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1.
2.
Analysis of the effect of medium and membrane conductance on the amplitude and kinetics of membrane potentials induced by externally applied electric fields. 总被引:5,自引:2,他引:3
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The kinetics and amplitudes of membrane potential induced by externally applied electric field pulses are determined for a spherical lipid bilayer using a voltage-sensitive dye. Several experimental parameters were systematically varied. These included the incorporation of gramicidin into the membrane to alter its conductivity and the variation of the external electrolyte conductivity via changes in salt concentration. The ability of the solution to Laplace's equation for a spherical dielectric shell to quantitatively describe the membrane potential induced on a lipid bilayer could thus be critically evaluated. Both the amplitude and the kinetics of the induced potential were consistent with the predictions of this simple model, even at the extremes of membrane conductance or electrolyte concentration. The success of the experimental approach for this system encourages its application to more complex problems such as electroporation and the influences of external electric fields in growth and development. 相似文献
3.
The hydroxylations of d-camphor, norcamphor, pericyclocamphanone, and 5,5-difluorocamphor by cytochrome P-450cam have been examined using theoretical methods to identify and characterize properties which determine product specificity. Experimental results indicate that each molecule is hydroxylated with quite different regio-specificity when metabolized by P-450cam. This result is surprising in view of their overall structural similiarity. Herein we report the results of calculations on d-camphor and three of its analogues which suggest that all of these molecules should, when metabolized by P-450cam, form hydroxylation products and predict the product distribution for each. Our conclusions are based on two fundamental criteria which are consistent with a generally accepted radical mechanism in determining product specificity in these molecules: 1) relative heats of formation of the radicals formed by abstracting a hydrogen, and 2) orientation of the substrate molecule with respect to the putative active oxygen species bound to iron. Our results explain the experimental observations for camphor and 5,5-difluorocamphor but disagree with original published results for norcamphor and pericyclocamphanone. In light of our results, new experiments have been performed for norcamphor and the original data reexamined for pericyclocamphanone. Our predictions have recently been experimentally confirmed for norcamphor, and unpublished data (Dr. S. Sligar) suggest that the same is true for pericyclocamphanone. 相似文献
4.
Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth. 总被引:9,自引:0,他引:9
Our study explores the mechanisms behind neurite galvanotropism. Using phase, differential interference contrast and ratiometric fluorescence microscopy, we reveal four responses of N1E-115 mouse neuroblastoma cells to 0.1-1.0 mV/microns uniform DC electric fields: cathode-directed neurite initiation and elongation, cathode-biased growth cone filopodial protrusions, transient cathode-localized calcium increases, and persistent cathode-localized membrane depolarizations. These newly demonstrated events are temporally and spatially correlated, suggesting that they are causally related. The calcium increases are prevented by calcium channel blockers and by the removal of extracellular calcium. We therefore propose that the observed field-induced membrane depolarizations activate voltage-dependent calcium channels, resulting in cathode-localized calcium influx. This, in turn, may initiate the observed cathode-biased growth cone filopodial protrusions, followed by the cathode-directed neurite elongation. 相似文献
5.
6.
Purification and pore-forming activity of two hydrophobic polypeptides from the secretion of the Red Sea Moses sole (Pardachirus marmoratus) 总被引:5,自引:0,他引:5
A new column chromatography procedure, based on ion exchange, chromatofocusing, and reverse phase high pressure liquid chromatography was employed to isolate the two main proteinaceous, toxic, cytolytic, pore-forming factors from the secretion of the Red Sea Moses sole Pardachirus marmoratus. Pardaxin I, comprising 10% of the gland secretion proteins, was shown to be 5-10 times more toxic, cytolytic, and active in membrane pore formation than pardaxin II (8% of gland secretion proteins). Gel electrophoresis, amino acid analysis, and NH2-terminal amino acid sequence reveals a high degree of homogeneity and resemblance between the two toxins. They are rich in aspartic acid, serine, glycine, and alanine and devoid of arginine, tyrosine, and tryptophan. Their NH2-terminal residue sequence was found to be NH2-Gly-Phe-Phe. Their hydrophobicity is evident from chromatographic behavior on a hydrophobic matrix, presence of 9 successive hydrophobic residues at the NH2 terminus, and a decrease in drop size during elution of active fractions during chromatographic purification. The minimal molecular weight of pardaxin I is about 3500 as determined by sodium dodecyl sulfate gel electrophoresis and amino acid analyses. It is composed of 35 amino acids and is free of carbohydrate and sialic acid residues. Mass spectrometry of the ethyl acetate extract of the gland secretion and purified toxin reveals the presence of sterols in the secretion but their absence in the purified toxins. Pardaxin I was iodinated without affecting its chemical and pore-forming properties. It binds to liposomes of different phospholipid compositions. In hyperpolarized unilamellar liposomes, pardaxin I produced a fast, nonspecific permeabilization and in multilamellar liposomes, a slow, cation-specific pore. It is suggested that pardaxins exert their effects due to their hydrophobic and pore-formation properties. 相似文献
7.
Fluorometric analysis of transferable membrane pores 总被引:4,自引:0,他引:4
When pore-forming factors insert into the hyperpolarized membranes of lipid vesicles, ion gradients are rapidly equilibrated, effecting complete depolarization. This process can be conveniently followed with a potentiometric cyanine dye. The generality of the method is demonstrated by applications to three diverse materials. The well-studied gramicidin channel is used to demonstrate that the method is sensitive down to concentrations of 10(-12)M. An extract from the shark repellent skin secretion of the Red Sea flatfish displays activity in the assay and is used to demonstrate the potential of the method to elucidate some of the characteristics of the pore, including its molecularity. That membrane-active factors can be detected and assayed in crude preparations is demonstrated with an impure extract of "amoebapore" from Entamoeba histolytica. In addition, variation of the buffer composition surrounding the vesicles can provide information about the ion selectivity of the pore under investigation. 相似文献
8.
In order to study intact red cell membrane skeletons, we developed two methods for the electron microscopic study of stress-ruptured and partially opened glutaraldehyde pre-fixed Triton shells of red cell ghosts. Both methods showed continuous networks of fine filaments decorated and apparently crosslinked by elongated particles. Our electron micrographs show a filamentous component that is morphologically consistent with F-actin. Quantitative considerations, including the dimensions of the elongated particles and published estimates of the number of particles in situ, suggest that compact spectrin decamers or dodecamers may exist in intact red cells. Long filaments consistent with F-actin appear to be decorated with the putative spectrin particles. 相似文献
9.
Faster voltage-dependent activation of Na+ channels in growth cones versus somata of neuroblastoma N1E-115 cells.
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Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells. 相似文献
10.