Molecular Cloning of Human Plasma Membrane Phospholipid Scramblase

The rapid movement of phospholipids (PL) between plasma membrane leaflets in response to increased intracellular Ca2+ 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 ∼37-kDa protein in erythrocyte membrane that mediates Ca2+-dependent movement of PL between membrane leaflets, similar to that observed upon elevation of Ca2+ 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 C-terminal peptide was found to precipitate the ∼37-kDa red blood cell protein and absorb PL scramblase activity, confirming the identity of the cloned cDNA to erythrocyte PL scramblase. Ca2+-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 (∼104 molecules/cell) than in erythrocyte (∼103 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. The rapid movement of phospholipids (PL) between plasma membrane leaflets in response to increased intracellular Ca2+ 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 ∼37-kDa protein in erythrocyte membrane that mediates Ca2+-dependent movement of PL between membrane leaflets, similar to that observed upon elevation of Ca2+ 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 C-terminal peptide was found to precipitate the ∼37-kDa red blood cell protein and absorb PL scramblase activity, confirming the identity of the cloned cDNA to erythrocyte PL scramblase. Ca2+-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 (∼104 molecules/cell) than in erythrocyte (∼103 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. The plasma membrane phospholipids (PL) 1The abbreviations used are: PL, phospholipid(s); EST, expressed sequence tag; MBP, maltose binding protein; NBD-PC, 1-oleoyl-2-6(7nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl-sn-glycero-3-phosphocholine; OG, N-octyl-β-d-glucopyranoside; PC, phosphatidylcholine; PS, phosphatidylserine; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid. are normally asymmetrically distributed, with phosphatidylcholine (PC) and sphingomyelin located primarily in the outer leaflet, and the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine restricted to the cytoplasmic leaflet (1Schroit A.J. Zwaal R.F.A. Biochim. Biophys. Acta. 1991; 1071: 313-329Crossref PubMed Scopus (283) Google Scholar, 2Devaux P. Biochemistry. 1991; 30: 1163-1173Crossref PubMed Scopus (683) Google Scholar). An increase in intracellular Ca2+ due to either cell activation, cell injury, or apoptosis causes a rapid bidirectional movement of the plasma membrane PL between leaflets, resulting in exposure of PS and phosphatidylethanolamine at the cell surface (1Schroit A.J. Zwaal R.F.A. Biochim. Biophys. Acta. 1991; 1071: 313-329Crossref PubMed Scopus (283) Google Scholar, 3Williamson P. Kulick A. Zachowski A. Schlegel R.A. Devaux P.F. Biochemistry. 1992; 31: 6355-6360Crossref PubMed Scopus (191) Google Scholar, 4Chang C.-P. Zhao J. Wiedmer T. Sims P.J. J. Biol. Chem. 1993; 268: 7171-7178Abstract Full Text PDF PubMed Google Scholar, 5Smeets E.F. Comfurius P. Bevers E.M. Zwaal R.F.A. Biochim. Biophys. Acta. 1994; 1195: 281-286Crossref PubMed Scopus (113) Google Scholar). This exposure of the plasma membrane aminophospholipids has been shown to promote assembly and activation of several key enzymes of the coagulation and complement systems, as well as to accelerate the clearance of injured or apoptotic cells by the reticuloendothelial system, suggesting that Ca2+-induced remodeling of plasma membrane PL is central to both vascular hemostatic and cellular clearance mechanisms (1Schroit A.J. Zwaal R.F.A. Biochim. Biophys. Acta. 1991; 1071: 313-329Crossref PubMed Scopus (283) Google Scholar, 6Bevers E.M. Comfurius P. Zwaal R.F. Blood Rev. 1991; 5: 146-154Crossref PubMed Scopus (162) Google Scholar, 7Sims P.J. Faioni E.M. Wiedmer T. Shattil S.J. J. Biol. Chem. 1988; 263: 18205-18212Abstract Full Text PDF PubMed Google Scholar, 8Wang R.H. Phillips Jr., G. Medof M.E. Mold C. J. Clin. Invest. 1993; 92: 1326-1335Crossref PubMed Scopus (102) Google Scholar, 9Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216Crossref PubMed Google Scholar). We recently reported isolation of a ∼37-kDa integral membrane protein from human erythrocytes that when reconstituted into liposomes mediated a Ca2+-dependent, bidirectional scrambling of PL between membrane leaflets mimicking the action of Ca2+at the endofacial surface of the erythrocyte membrane (10Bassé F. Stout J.G. Sims P.J. Wiedmer T. J. Biol. Chem. 1996; 271: 17205-17210Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 11Stout J.G. Bassé F. Luhm R.A. Weiss H.J. Wiedmer T. Sims P.J. J. Clin. Invest. 