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121.
122.
用闪光动力学光谱仪测量了酰化紫膜LB膜中M衰减速率的变化。酰化紫膜LB膜的衰减无论是悬浮液状态,还是LB膜中,均比未修饰的要慢。在温度为20℃时,酰化紫膜LB随着相对湿度的增加,M衰减加快。在相对湿度较低时(RH34—75%),变化较平缓,即M的衰减加快不明显;在相对湿度较高时(RH84—95%),M衰减明显加快。温度的变化则随相对湿度不同而不同。相对湿度较低时,随着温度的升高,M衰减加快;相对湿度较高时,M衰减反而减慢。酰化紫膜悬浮液的M衰减随着温度的升高而明显加快.这说明酰化紫膜LB膜中BR水合程度可能是直接影响M衰减的因素之一。 相似文献
123.
124.
Optical methods to measure membrane transport processes 总被引:6,自引:0,他引:6
A. S. Verkman 《The Journal of membrane biology》1995,148(2):99-110
125.
3-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a photoaffinity analog of ATP to label potential ATP receptors in ciliated cells. Like ATP, without photoactivation, BzATP stimulated the ciliary beat frequency in tissue culture up to threefold. Irradiation of intact cells in the presence of [-32P]BzATP followed by SDS-PAGE and autoradiography revealed two labeled proteins with molecular masses of 46 and 96 kDa (p46 and p96). Photolabeling of both proteins was susceptible to digestion with trypsin, implying that the labeled proteins are at least partially exposed on the extracellular surface of the plasma membrane. The dependence of 32P incorporation in both proteins on [-32P]BzATP concentration was similar. Labeling of p46 but not p96 required Ca2+ or Mg2+. Various nucleotides stimulated the ciliary frequency, and inhibited the photolabeling of p46 and p96. The rank order of apparent affinity for p46 is: ATP ÃDP>GTPS>ADP S, UTP, 2MeSATP, AMP-PNP >AMP-PCP>AMP>adenosine; for p96 it is: ADPADP S ATP AMP-PCP, AMP-PNP>GTPS AMP>2MeSATP, UTP, adenosine. The rank of stimulation of ciliary beat frequency is: ADPS, UTP 2MeSATP, GTPS, AMP-PNP, ATPADP>AMPPCP>adenosine>AMP. These results suggest the involvement of p46 in the stimulatory effect of extracellular ATP on the ciliary beat, as a P2 purinoceptor. On the other hand, p96 may represent a P2 purinoceptor or an ectonucleotidase.This work was supported by grants (to Z.P. and to V.S-B.) from the Fund for Basic Research administered by the Israel Academy of Science and Humanities. 相似文献
126.
Gap junctions, collections of membrane channels responsible for intercellular communication, contain two paired hemichannels (also called connexons). We have investigated conditions for splitting the membrane pair using urea. We have developed a protocol which consistently splits the gap junction samples with 60–90% efficiency. Our results indicate that hydrophobic forces are important in holding the two connexons together but that Ca2+ ions are also important in the assembly of the membrane pair. Greater yields and better structural integrity of split junctions were obtained with a starting preparation of gap junctions which had been detergent treated. Image analysis of edge views of single connexon layers reveal an asymmetry in the appearance of the cytoplasmic and extracellular surface. Cryo-electron microscopy and image analysis of split junctions show that the packing and structural detail of membranes containing arrays of single connexons are the same as for intact junctions, and that the urea treatment causes no gross structural changes in the connexon assembly.The antibodies used to analyze the protein composition in our samples were the generous gift of Dr. David Paul. We thank Dr. Camillo Peracchia for sending us a preprint of his chapter on molecular mechanisms of connexon gating and docking which helped guide our thinking about interconnexon forces and John Badger for information on divalent cation sites in protein structure. We thank Amani Thomas-Yusuf for technical assistance. The photographic expertise of Marie Craig is gratefully acknowledged. This work was funded by National Institutes of Health GM43217 to G.E.S. and GM18974 to D.A.G. 相似文献
127.
Inês A. C. Pereira Jean LeGall António V. Xavier M. Teixeira 《Journal of biological inorganic chemistry》1997,2(1):23-31
The high-molecular-mass cytochromes c (Hmcs) from the sulfate-reducing bacteria Desulfovibrio gigas and Desulfovibrio vulgaris (Hildenborough) were found to be strongly bound to the cytoplasmic membrane. After detergent solubilization they were shown to be water soluble and to be similar to those previously isolated from the soluble fractions in terms of N-terminal sequence, molecular mass, UV-visible and EPR spectroscopies. In D. gigas, higher amounts of Hmc can be obtained from the membranes than from the soluble fraction. This enabled further characterization of both cytochromes. The apparent heme reduction potentials of both Hmcs, determined at pH 7.5 through visible and EPR redox titrations, span a large range of redox potentials, approximately between 0 and –280?mV, and can be roughly divided into three groups: four to five hemes have E 0s of –30?mV to –100?mV, three to four hemes have E 0s around –170?mV, and seven to eight hemes have a lower E 0 of –250 to –280?mV. Several of these redox potentials are strongly pH dependent. Mössbauer studies of oxidized and reduced D. vulgaris Hmc show that this protein contains two high-spin hemes in both oxidation states. The rate of reduction of both Hmcs with the periplasmic hydrogenases from the corresponding organisms is extremely slow. 相似文献
128.
