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31.
Alpha-L-fucosidase was purified from the bull seminal plasma by chromatography on DEAE-disk, octyl sepharose hydrophobic column and HPLC. The enzyme appeared to be pure as judged by the polyacrylamide gel electrophoresis both under the nondenaturing and denaturing conditions. The pure enzyme promoted the acrosome reaction of guinea pig spermatozoa in vitro. This is the first report showing that an acrosomal enzyme induces acrosome reaction which is an essential pre-requisite for the gamete interaction and fertilization.  相似文献   
32.
Target-sensitive immunoliposomes: preparation and characterization   总被引:2,自引:0,他引:2  
R J Ho  B T Rouse  L Huang 《Biochemistry》1986,25(19):5500-5506
A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   
33.
Action of polyethylene glycol on the fusion of human erythrocyte membranes   总被引:5,自引:0,他引:5  
Summary Factors affecting the polyethylene glycol (PEG)-induced membrane fusion were examined. Human erythrocyte membrane ghosts, cytoskeleton-free vesicles budded from erythrocytes, mechanically disrupted erythrocyte vesicles, and recombinant vesicles from glycophorin and egg phosphatidylcholine were used as models. Fusion was monitored by darkfield light microscopy and by freeze-fracture electron microscopy. Osmotic swelling was found necessary for fusion between membrane ghosts following PEG treatment. The sample with the highest fusion percentage was sealed ghosts incubated in hypotonic media after at least 5 min of treatment in <25% PEG. At similar osmolarity, glycerol, dextran and PEG produced progressively more pronounced intramembranous particle (IMP) patching, correlating with their increasing fusion percentages. The patching of IMP preceded cell-cell contact, and occurred without direct PEG-protein interaction. The presence of cytoskeletal elements in small vesicles had no significant effect on fusion, nor on the aggregation of intramembranous particle (IMP) upon PEG treatment. Disrupting the membrane by lysolecithin, dimethylsulfoxide, retinol or mild sonication resulted in the fragmentation of ghosts without an increase in fusion percentage. The purity of the commercial PEG used had no apparent effect on fusion. We concluded that the key steps in PEG-induced fusion of cell membrane are the creation of IMP-free zones, and the osmotic swelling of cells after the formation of bilayer contacts during the PEG treatment. Cell cytoskeleton affects PEG-induced fusion only to the extent of affecting IMP patching.  相似文献   
34.
T6 DNA topoisomerase has been purified from bacteriophage T6 infected Escherichia coli. Unlike the T4 DNA topoisomerase which has three subunits, it consists of two subunits of molecular weights 75,000 and 51,000. They are the products of T6 genes 39 and 52, respectively. The purified T6 enzyme can stimulate in vitro T6 DNA replication. It has an ATP-dependent DNA relaxation activity similar to the T4 enzyme. Either ATP or dATP can be used in both reactions. Using a "Western blotting" and radioimmuno-detection methods, we show that T6 39 subunit contains protein sequences specified by both the T4 39 and 60 genes. The 52-proteins of both phages appear to be identical. The T4 and T6 topoisomerase genes represent a naturally occurring example of gene separation or fusion.  相似文献   
35.
PRCII is an avian retrovirus whose oncogene (v-fps) induces fibrosarcomas in birds. The viral gene v-fps arose by transduction of an undetermined portion of a cellular gene known as c-fps. PRCII is weakly oncogenic when compared with Fujinami sarcoma virus, another transforming virus containing v-fps. As a first step in the elucidation of the molecular basis for the decreased virulence of PRCII, we have determined the entire nucleotide sequence of v-fps in the PRCII genome. The v-fps domain in PRCII encodes a polypeptide with a molecular weight of ca. 60,500 fused to a portion of the polyprotein encoded by the viral structural gene gag. The hybrid gag-fps polyprotein of PRCII would have a molecular weight of ca. 98,100, in accord with results of previous studies of the protein encoded by the PRCII genome. The leftward junctions between fps and gag in Fujinami sarcoma virus and PRCII are located at the same position in fps, but at different positions in gag. A sequence of 1,020 nucleotides, bounded by direct repeats of 6 nucleotides, is present in v-fps of Fujinami sarcoma virus but absent from PRCII. Our data should permit further explorations of the relationship between structure and function in the transforming protein encoded by v-fps.  相似文献   
36.
Formation and properties of cell-size lipid bilayer vesicles   总被引:4,自引:2,他引:2       下载免费PDF全文
Hydration of single or mixed phospholipids or lipid protein mixtures at low ionic strength results in the formation of a population of large, solvent free, single bilayer vesicles with included volumes of up to 300 microliters/mumol lipid. Their size ranges from 0.1 to 300 microns and they can be sorted out according to size by centrifugation. When formed in distilled water their internal solution has a conductivity of 20-50 microseconds/cm-1, an osmolarity of 0.5-5 mOsM, and a density of 1.0005-1.001. The osmotic pressure produced by the internal solutes cause a surface stress of 25 dyn/cm for a 20-microns vesicle. Their elastic constant ranges from 75-150 dyn/cm. During formation they can internalize particles such as latex beads or cell nuclei. They can be impaled with microelectrodes, or patch clamped. They can also be sealed to a small Vaseline-treated hole in a thin partition between two aqueous compartments. Sealing occurs in two stages. In the first stage sealing resistance is similar to that seen with patch-clamp pipettes. In the second stage, a much tighter seal is obtained. After sealing, the smaller portion of the sealed vesicle can be selectively broken by an electric shock leaving a single membrane across the hole. The capacitance and resistance of such membranes, in the presence of 10 mM NaCl, are approximately 0.7 microF/cm2 and 10(8) omega cm2 for pure lipid vesicles. Gramicidin increases the membrane conductance and monazomycin induces voltage-dependent gating thus providing further evidence that the vesicles are bounded by a single bilayer.  相似文献   
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Bacterial LPS and TNF induce vascular endothelial cells to express a variety of response molecules. LPS that is partially deacylated (dLPS) by a human neutrophil enzyme blocks the ability of LPS, but not TNF, to augment one of these responses, the expression of endothelial cell surface molecules that promote neutrophil adherence (J. Exp. Med. 1987; 165:1393-1402). We show that dLPS can inhibit the ability of LPS, but not TNF, to elicit the expression of plasminogen activator inhibitor-1 (PAI-1), prostacyclin, and PGE2 by human umbilical vein endothelial cells. dLPS also prevented the accumulation of specific PAI-1 mRNA in response to LPS, but not to TNF. Neither the LPS- or TNF-induced expression of PAI-1 nor the dLPS inhibition of the LPS response was mediated by prostanoids. These results indicate that dLPS can specifically block a variety of endothelial cell responses to LPS and provide support for the hypotheses 1) that dLPS and LPS may interact with a common target molecule on or in endothelial cells, and 2) that dLPS, produced by enzymatic deacylation of LPS in vivo, could inhibit endothelial cell stimulation by LPS and thereby limit the host inflammatory response to invasive gram-negative bacteria.  相似文献   
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