1997; 99: 2232-2238Crossref PubMed Scopus (71) Google Scholar). Evidence for protein(s) in platelet that mediates a similar “PL scramblase” function when incorporated into liposomes has also been reported (12Comfurius P. Williamson P. Smeets E.F. Schlegel R.A. Bevers E.M. Zwaal R.F.A. Biochemistry. 1996; 35: 7631-7634Crossref PubMed Scopus (103) Google Scholar). Here we report the cDNA cloning and deduced structure of the PL scramblase from human erythrocyte and show evidence that this same protein is expressed in human platelet and various other cell lines and tissues where plasma membrane PL scramblase activity has been observed. Egg yolk PC, brain PS, and 1-oleoyl-2-6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl-sn-glycero-3-phosphocholine (NBD-PC) were obtained from Avanti Polar Lipids. Expressed sequence tag (EST) clone with GenBank™ accession number gb AA143025 was obtained through American Type Culture Collection (ATCC 962235). All restriction enzymes and amylose resin were from New England BioLabs, Inc. KlenTaq polymerase was from CLONTECH Laboratories, wheat germ agglutinin Sepharose was from Sigma, isopropyl-β-d-thiogalactopyranoside was from Eastman Kodak, factor Xa was from Hematologic Technologies, and Bio-Beads SM-2 were from Bio-Rad. N-Octyl-β-d-glucopyranoside (OG) and Glu-Gly-Arg chloromethyl ketone were from Calbiochem. Sodium dithionite (Na2S2O4, Sigma) was freshly dissolved in 1 m Tris, pH 10, at a concentration of 1 m. PL scramblase was purified as described previously (10Bassé F. Stout J.G. Sims P.J. Wiedmer T. J. Biol. Chem. 1996; 271: 17205-17210Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 11Stout J.G. Bassé F. Luhm R.A. Weiss H.J. Wiedmer T. Sims P.J. J. Clin. Invest. 1997; 99: 2232-2238Crossref PubMed Scopus (71) Google Scholar), 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. The breakthrough material was concentrated and exchanged into 20 mm Tris, 0.1 mm EGTA, 0.02% Nonidet P-40, pH 7.4, and subjected to SDS-PAGE under reducing conditions in a 10% NuPAGE gel (Novex, San Diego, CA). The band at ∼37 kDa was visualized with 0.1% Brilliant Blue R-250 and excised for amino acid analysis and sequencing (University of Michigan Protein and Carbohydrate Structure Facility). 450 pmol of this protein was subjected to in situ cleavage with 10 mg/ml CNBr in 70% formic acid, the cleaved peptides were extracted into 60% acetonitrile, 10% trifluoroacetic acid, dried in a speed vacuum, and resolved by SDS-PAGE and electroblotted onto sequencing grade polyvinylidene difluoride. Peptides were observed by staining with Coomassie Blue, excised, and subjected to microsequencing using Edman chemistry on a model 494 Applied Biosystems sequencer run with standard cycles, yielding the sequence PAPQPPLNCPPGLEYLSQIDQILIHQQIELLE. This same sequence was partially confirmed in a second preparation of PL scramblase purified from erythrocyte ghosts and subjected to internal peptide sequencing as above. A BLAST homology search revealed 100% identity to the translation product of EST clone gb AA143025 with no significant sequence homology to any protein in available data bases. The 568-bp insert of EST clone gb AA143025 was labeled with α-32PdCTP by Random Primed DNA Labeling Kit (Boehringer Mannheim) and used to screen a cDNA library derived from human erythroleukemic cell line K-562 in λgt11 (CLONTECH). Escherichia coli strain Y1090r was transformed by K-562 cDNA library (4.86 × 105 pfu) and poured on 27 agarose plates (15-cm diameter, 18,000 plaque-forming unit/plate). Plaques were lifted onto Hybond-N Nylon membranes (Amersham Corp.). After UV-cross-linking and prehybridization in a solution composed of 5 × Denhardt, 5 × SSC, 1% SDS, and 200 μg/ml herring sperm DNA for 3 h at 68 °C, the membranes were hybridized in the same solution containing 5 ng/ml 32P-labeled probe for 16 h at 68 °C. The membranes were washed once with 1 × SSC, 0.1% SDS, then three times with 0.2 × SSC, 0.1% SDS for 20 min at 65 °C, and exposed to x-ray film. Secondary plaque lifts and hybridization were carried out on 32 strongly positive plaques at a density of 50–100 plaques/plate. Single positive and well isolated plaques were picked and amplified. The length of cDNA insert was examined by PCR with λgt11 reverse and forward primers. Six