showed that the dissipation of the proton gradient of secretory granules rather than their exocytosis caused MβCD-evoked decrease in platelet acidification. Thus, the depletion of plasma membrane cholesterol in the presence of MβCD changed the functional state of platelets affecting storage capacity of secretory granules but did not evoke glutamate release from platelets. 相似文献
95.
Thermoluminescence experiments have been carried out to study the effect of a transmembrane proton gradient on the recombination properties of the S2 and S3 states of the oxygen evolving complex with QA
- and QB
-, the reduced electron acceptors of Photosystem II. We first determined the properties of the S2QA
- (Q band), S2QB
- and S3QB
- (B bands) recombinations in the pH range 5.5 to 9.0, using uncoupled thylakoids. The, a proton gradient was created in the dark, using the ATP-hydrolase function of ATPases, in coupled unfrozen thylakoids. A shift towards low temperature of both Q and B bands was observed to increase with the magnitude of the proton gradient measured by the fluorescence quenching of 9-aminoacridine. This downshift was larger for S3QB
- than for S2QB
- and it was suppressed by nigericin, but not by valinomycin. Similar results were obtained when a proton gradient was formed by photosystem I photochemistry. When Photosystem II electron transfer was induced by a flash sequence, the reduction of the plastoquinone pool also contributed to the downshift in the absence of an electron acceptor. In leaves submitted to a flash sequence above 0°C, a downshift was also observed, which was supressed by nigericin infiltration. Thus, thermoluminescence provides direct evidence on the enhancing effect of lumen acidification on the S3S2 and S2S1 reverse-transitions. Both reduction of the plastoquinone pool and lumen acidification induce a shift of the Q and B bands to lower temperature, with a predominance of lumen acidification in non-freezing, moderate light conditions.Abbreviations 9-AA
9-aminoacridine
- EA
activation energy
- F0
constant fluorescence level
- FM
maximum fluorescence, when all PS-II centers are closed
- FV
variable fluorescence (FM–F0)
- PS I, PS II
Photosystem I, photosystem II
- PQ
plastoquinone
- TL
thermoluminescence 相似文献
96.
Rodríguez-González I Marín C Vargas F Córdova O Barrera M Gutiérrez-Sánchez R Alunda JM Sánchez-Moreno M 《Experimental parasitology》2006,112(1):44-51
Eight Leishmania promastigotes were isolated from different geographical areas: three (LP1, LP2, and LP3) from the provincial department La Libertad and the fourth (LP4) from the department of Cajamarca (northern Peru); another three (LM1, LM2, and LM3) in the province of Campeche (Mexico); and the last (LS1) from a clinical case of a dog in Madrid (Spain). The isolates were characterized by carbohydrate cell-surface residues using agglutinations with four purified lectins, by isoenzyme analysis using different isoenzymes, by analysis of kinetoplast DNA (kDNA) restriction fragment length polymorphism using four different restriction endonucleases and by the final metabolite patterns after in vitro culture. These isolates were compared with four reference strains and typified as: Leishmania (Leishmania) donovani, two strains of L. (L.) infantum, and one species of L. (Viania) peruviana. According to our results and the statistical study, the Peruvian isolates represent three different strains: one would be L. (V.) peruviana, another the strain isolated in Cajamarca (LP4) and the third would include the three strains from the department of La Libertad (LP1, LP2, and LP3), these latter three isolates being phylogenetically closer to the reference strain L. (L.) donovani. Meanwhile, the three isolates from Mexico form a group with close phylogenetic relationships to each other. The isolate from Spain belongs to the species L. (L.) infantum. Thus, a close correlation was drawn between the identity of each strain and its geographical origin. 相似文献
97.
钙调蛋白(CaM)是一种多功能调节蛋白,它含有4个Ca~(2+)结合域.晶体研究表明所有Ca~(2+)都与主链氧原子及酸性残基侧链氧原子配位,但Ca~(2+)的配位层中是否有水分子存在尚未确定.木文利用核磁共振技术,以Mn~(2+)为探针,通过测定水质子的核磁弛豫速率T_(1P)_(-1)建立了有关Ca~(2+)配位层中水分子数目的模型,该模型指出Cam中高、低亲和位上Ca~(2+)结合水的能力不同,高亲和位上Ca~(2+)的配位层中没有水分子存在,而低亲和位上Ca~(2+)配位层中含两个水分子. 相似文献
98.
