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Query: UNIPROT:Q9NRP7 (fused)
 58,367 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ca2+ facilitated the fusion by Sendai virus of Friend erythroleukaemic cells and Ehrlich ascites tumour cells but not that of hepatoma tissue culture cells. In the absence of Ca2+ Sendai virus caused the complete depletion of ATP and abolished protein synthesis in Friend erythro-leukaemic cells fused with each other. Addition of high concentrations of Ca2+ (10-20 mM) partially protected the cells from ATP depletion. After a further incubation of cells in complete medium plus 0.2 mM adenine, ATP levels and protein synthesis were restored to 60-85% of those of the untreated control. The protective effect of Ca2+ was used to improve the ultramicroinjection method which involves the fusion of human erythrocyte ghosts with cells. When human erythrocyte ghosts containing high K+ were fused with Friend erythroleukaemic cells in the presence of 10 mM Ca2+ ATP levels and protein synthesis after recovery were about 60-85% of the control. Friend erythroleukaemic cells subjected to ultramicroinjection under these conditions had a cloning efficiency of 75-95% of that of the untreated controls. In these experiments 70-100% of the cells had fused with ghosts. Induction of haemoglobin synthesis by dimethylsulphoxide was unimpaired in cells subjected to ultramicroinjection under the same conditions.
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PMID:Role of Ca2+ in preserving viability of Friend erythroleukaemic cells after ultramicroinjection. 20 56

Previous work had demonstrated the coupling of a beta-adrenergic receptor on an erythrocyte with the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of a tissue culture cell when the two cells were fused by Sendai virus. The validity of this finding for animal tissues in general, for membrane preparations, and for peptide hormone receptors could hitherto not be assessed. Available fusion procedures worked efficiently only with certain intact cells from tissue culture and with erythrocytes. In the present work a membrane fusion method was developed that causes the transfer of the glucagon receptor from purified rat liver membranes to Friend erythroleukemia cells; even direct transfer to a membrane fraction prepared from Friend cells became feasible. It can therefore be concluded that a peptide hormone receptor in a normal tissue membrane has properties similar to those demonstrated for a beta-adrenergic receptor in an erythrocyte: it exists in the membrane as a dissociable independent unit that can readily couple with the adenylate cyclase of a foreign cell. The efficiency of the membrane fusion procedure is due to the combined action of polyethylene glycol, phospholipids, stearylamine, and ATP in a salt medium. The method promises to be applicable to membranes of various cells and tissues, and it can probably be used to analyze hormone receptors and adenylate cyclase systems in states of malfunction by transfer to their respective counterpart in a normal cell membrane. Studies in biochemical hybridization of membrane components need not be limited to hormone activation of adenylate cyclase. With the aid of the membrane fusion method, this approach could be applied to any dissociable multicomponent system in biological membranes.
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PMID:Transfer of glucagon receptor from liver membranes to a foreign adenylate cyclase by a membrane fusion procedure. 22 Jun 8

The interaction between [131I]-thyroglobulin liposomes and thyroidal lysosomes was used as an in vitro model system for analyzing the relation of colloid droplets to lysosomes in follicular cells. The rates of hydrolysis of [131I]-thyroglobulin in a liposome-lysosome system (Lipo-Lyso system) and a thyroglobulin-lysosome system (TG-Lyso system) were compared. Hydrolysis of thyroglobulin in the Lipo-Lyso system increased hyperbolically and was greater than that in the TG-Lyso system for about 10 h. Liposomal thyroglobulin was not degraded by the 20,000 X g supernatant obtained on disruption of the lysosomal fraction. On varying the pH of the incubation medium, the highest activity was observed under acidic conditions in both systems. Under neutral or weakly alkaline conditions, the Lipo-Lyso system still showed 50% of the maximal hydrolytic activity, while the TG-Lyso system showed no activity. ATP and anaerobic conditions had no effect on either system. Cysteine (5 X 10-2M) and p-chloromercuribenzene sulfonic acid (PCMBS, 10-3 M) had not influence on hydrolysis in the Lipo-Lyso system, but in the TG-Lyso system cysteine greatly increased, and PCMBS significantly reduced the rate of hydrolysis. Dibutyryl cyclic AMP had no effect. Chlorpromazine (Cpz) decreased liposomal thyroglobulin hydrolysis in a concentration-dependent manner. In the TG-Lyso system, concentrations of 10-4M Cpz had no effect. These results strongly suggest that liposomes rapidly fused with lysosomes, providing optimal conditions for hydrolysis of thyroglobulin.
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PMID:Study of the mechanism of thyroid hormone secretion in an in vitro model system: indirect evidence for fusion of lysosomes with thyroglobulin liposomes. 23 19