In the giant-celled marine algae Valonia utricularis the turgor-sensing mechanism of the plasmalemma and the role of the tonoplast in turgor regulation is unknown because of
the lack of solid data about the individual electrical properties of the plasmalemma and the vacuolar membrane. For this reason,
a vacuolar perfusion technique was developed that allowed controlled manipulation of the vacuolar sap under turgescent conditions
(up to about 0.3 MPa). Charge-pulse relaxation studies on vacuolarly perfused cells at different turgor pressure values showed
that the area-specific resistance of the total membrane barrier (tonoplast and plasmalemma) exhibited a similar dependence
on turgor pressure as reported in the literature for nonperfused cells: the resistance assumed a minimum value at the physiological
turgor pressure of about 0.1 MPa. The agreement of the data suggested that the perfusion process did not alter the transport
properties of the membrane barrier.
Addition of 16 μm of the H+-carrier FCCP (carbonylcyanide p-trifluoromethoxyphenyhydrazone) to the perfusion solution resulted in a drop of the total membrane potential from +4 mV to
−22 mV and in an increase of the area-specific membrane resistance from 6.8 × 10−2 to 40.6 × 10−2Ωm2. The time constants of the two exponentials of the charge pulse relaxation spectrum increased significantly. These results
are inconsistent with the assumption of a high-conductance state of the tonoplast (R. Lainson and C.P. Field, J. Membrane Biol.
29:81–94, 1976).
Depending on the site of addition, the pore-forming antibiotics nystatin and amphotericin B affected either the time constant
of the fast or of the slow relaxation (provided that the composition of the perfusion solution and the artificial sea water
were replaced by a cytoplasma-analogous medium). When 50 μm of the antibiotics were added externally, the fast relaxation process disappeared. Contrastingly, the slow relaxation process
disappeared upon vacuolar addition. The antibiotics cannot penetrate biomembranes rapidly, and therefore, the findings suggested
that the fast and slow relaxations originated exclusively from the electrical properties of the plasmalemma and the tonoplast
respectively. This interpretation implies that the area-specific resistance of the tonoplast is significantly larger than
that of the plasmalemma (consistent with the FCCP data) and that the area-specific capacitance of the tonoplast is unusually
high (6.21 × 10−2 Fm−2 compared to 0.77 × 10−2 Fm−2 of the plasmalemma). Thus, we have to assume that the vacuolar membrane of V. utricularis is highly folded (by a factor of about 9 in relation to the geometric area) and/or contains a fairly high concentration of
mobile charges of an unknown electrogenic ion carrier system.
Received: 22 October 1996/Revised: 16 January 1997 相似文献
129.
W. H. H. Krueger G. E. Gonye D. L. Madison K. E. Murray M. Kumar N. Spoerel S. E. Pfeiffer 《Journal of neurochemistry》1997,69(4):1343-1355
Abstract: Although the myelin membrane contains only a small set of major proteins, more sensitive assays indicate the presence of a plethora of uncharacterized proteins. We have used an antibody perturbation approach to reversibly block the differentiation of prooligodendroblasts into myelinating cells, and, in combination with a differential screening procedure, identified novel mRNAs that are activated during this period. One cDNA, TPO1, recognizes a 5.5-kb mRNA that is strongly up-regulated in oligodendrocytes after release of the differentiation block and that is expressed at high levels in brain tissue during active myelination. This cDNA represents at least two mRNAs differing from each other in their 5'-termini. The TPO1 cDNA contains an open reading frame of 1,380 bp, encoding a protein of 51.8 kDa with a predicted pl of 9.1 that contains two regions homologous to nonclassic zinc finger motifs. Subcellular localization studies suggest the enriched presence of TPO1 in spherical structures along the major cytoplasmic processes of oligodendrocytes. TPO1, along with homologues expressed in testis, placenta, and PC12 cells, form a novel family of proteins with multiple hydrophobic domains possibly serving as membrane spanning regions. We postulate that in oligodendrocytes, TPO1 encodes a protein factor involved in myelin biogenesis. 相似文献
130.
Characterization of the Palmitoylation Domain of SNAP-25 总被引:5,自引:2,他引:3
Abstract: SNAP-25 (synaptosomal associated protein of 25 kDa) is a neural specific protein that has been implicated in the synaptic vesicle docking and fusion process. It is tightly associated with membranes, and it is one of the major palmitoylated proteins found in neurons. The functional role of palmitoylation for SNAP-25 is unclear. In this report, we show that the palmitate of SNAP-25 is rapidly turned over in PC12 cells, with a half-life of ∼3 h, and the half-life for the protein is 8 h. Mutation of Cys to Ser at positions 85, 88, 90, and 92 reduced the palmitoylation to 9, 21, 42, and 35% of the wild-type protein, respectively. Additional mutations of either Cys85,88 or Cys90,92 nearly abolished palmitoylation of the protein. A similar effect on membrane binding for the mutant SNAP-25 was observed, which correlated with the degree of palmitoylation. These results suggest that all four Cys residues are involved in palmitoylation and that membrane association of SNAP-25 may be regulated through dynamic palmitoylation. 相似文献