clones with cDNA inserts of >1.4 kilobase pairs were selected for DNA sequencing. DNA was sequenced on an Applied Biosystems DNA Sequencer model 373 Stretch using PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Perkin-Elmer) and combinations of vector and insert sequence primers. To express PL scramblase as a fusion protein with maltose binding protein (MBP), cDNA encoding PL scramblase was cloned into pMAL-C2 (New England BioLabs). PCR was performed on a full-length clone using the primers 5′-TCAGAATTCGGATCCATGGACAAACAAAACTCACAGATG-3′ with anEcoRI site before the ATG start codon and 5′-GCTTGCCTGCAGGTCGACCTACCACACTCCTGATTTTTGTTCC-3′ with aSalI site after the stop codon. KlenTaq polymerase (CLONTECH) was used to ensure high fidelity amplification. The PCR product was digested with EcoRI andSalI and isolated by electrophoresis on 1% low melting agarose gel and purification with Wizard kit (Promega). The amplified cDNA was cloned into pMAL-C2 vector digested with EcoRI and SalI, immediately 3′ of MBP. This construct was amplified in E. coli strain TB1, and the sequence of the cDNA insert of plasmids from single colonies was confirmed. 10 ml of E. coli TB1 transformed with scramblase cDNA-pMAL-C2 were used to inoculate 1 liter of rich LB containing 2 mg/ml glucose, 100 μg/ml ampicillin, and the bacteria were allowed to grow for about 4 h at 37 °C. When A 600reached ∼0.5, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.3 mm. After 2 h of incubation at 37 °C, the cells were centrifuged at 4000 for 20 The cell was in ml of 20 mm Tris, 200 mm NaCl, 1 mm 1 mm 1 mm and subjected to a After × on and at × for 1 the was to 10 ml of amylose The was washed with 20 of and the fusion protein with the same containing 10 mm of scramblase protein with factor Xa was performed at of and by to MBP, the product of this is the PL scramblase translation product containing the to into was performed as described previously (10Bassé F. Stout J.G. Sims P.J. Wiedmer T. J. Biol. Chem. 1996; 271: 17205-17210Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 11Stout J.G. Bassé F. Luhm R.A. Weiss H.J. Wiedmer T. Sims P.J. J. Clin. Invest. 1997; 99: 2232-2238Crossref PubMed Scopus (71) Google Scholar). a of and PS was dried under a of and in 100 mm Tris, 100 mm 0.1 mm EGTA, pH Protein to reconstituted were added to the liposomes at a final concentration of 4 mg/ml in the of mm and at 4 against 200 of containing 1 Bio-Beads To PL scramblase from MBP, the were for 3 h at in the of factor The was by the of 100 Glu-Gly-Arg chloromethyl of the was by the were labeled in the outer leaflet by the of final PL activity was as described previously (10Bassé F. Stout J.G. Sims P.J. Wiedmer T. J. Biol. Chem. 1996; 271: 17205-17210Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 11Stout J.G. Bassé F. Luhm R.A. Weiss H.J. Wiedmer T. Sims P.J. J. Clin. Invest. 1997; 99: 2232-2238Crossref PubMed Scopus (71) Google Scholar). labeled with were for 2 h at 37 in in the or the of 2 mm were in containing 4 mm and to a at °C. was 20 mm dithionite was and the was for a of The in observed in the of was to Ca2+-induced in located in the outer leaflet (10Bassé F. Stout J.G. Sims P.J. Wiedmer T. J. Biol. Chem. 1996; 271: 17205-17210Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 11Stout J.G. Bassé F. Luhm R.A. Weiss H.J. Wiedmer T. Sims P.J. J. Clin. Invest. 1997; 99: 2232-2238Crossref PubMed Scopus (71) Google Scholar, Biochemistry. 1991; 30: PubMed Scopus Google Scholar). was using by F. of The peptide to amino of the of PL scramblase with an added was and to Blood to this protein was in and the fraction was isolated on protein was isolated by on to peptide was This was used for and of PL scramblase and PL scramblase purified from human erythrocytes was with by gel and the protein was °C, with either an of mg/ml in 150 mm NaCl, 10 mm MOPS, mm OG, pH or no as The was with protein and washed and protein were resolved by SDS-PAGE under reducing were visualized by To to this peptide the activity with the purified erythrocyte PL scramblase protein, the after were reconstituted in liposomes for activity performed as described above. erythrocyte PL scramblase for 2 × washed 2 × erythrocyte pmol of purified recombinant PL scramblase by factor Xa of the PL fusion and 0.3 pmol of PL scramblase purified from human erythrocyte were by in of containing 10% SDS, and 1 mm and protein were resolved by After to the membrane was with 1 μg/ml