Small phospholipid vesicles (liposomes) fuse upon calcium addition as demonstrated by electron microscopy, light absorbance increases, and mixing of original liposome contents within the boundaries of the fused liposome. The integrity of the fusion event is demonstrated by a novel assay based on the luminescence of firefly extract when mixed with ATP. Subsequent addition of valinomycin or the calcium ionophore A23187 leads to further fusion as shown by electron microscopy, light microscopy, and additional absorbance increase. Concomitant with this second absorbance increase is an increase in the amount of calcium that associates with the liposomes. This increased calcium association is more than can be accounted for by equilibration of 5 mM Ca2+ across the membrane and must indicate exposure of extra calcium binding sites. Binding of calcium to the inner side of the membrane may catalyze the second stage of liposome fusion.
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PMID:The role of calcium in fusion of artificial vesicles. 34 98

In order to study the conductances of the Sarcoplasmic Reticulum (SR) membrane, microsomal fractions from cardiac SR were isolated by differential and sucrose gradient centrifugations and fused into planar lipid bilayers (PLB) made of phospholipids. Using either KCl or K-gluconate solutions, a large conducting K+ selective channel was characterized by its ohmic conductance (152 pS in 150 mM K+), and the presence of short and long lasting subconducting states. Its open probability Po increased with depolarizing voltages, thus supporting the idea that this channel might allow counter-charge movements of monovalent cations during rapid SR Ca2+ release. An heterogeneity in the kinetic behavior of this channel would suggest that the cardiac SR K+ channels might be regulated by cytoplasmic, luminal, or intra SR membrane biochemical mechanisms. Since the behavior was not modified by variations of [Ca2+] nor by the addition of soluble metabolites such as ATP, GTP, cAMP, cGMP, nor by phosphorylation conditions on both sides of the PLB, a specific interaction with a SR membrane component is postulated. Another cation selective channel was studied in asymmetric Ca2+, Ba2+ or Mg(2+)-HEPES buffers. This channel displayed large conductance values for the above divalent cations 90, 100, and 40 pS, respectively. This channel was activated by microM Ca2+ while its Ca2+ sensitivity was potentiated by millimolar ATP. However Mg2+ and calmodulin modulated its gating behavior. Ca2+ releasing drugs such as caffeine and ryanodine increased its Po. All these features are characteristics of the SR Ca2+ release channel. The ryanodine receptor which has been purified and reconstituted into PLB, may form a cation selective pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Reconstitution and regulation of cation-selective channels from cardiac sarcoplasmic reticulum. 128 Dec 62

Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli is conferred by the conjugative R-factor R773, which encodes an ATP-driven anion extrusion pump. The ars operon is composed of three structural genes, arsA, arsB, and arsC. Although transcribed as a single unit, the three genes are differentially expressed as a result of translational differences, such that the ArsA and ArsC proteins are produced in high amounts relative to the amount of ArsB protein made. Consequently, biochemical characterization of the ArsB protein, which is an integral membrane protein containing the anion-conducting pathway, has been limited, precluding studies of the mechanism of this oxyanion pump. To overexpress the arsB gene, a series of changes were made. First, the second codon, an infrequently used leucine codon, was changed to a more frequently utilized codon. Second, a GC-rich stem-loop (delta G = -17 kcal/mol) between the third and twelfth codons was destabilized by changing several of the bases of the base-paired region. Third, the re-engineered arsB gene was fused 3' in frame to the first 1458 base pairs of the arsA gene to encode a 914-residue chimeric protein (486 residues of the ArsA protein plus 428 residues of the mutated ArsB protein) containing the entire re-engineered ArsB sequence except for the initiating methionine. The ArsA-ArsB chimera has been overexpressed at approximately 15-20% of the total membrane proteins. Cells producing the chimeric ArsA-ArsB protein with an arsA gene in trans excluded 73AsO2- from cells, demonstrating that the chimera can function as a component of the oxyanion-translocating ATPase.
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PMID:Construction of a chimeric ArsA-ArsB protein for overexpression of the oxyanion-translocating ATPase. 128 74

