Scott syndrome erythrocytes contain a membrane protein capable of mediating Ca2+-dependent transbilayer migration of membrane phospholipids.

Stout, James G.; Bassé, François; Luhm, Robert A.; Weiss, Harvey J.; Wiedmer, Therese; Sims, P.

doi:10.1172/jci119397

Cited by 69 publications

(69 citation statements)

References 37 publications

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“…PL Scramblase Isolation and Amino Acid Sequencing-PL scramblase was purified as described previously (10,11), with the following modifications. The active fraction eluting from Mono S was concentrated and exchanged into 150 mM NaCl, 20 mM Tris, 0.1 mM EGTA, 0.1% Nonidet P-40, pH 7.4, and absorbed against 5 ml of wheat germ agglutinin-Sepharose to remove trace contaminating glycophorins.…”

Section: Methodsmentioning

confidence: 99%

“…Reconstitution of PL Scramblase or Scramblase Fusion Protein into Proteoliposomes-Reconstitution into proteoliposomes was performed essentially as described previously (10,11). Briefly, a mixture of PC and PS (9:1 molar ratio) was dried under a stream of nitrogen and resuspended in 100 mM Tris, 100 mM KCl, 0.1 mM EGTA, pH 7.4 (Tris buffer).…”

Section: Methodsmentioning

confidence: 99%

“…PL Scramblase Activity-PL Scramblase activity was measured as described previously (10,11). Routinely, proteoliposomes labeled with NBD-PC were incubated for 2 h at 37°C in Tris buffer in the presence or the absence of 2 mM CaCl 2 .…”

Section: Methodsmentioning

confidence: 99%

“…Proteoliposomes were diluted 25-fold in Tris buffer containing 4 mM EGTA and transferred to a stirred fluorescence cuvette at 23°C. Initial fluorescence was recorded (SLM Aminco 8000 spectrofluorimeter; excitation, 470 nm; emission, 532 nm), 20 mM dithionite was added, and the fluorescence was continuously monitored for a total of 120 s. The difference in nonquenchable fluorescence observed in the presence versus the absence of CaCl 2 was attributed to Ca 2ϩ -induced change in NBD-PC located in the outer leaflet (10,11,13). Ionized [Ca 2ϩ ] was calculated using FreeCal version 4.0 software (generously provided by Dr. Lawrence F. Brass, University of Pennsylvania, Philadelphia, PA).…”

Section: Methodsmentioning

confidence: 99%

“…We recently reported isolation of a ϳ37-kDa integral membrane protein from human erythrocytes that when reconstituted into liposomes mediated a Ca 2ϩ -dependent, bidirectional scrambling of PL between membrane leaflets mimicking the action of Ca 2ϩ at the endofacial surface of the erythrocyte membrane (10,11). Evidence for protein(s) in platelet that mediates a similar "PL scramblase" function when incorporated into liposomes has also been reported (12).…”

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confidence: 95%

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Molecular Cloning of Human Plasma Membrane Phospholipid Scramblase

Zhou¹,

Zhao

Stout

et al. 1997

Journal of Biological Chemistry

377

182

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The rapid movement of phospholipids (PL) between plasma membrane leaflets in response to increased intracellular Ca 2؉ is thought to play a key role in expression of platelet procoagulant activity and in clearance of injured or apoptotic cells. We recently reported isolation of a ϳ37-kDa protein in erythrocyte membrane that mediates Ca 2؉ -dependent movement of PL between membrane leaflets, similar to that observed upon elevation of Ca 2؉ in the cytosol (Bassé , F., Stout, J. G., Sims, P. J., and Wiedmer, T. (1996) J. Biol. Chem. 271, 17205-17210). Based on internal peptide sequence obtained from this protein, a 1,445-base pair cDNA was cloned from a K-562 cDNA library. The deduced ''PL scramblase'' protein is a proline-rich, type II plasma membrane protein with a single transmembrane segment near the C terminus. Antibody against the deduced Cterminal peptide was found to precipitate the ϳ37-kDa red blood cell protein and absorb PL scramblase activity, confirming the identity of the cloned cDNA to erythrocyte PL scramblase. Ca 2؉ -dependent PL scramblase activity was also demonstrated in recombinant protein expressed from plasmid containing the cDNA. Quantitative immunoblotting revealed an approximately 10-fold higher abundance of PL scramblase in platelet (ϳ10 4 molecules/cell) than in erythrocyte (ϳ10 3 molecules/ cell), consistent with apparent increased PL scramblase activity of the platelet plasma membrane. PL scramblase mRNA was found in a variety of hematologic and nonhematologic cells and tissues, suggesting that this protein functions in all cells.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 95%

See 3 more Smart Citations

Molecular Cloning of Human Plasma Membrane Phospholipid Scramblase

Zhou¹,

Zhao

Stout

et al. 1997

Journal of Biological Chemistry

377

182

View full text Add to dashboard Cite

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Phosphatidylserine Exposure in Hemoglobinopathies

Kuypers¹,

Soupène²

2011

Transmembrane Dynamics of Lipids

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Involvement of ABCB1 and ABCC1 transporters in sea urchin Echinometra lucunter fertilization

Silva-Neta¹,

Torrezan²,

Leite³

et al. 2012

Molecular Reproduction Devel

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Fertilization is an ordered sequence of cellular interactions that promotes gamete fusion to form a new individual. Since the pioneering work of Oskar Hertwig conducted on sea urchins, echinoderms have contributed to the understanding of cellular and molecular aspects of the fertilization processes. Studies on sea urchin spermatozoa reported the involvement of a plasma membrane protein that belongs to the ABC proteins superfamily in the acrosome reaction. ABC transporters are expressed in membranes of eukaryotic and prokaryotic cells, and are associated with the transport of several compounds or ions across biomembranes. We aimed to investigate ABCB1 and ABCC1 transporter activity in sea urchin spermatozoa and their involvement in fertilization. Our results indicate that Echinometra lucunter spermatozoa exhibit a low intracellular calcein accumulation (18.5% stained cells); however, the ABC blockers reversin205, verapamil, and MK571 increased dye accumulation (93.0-96.6% stained cells). We also demonstrated that pharmacologically blocking ABCB1 and ABCC1 decreased spermatozoa fertilizing capacity (70% inhibition), and this phenotype was independent of extracellular calcium. These data suggest that functional spermatozoa ABCB1 and ABCC1 transporters are crucial for a successful fertilization. Additional studies must be performed to investigate the involvement of membrane lipid homeostasis in the fertilization process.

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Scott syndrome erythrocytes contain a membrane protein capable of mediating Ca2+-dependent transbilayer migration of membrane phospholipids.

Cited by 69 publications

References 37 publications

Molecular Cloning of Human Plasma Membrane Phospholipid Scramblase

Molecular Cloning of Human Plasma Membrane Phospholipid Scramblase

Phosphatidylserine Exposure in Hemoglobinopathies

Involvement of ABCB1 and ABCC1 transporters in sea urchin Echinometra lucunter fertilization

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