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1.
Three polyethyleneimine-metal complexes were synthesized and cytochemically tested for demonstration of negatively charged sites. For this purpose rat cerebral cortex synaptosomes were used. It was established that both types of polyethyleneimine-copper complexes labeled the synaptosomal membrane and synaptic vesicles with electron-dense granules, whereas the polyethyleneimine-lead complex marked the anionic sites with amorphous electron-dense material. 相似文献
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Sequence comparisons of members of the myosin superfamily show a high degree of charge conservation in a surface exposed helix (Dictyostelium discoideum myosin II heavy chain residues S510 to K546). Most myosins display a triplet of acidic residues at the equivalent positions to D. discoideummyosin II residues D530, E531, and Q532. The high degree of charge conservation suggests strong evolutionary constrain and that this region is important for myosin function. Mutations at position E531 were shown to strongly affect actin binding [Giese, K. C., and Spudich, J. A. (1997) Biochemistry 36, 8465-8473]. Here, we used steady-state and transient kinetics to characterize the enzymatic competence of mutant constructs E531Q and Q532E, and their properties were compared with those of a loop 2 mutant with a 20 amino acid insertion containing 12 positive charges (20/+12) [Furch et al. (1998) Biochemistry 37, 6317-6326], double mutant Q532E(20/+12), and the native motor domain constructs. Our results confirm that charge changes at residues 531 and 532 primarily affect actin binding with little change being communicated to the nucleotide pocket. Mutation D531Q reduces actin affinity (K(A)) 10-fold, while Q532E leads to a 5-fold increase. The observed changes in K(A)() stem almost exclusively from variations in the dissociation rate constant (k(-A)), with the introduction of a single negative charge at position 532 having the same effect on k(-A) as the introduction of 12 positive charges in the loop 2 region. 相似文献
3.
Björn Sandström 《Histochemistry and cell biology》1971,26(3):197-200
Summary A phosphatase activity similar to that of the plasma membrane is demonstrated at the inside of the membrane, which delimits the lipid-containing lysosomes of chicken liver parenchymal cells. This is taken as a definite confirmation of their endocytotic origin.It should be possible to demonstrate endocytosis vesicles in other cells in this way. It is, however, pointed out, that cells with a weak plasma membrane activity may require particular attention to certain steps in the technical procedure.This study was supported in part by a grant from the Swedish Medical Research Council. 相似文献
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W Lissens R Vrijsen R J Sijens I Liebaers A Boeyé 《Biochimica et biophysica acta》1985,831(3):281-287
Conventional rabbit antibodies and mouse monoclonal antibodies were raised to alpha-L-fucosidase purified from human placenta. Four monoclonal antibodies were studied, of which only one (A) was able to immunoprecipitate the fucosidase activity completely. Two antibodies (B and C) precipitated 65% and one (D) 35% of the activity. The enzyme precipitated by the monoclonal antibodies remained fully active, whereas the enzyme precipitated by conventional antibodies was partly inactivated. As shown by the method of successive immunoprecipitations, the monoclonal antibodies B and C recognized the same set of placental fucosidase molecules, and D a subset thereof. The purified fucosidase also yielded two components after gel electrophoresis in nondenaturing conditions, and the slower component corresponded to the set recognized by antibodies B and C. The fucosidase extracted from different tissues and serum was studied by immunoprecipitation. In all cases, the enzyme was completely precipitated by monoclonal antibody A. Two patterns were found with B, C and D: either part of the activity was precipitated by these antibodies (leucocytes, placenta, brain, liver, spleen, thymus) or B, C and D failed to precipitate any of the enzyme (serum, heart, kidney, testes). 相似文献
6.
C L Schauf 《Biophysical journal》1983,42(3):225-231
In Myxicola giant axons, the total amount of intramembrane charge available to move over the range -80 to + 120 mV decreases by 23% when the external pH is reduced from 7.3 to 5.5. The remaining charge moves more slowly at the onset of depolarizing pulse, but the rate of charge movement at the end of the pulse is unchanged. In contrast to acidic external pH, intramembrane charge movements are insensitive to alkaline external pH or any change in the internal pH. The results are consistent with a hypothesis in which a portion of the initial outward gating current consists of titratable negative charges moving inward. 相似文献
7.
The distribution of negative surface charges on mammalian spermatozoa 总被引:10,自引:0,他引:10
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If anti-sera are combined with native whole blood by an in-vitro technique, immunocomplexes are formed. They are fixed to erythrocytes and can be made visible by a scanning electron microscope on blood smears especially pretreated. 相似文献
9.