of and the was with using Protein were upon density at using of (MBP), and PL scramblase in human platelet and erythrocyte membranes was by immunoblotting of the with to of purified scramblase fusion and human cell line membranes were obtained from The were with (CLONTECH) at 68 for min and hybridized with containing 5 PL scramblase cDNA probe at 68 for 1 then and exposed to x-ray film. After the were and hybridized with 32P-labeled cDNA probe using PL scramblase was purified from human erythrocyte membranes and cleaved with and Edman was performed on a peptide to 32 of peptide sequence This peptide the to the site of was in the translation product of a 568-bp EST clone in by the number other significant to this sequence were in any protein data The EST clone was used to screen a human K-562 cell cDNA library. 32 positive clones by plaque clones were sequenced yielding of cDNA The a protein a sequence that with a of a of and a single transmembrane near the C in with the observed for the ∼37-kDa protein band we as PL scramblase in human erythrocyte membrane (10Bassé F. Stout J.G. Sims P.J. Wiedmer T. J. Biol. Chem. 1996; 271: 17205-17210Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). the deduced protein sequence is for high homology to significant to protein or with the of a single protein C site The that PL scramblase function is mediated by a has previously been on an observed in PL scrambling activity in erythrocytes of J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the protein sequence of revealed a strongly of the transmembrane consistent with a type II plasma membrane of the from the cytoplasmic membrane leaflet, a The of this protein is consistent with the of PL scramblase in the erythrocyte where function is to at the endofacial surface of the membrane P. Kulick A. Zachowski A. Schlegel R.A. Devaux P.F. Biochemistry. 1992; 31: 6355-6360Crossref PubMed Scopus (191) Google E.F. Comfurius P. Bevers E.M. Zwaal R.F.A. Biochim. Biophys. Acta. 1994; 1195: 281-286Crossref PubMed Scopus (113) Google Scholar, F. Stout J.G. Sims P.J. Wiedmer T. J. Biol. Chem. 1996; 271: 17205-17210Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 11Stout J.G. Bassé F. Luhm R.A. Weiss H.J. Wiedmer T. Sims P.J. J. Clin. Invest. 1997; 99: 2232-2238Crossref PubMed Scopus (71) Google Scholar, P. Bevers E.M. Smeets E.F. Comfurius P. Schlegel R.A. Zwaal R.F.A. Biochemistry. PubMed Scopus Google Scholar, D.L. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). To that the cDNA we cloned from the K-562 cDNA library the same protein purified as PL scramblase from human erythrocyte we a against the deduced C from the of the cloned cDNA shown in this the ∼37-kDa red cell protein we as PL scramblase and also absorbed the activity in this isolated erythrocyte membrane protein also from 2 we observed the of PL scramblase to a of The apparent of this protein to for the reported rapid of activity observed in to PL scramblase from platelet (12Comfurius P. Williamson P. Smeets E.F. Schlegel R.A. Bevers E.M. Zwaal R.F.A. Biochemistry. 1996; 35: 7631-7634Crossref PubMed Scopus (103) Google Scholar). PL scramblase was expressed in E. coli as fusion protein with MBP, purified by amylose and incorporated into liposomes for of PL scramblase When incorporated into the recombinant protein mediated a Ca2+-dependent movement of mimicking the activity of PL scramblase isolated from PL scramblase activity was observed both for the scramblase fusion protein and for recombinant PL scramblase from through with factor Xa no activity was observed for protein of the pMAL-C2 translation product the PL scramblase cDNA The PL activity of recombinant PL scramblase expressed and purified from E. coli was approximately of that observed for the protein purified from the erythrocyte is due to of the recombinant for activation was approximately for recombinant protein purified from E. coli for the protein, the that or an in cells the Ca2+ binding site (10Bassé F. Stout J.G. Sims P.J. Wiedmer T. J. Biol. Chem. 1996; 271: 17205-17210Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 11Stout J.G. Bassé F. Luhm R.A. Weiss H.J. Wiedmer T. Sims P.J. J. Clin. Invest. 1997; 99: 2232-2238Crossref PubMed Scopus (71) Google Scholar). to activation by the of PL in erythrocytes is upon of the leaflet to pH of a response that is also observed in containing PL scramblase purified from erythrocyte membranes J.G. Bassé F. Luhm R.A. Weiss H.J. Wiedmer T. Sims P.J. J. Clin. Invest. 