Using polyethylene glycol-mediated fusion of ATP-ase-enriched (native) microsomes with red blood cells, we have delivered sarcoplasmic reticulum (SR) Ca-ATPase and kidney Na,K-ATPase into the mammalian erythrocyte membrane. Experiments involving delivery of the SR Ca-ATPase into human red cells were first carried out to assess the feasibility of the fusion protocol. Whereas there was little detectable 45Ca2+ uptake into control cells in either the absence or presence of extracellular ATP, a marked time-dependent uptake of 45Ca2+ was observed in the presence of ATP in cells fused with SR Ca-ATPase. Comparison of the kinetics of uptake into microsome-fused cells versus native SR vesicles supports the conclusion of true delivery of pumps into the red cell membrane. Thus, the time to reach steady state was more than two orders of magnitude longer in the (large) cells versus the native SR vesicles. Na,K-ATPase from dog and rat kidney microsomes were fused with red cells of humans, sheep, and dogs. Using dog kidney microsomes fused with dog red cells which are practically devoid of Na,K-ATPase, functional incorporation of sodium pumps was evidenced in ouabain-sensitive Rb+ uptake and Na+ efflux energized by intracellular ATP, as well as in ATP-stimulated Na+ influx and Rb+ efflux from inside-out membrane vesicles prepared from the fusion-treated cells. From analysis of the biphasic kinetics of ouabain-sensitive Na+ efflux under conditions of limited intracellular Na+ concentration, it is concluded that the kidney pumps are incorporated into a relatively small fraction (approximately 15%) of the red cells. This system provides a uniquely useful system for studying the behavior of native sodium pumps in a compartment (red cell) of small surface/volume ratio. The newly incorporated native kidney pumps, while of the same isoform as the endogenous red cell pump, behave differently from the endogenous red cell sodium pump with respect to their very low "uncoupled" Na+/O flux activity.
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PMID:Delivery of ion pumps from exogenous membrane-rich sources into mammalian red blood cells. 131 86

Two ATP-binding domains are found in members of the family of ATP-dependent transport proteins, which includes P-glycoprotein and cystic fibrosis transmembrane conductance regulator. To investigate the involvement of the two ATP-binding domains in the ATPase activity of P-glycoprotein, full-length and the 5'-half of human MDR1 cDNA, which encodes P-glycoprotein, were fused with the Escherichia coli lacZ gene and expressed in NIH3T3 cells. Immunoprecipitated full-length P-glycoprotein beta-galactosidase showed ATPase activity with apparent specific activity of 180 nmol/mg/min, a value higher than previously reported, in the presence of phospholipids, suggesting that stabilization of the transmembrane domains is necessary for ATP hydrolysis. N-terminal half P-glycoprotein-beta-galactosidase also showed ability to hydrolyze ATP but with slightly lower specific activity. Both ATPase activities showed similar characteristics when the effect of several inhibitors was analyzed, indicating that the N-terminal ATP-binding domain contains all residues necessary to hydrolyze ATP without interacting with the C-terminal ATP-binding domain.
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PMID:P-glycoprotein. ATP hydrolysis by the N-terminal nucleotide-binding domain. 134 41

Two new fused indoles were found to overcome multidrug resistance in P388/Adr cells in vitro. These agents potentiated the cytotoxicity of the antitumor drugs Adriamycin, vinblastine, and vincristine in multidrug-resistant cells with no effect on drug-sensitive parent P388 cells. They significantly increased the ATP-dependent accumulation of [3H]-vinblastine and inhibited efflux of the labeled drug from resistant cells. These compounds also inhibited photoaffinity labeling of P-glycoprotein by [3H]azidopine in P388/Adr cells and membranes isolated from these cells. In addition, the calcium antagonist activity of these compounds was very weak compared with that of verapamil. These data suggest that the compounds reported here may specifically overcome multidrug resistance without the serious hypotensive effects associated with calcium antagonists and that this activity may be independent of their ability to block calcium transport.
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PMID:Reversal of multidrug resistance by two novel indole derivatives. 135 8

Membrane vesicles, prepared from mouse NIH-3T3 fibroblasts and Chinese hamster ovary cells expressing high levels of cystic fibrosis transmembrane conductance regulator (CFTR), were fused with Mueller-Rudin planar lipid bilayers. Upon addition of the catalytic subunit of cAMP-dependent protein kinase and ATP, low conductance Cl(-)-selective ion channels were observed in 10 of 16 experiments. The channels had a linear current-voltage relationship and a unitary conductance of approximately 6.5 pS. The channels were more permeable to Cl- than to I- and showed no appreciable time-dependent voltage activation. In contrast, addition of cAMP-dependent protein kinase and ATP to lipid bilayers fused with vesicles prepared from mock transfected (n = 14) cells failed to activate Cl- channels. These data support the conclusion that CFTR is a Cl- channel. They indicate that it can be reconstituted in a planar lipid bilayer and that the biophysical and regulatory properties are very similar to those observed in the native cell membrane. These data also argue against the requirement for loosely associated factors for regulation or function of the channel.
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PMID:Cyclic AMP-dependent protein kinase activation of cystic fibrosis transmembrane conductance regulator chloride channels in planar lipid bilayers. 137 3


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