Summary On the basis of a model presented in a previous paper (Hook and Hildebrand, 1979) the influence of external cation concentrations [K+]0, [Ca2+]0 and of membrane voltage Vm (i.e. the actual potential difference between the two membrane faces) on the locomotor behavior of Paramecium is theoretically analyzed. In an extended model system we discuss the negative feedback of intraciliary calcium [Ca2+]i on the excitability of the ciliary membrane. While a fast blocking of Ca channels is mediated by increased [Ca2+]i and accounts for the short duration of action potentials, a slow [Ca2+ ]i-dependent denaturation of channel molecules is assumed to determine excitability changes of Paramecium on a long time scale.It is emphasized that the duration of long-lasting ciliary reversal which reflects the excitability is not a direct function of the cation ratio Ju [K+]0/[Ca2+]
0
1/2
but rather of the membrane potential Vm.Introduction of negative surface charges can well explain why for a series of different [K+]0, [Ca2+]0 but constant Ja value the excitability is unchanged despite corresponding shifts in measured membrane potentials. 相似文献
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Abnormal spermiogenesis in sterile pink-eyed dilution mutants results in spermatozoa with bizarre sperm heads. The spermatozoa of normal mice bind colloidal iron hydroxide (CIH) along the length of the tail, yet the spermatozoa of pink-eyed sterile mice show a great reduction in ability to bind CIH. This implies a loss of negative surface charges. The group(s) responsible for the charges are sensitive to methylation but resistant to neuraminidase treatment, even after deacetylation with alkaline treatment. The membrane components containing the negatively charged groups may be neuraminidase-resistant forms of gangliosides. 相似文献
12.
The interaction between various polycations and cultured glomerular epithelial cells was studied by cell electrophoresis. It was shown that the glomerular epithelial cell presents a negatively charged surface which imparts a zeta potential of -29.0 +/- 1.5 mV at the peripheral layer of the plasma membrane. The pH at which the GEC charge became 50% reduced (pKa) was determined to be 3.0. A variety of polycations of various sizes and fixed and flexible geometries were tested for their capacity to neutralize the cell charge. All the polycations except cytochrome c and lysozyme were capable of completely neutralizing the cell. Cytochrome c could maximally neutralize only 50% of charge and lysozyme only 72% of charge. However, reduced and 'relaxed' molecules of cytochrome c and lysozyme efficiently neutralized the cell surface, as did larger sized 'flexible' polylysines. On the basis of these findings, we conclude that all polycations are not equal in their capacity to neutralize the cell surface. Flexible molecules in contrast to molecules with rigid structures were more effective in neutralizing the cell. This may likely be due to the exposure and availability of more cationic groups in a flexible molecule which results in stabilization of interaction with cells. 相似文献
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The amount and distribution of negatively charged sites of different cells (human fibroblasts, B16 melanoma cells, a human lymphoid leukemic cell type) were investigated. In the non-irradiated fibroblasts and B16 melanoma cells the negatively charged sites were localized mainly on apical and lateral surfaces as well as in a polarized manner. However, negatively charged sites on the control human lymphoid leukemic cells often have patched distribution. It was demonstrated that the amount and distribution of negatively charged sites on primary human fibroblasts and B16 melanoma cells changed upon ionizing radiation. However, the topology of negative charges on investigated human leukemic cell membrane did not change. 相似文献
15.
A synthetic barium 5-bromoindol-3-yl-β-d-glucuronate was used as the substrate for demonstration of β-glucuronidase in disk electrophoresis. The advantages of this substrate is that it affords a precise enzyme localization with no diffusion, and, also, a simple and direct method for demonstration of this enzyme without the need for a coupling reaction. 相似文献
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The gate of mitochondrial porin channel is controlled by a number of negative and positive charges 总被引:1,自引:0,他引:1
Negatively charged carboxyl groups of mitochondrial porin have been converted into positively charged ones by means of reaction with water-soluble carbodiimide in the presence of ethylenediamine. Properties of channels formed in a planar lipid bilayer by native and modified porins are compared. Amidation has only little influence on the porin channel-forming activity as well as on the open-state conductance of the channel. However, the modification results in a significant enhancement of the voltage dependence of the channel gating and in an increase of the anionic selectivity. It is suggested that the voltage sensor of the porin channel gate is composed of a number of negative (greater than 14) and positive (greater than 22) charges. 相似文献
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Moise Bendayan 《The Histochemical journal》1981,13(5):699-710
Summary Nucleic acids can be specifically localized at the electron microscope level by means of enzyme-gold complexes. Two enzymes RNAase A and DNAase I were labelled with colloidal gold, and the enzyme-gold complexes obtained applied on thin sections of glutaraldehyde-fixed and Epon-embedded tissues. Using RNAase-gold, the rough endoplasmic reticulum and the nucleolus of different cells appeared densely labelled. With the DNAase-gold the labelling was present over the euchromatin and the mitochondria. The quantitative evaluation, performed on different cellular compartments of the pancreatic acinar cells, confirmed the qualitative observations and ascertained the specificity of the labelling. The application of this technique, for the demonstration of nucleic acids in different tissues, is illustrated. 相似文献