1997; 99: 2232-2238Crossref PubMed Scopus (71) Google Scholar). A similar activation of PL function was also by recombinant PL scramblase purified from E. coli to the role of PL scramblase in PS exposure following cell and upon of red the of to aminophospholipids the plasma membrane is thought to play a central role in the of for plasma R.F.A. A.J. 1997; PubMed Google Scholar). incubation with Ca2+ causes a in movement of plasma membrane PL in both and the apparent of PL in platelet that in erythrocyte by approximately either a higher abundance of PL scramblase or the action of in platelet with PL scrambling function Zachowski A. Devaux P.F. F. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, C. Devaux P.F. Zachowski A. F. Biochemistry. 1996; 35: PubMed Scopus Google Scholar). Zwaal and recently reported evidence for the of protein(s) in platelet with similar to that of PL scramblase we isolated from erythrocyte (10Bassé F. Stout J.G. Sims P.J. Wiedmer T. J. Biol. Chem. 1996; 271: 17205-17210Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 11Stout J.G. Bassé F. Luhm R.A. Weiss H.J. Wiedmer T. Sims P.J. J. Clin. Invest. 1997; 99: 2232-2238Crossref PubMed Scopus (71) Google Scholar, P. Williamson P. Smeets E.F. Schlegel R.A. Bevers E.M. Zwaal R.F.A. Biochemistry. 1996; 35: 7631-7634Crossref PubMed Scopus (103) Google Scholar). To the protein we in the erythrocyte membrane is also found in we with against PL scramblase shown in this a single protein in platelet with similar to the ∼37-kDa PL scramblase in Based on immunoblotting with we approximately in platelet in consistent with the increased PL scramblase activity and procoagulant function observed for human to platelet and red blood PL scramblase activity has been observed in other and this Ca2+-induced response is thought to central to the rapid movement of PS and phosphatidylethanolamine from plasma membrane leaflet to the surface of and a variety of injured and apoptotic cells R.F.A. A.J. 1997; PubMed Google Scholar). The resulting exposure of PS at the cell surface is thought to play a key role in of cells by the reticuloendothelial system, in to activation of both the plasma complement and coagulation R.H. Phillips Jr., G. Medof M.E. Mold C. J. Clin. Invest. 1993; 92: 1326-1335Crossref PubMed Scopus (102) Google Scholar, 9Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216Crossref PubMed Google Scholar, R.F.A. A.J. 1997; PubMed Google Scholar). the in evidence for a platelet membrane protein to accelerate of PL between membrane leaflets at increased has been reported (12Comfurius P. Williamson P. Smeets E.F. Schlegel R.A. Bevers E.M. Zwaal R.F.A. Biochemistry. 1996; 35: 7631-7634Crossref PubMed Scopus (103) Google Scholar), similar to the role of PL scramblase in red blood cells (10Bassé F. Stout J.G. Sims P.J. Wiedmer T. J. Biol. Chem. 1996; 271: 17205-17210Abstract Full Text Full Text PDF PubMed Scopus (269) Google J.G. Bassé F. Luhm R.A. Weiss H.J. Wiedmer T. Sims P.J. J. Clin. Invest. 1997; 99: 2232-2238Crossref PubMed Scopus (71) Google Scholar). was of to mRNA for this protein is expressed in cells where PL activity has been observed. shown by with PL scramblase cDNA revealed of and in all tissues and cell lines and cell line in the abundance of is the of to was expression in and the lines and was in and to the transformed cell lines mRNA for PL scramblase was also confirmed in human cells data that the same protein as of the erythrocyte membrane PL also a similar role in the plasma membrane of and other for this role of PL scramblase analysis of a cell line that is in this a to an of plasma membrane PL scramblase erythrocytes and other cells in PL scramblase activity were found to of the PL scramblase protein J.G. Bassé F. Luhm R.A. Weiss H.J. Wiedmer T. Sims P.J. J. Clin. Invest. 1997; 99: 2232-2238Crossref PubMed Scopus (71) Google Scholar). J. J. G. Stout, P. J. Sims, and T. Wiedmer, the apparent in cells of PL scramblase when PL scramblase protein from cells was purified and reconstituted in containing PL, Ca2+-dependent PL activity J.G. Bassé F. Luhm R.A. Weiss H.J. Wiedmer T. Sims P.J. J. Clin. Invest. 1997; 99: 2232-2238Crossref PubMed Scopus (71) Google Scholar). This that in to the by intracellular the activity of PL scramblase in the plasma membrane is by other as membrane or cytoplasmic The of J. and T. is The of C. in protein sequencing and in peptide sequencing is also