Visible absorption and CD spectral and potentiometric studies on the His- and Tyr-containing ternary copper(II) complexes Cu(A)(L-B), where A refers to L-His, D-His, or L-Tyr and B to Lys, Tyr, Trp, Phe, Ala, Val, Arg, Glu, Asn, Gln, Ser, or Thr, were made to study ligand-ligand interactions in the complexes. While the CD spectral magnitudes in the d—d region are additive in the absence of side chain interactions and can be estimated from the magnitudes for the ternary systems involving DL-A or DL-B, deviation from the additivity was observed for Cu(L-His)(L-B) (B = LysH, Tyr, Trp, or Phe) and Cu(L-Tyr)(L-Trp). From the stability constants determined at 25 °C and I = 0.1 M (KNO3), the equilibrium constants, K, for the following hypothetical equilibria were calculated to be large (0.14–0.60) for formation of Cu(L-/D-His)(L-B)(B = Tyr or Trp) and Cu(D-His)(L-Phe) with Cu(en)(L-Ala) as standard: The positive values indicate the stabilization due to the stacking between the imidazole ring of His and the aromatic side chain of L-B. Solvent dependence of the CD spectra for Cu(L-His)(L-LysH) and Cu(L-His) L-Trp) further supported the existence of the intramolecular electrostatic and hydrophobic interactions. 相似文献
99.
The proton pumping activity of the tonoplast (vacuolar membrane) H+ -ATPase and H+ -pyrophosphatase (H+ -PPase) has been studied on a tonoplast-enriched microsomal fraction and on intact vacuoles isolated from a heterotrophic cell suspension culture of Chenopodium rubrum L. in the presence of the lysosphingolipids D-sphingosine, psychosine (galactosylsphingosine) and lysosulfatide (sulfogalactosyl-sphingosine). Sphingosine strongly stimulates (Ka = 0.16 μ M ) the PPase activity, assayed both as ΔpH formation across the tonoplast vesicle membrane, and as reversible clamp current measured by the whole-vacuolar mode of the patch-clamp technique. Psychosine showed a minor, and lysosulfatide no stimulatory effect. No effect upon the ATPase activity has been observed. No sphingosine-induced change could be observed in the affinity of the PPase for its substrate (apparent Km = 10 μ M MgPPi). We tentatively conclude that sphingosine, which is known as a potent inhibitor of the protein kinase C in animal cells, may be a regulator of the plant vacuolar PPase. 相似文献
100.
Gaoqiang Yang Shule Yu Zhenye Kang Yifan Li Guido Bender Bryan S. Pivovar Johney B. Green David A. Cullen Feng‐Yuan Zhang 《Liver Transplantation》2020,10(16)
Low electron/proton conductivities of electrochemical catalysts, especially earth‐abundant nonprecious metal catalysts, severely limit their ability to satisfy the triple‐phase boundary (TPB) theory, resulting in extremely low catalyst utilization and insufficient efficiency in energy devices. Here, an innovative electrode design strategy is proposed to build electron/proton transport nanohighways to ensure that the whole electrode meets the TPB, therefore significantly promoting enhance oxygen evolution reactions and catalyst utilizations. It is discovered that easily accessible/tunable mesoporous Au nanolayers (AuNLs) not only increase the electrode conductivity by more than 4000 times but also enable the proton transport through straight mesopores within the Debye length. The catalyst layer design with AuNLs and ultralow catalyst loading (≈0.1 mg cm?2) augments reaction sites from 1D to 2D, resulting in an 18‐fold improvement in mass activities. Furthermore, using microscale visualization and unique coplanar‐electrode electrolyzers, the relationship between the conductivity and the reaction site is revealed, allowing for the discovery of the conductivity‐determining and Debye‐length‐determining regions for water splitting. These findings and strategies provide a novel electrode design (catalyst layer + functional sublayer + ion exchange membrane) with a sufficient electron/proton transport path for high‐efficiency electrochemical energy conversion devices. 相似文献