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Therese Wiedmer - One of the best experts on this subject based on the ideXlab platform.
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palmitoylation of phospholipid scramblase is required for normal function in promoting ca2 activated transbilayer movement of membrane Phospholipids
Biochemistry, 1998Co-Authors: Ji Zhao, Therese Wiedmer, Quansheng Zhou, Peter J SimsAbstract:Accelerated transbilayer movement of plasma membrane Phospholipids (PL) plays a central role in the initiation of plasma clotting and in phagocytic clearance of injured or apoptotic cells. We recen...
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identity of a conserved motif in phospholipid scramblase that is required for ca2 accelerated transbilayer movement of membrane Phospholipids
Biochemistry, 1998Co-Authors: Quansheng Zhou, Therese WiedmerAbstract:Accelerated transbilayer movement of plasma membrane Phospholipids (PL) upon elevation of Ca2+ in the cytosol plays a central role in the initiation of plasma clotting and in phagocytic clearance of injured or apoptotic cells. We recently identified a human erythrocyte membrane protein that induces rapid transbilayer movement of PL at elevated Ca2+. We also presented evidence that this PL scramblase is expressed in a variety of other cells and tissues where transbilayer movement of plasma membrane PL is promoted by intracellular Ca2+ [Zhou, Q., et al. (1997) J. Biol. Chem. 272, 18240-18244]. We have now cloned murine PL scramblase for comparison with the human polypeptide. Both human and murine PL scramblase are acidic proteins (pI = 4.9) with a predicted inside-outside (type 2) transmembrane segment at the carboxyl-terminus. Whereas human PL scramblase (318 AA) terminates in a short exoplasmic tail, murine PL scramblase (307 AA) terminates in the predicted membrane-inserted segment. The aligned polypeptide sequences reveal 65% overall identity, including near identity through 12 residues of an apparent Ca2+ binding motif (D[A/S]DNFGIQFPLD) spanning codons 273-284 (human) and 271-282 (murine), respectively. This conserved sequence in the cytoplasmic domain of PL scramblase shows similarity to Ca2+-binding loop motifs previously identified in known EF hand structures. Recombinant murine and human PL scramblase were each expressed in Escherichia coli and incorporated into proteoliposomes. Measurement of transbilayer movement of NBD-labeled PL confirmed that both proteins catalyzed Ca2+-dependent PL flip-flop similar to that observed for the action of Ca2+ at the cytoplasmic face of plasma membranes. Mutation of residues within the putative EF hand loop of human PL scramblase resulted in loss of its PL mobilizing function, suggesting that these residues directly participate in the Ca2+-induced active conformation of the polypeptide.
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molecular cloning of human plasma membrane phospholipid scramblase a protein mediating transbilayer movement of plasma membrane Phospholipids
Journal of Biological Chemistry, 1997Co-Authors: Quansheng Zhou, James G Stout, Ji Zhao, Robert A Luhm, Therese WiedmerAbstract:Abstract 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 (Basse, 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.
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molecular cloning of human plasma membrane phospholipid scramblase a protein mediating transbilayer movement of plasma membrane Phospholipids
Journal of Biological Chemistry, 1997Co-Authors: Quansheng Zhou, James G Stout, Therese Wiedmer, Ji Zhao, Robert A Luhm, Peter J SimsAbstract: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 approximately 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 (Basse, 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 approximately 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 ( approximately 10(4) molecules/cell) than in erythrocyte ( approximately 10(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.
Quansheng Zhou - One of the best experts on this subject based on the ideXlab platform.
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palmitoylation of phospholipid scramblase is required for normal function in promoting ca2 activated transbilayer movement of membrane Phospholipids
Biochemistry, 1998Co-Authors: Ji Zhao, Therese Wiedmer, Quansheng Zhou, Peter J SimsAbstract:Accelerated transbilayer movement of plasma membrane Phospholipids (PL) plays a central role in the initiation of plasma clotting and in phagocytic clearance of injured or apoptotic cells. We recen...
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identity of a conserved motif in phospholipid scramblase that is required for ca2 accelerated transbilayer movement of membrane Phospholipids
Biochemistry, 1998Co-Authors: Quansheng Zhou, Therese WiedmerAbstract:Accelerated transbilayer movement of plasma membrane Phospholipids (PL) upon elevation of Ca2+ in the cytosol plays a central role in the initiation of plasma clotting and in phagocytic clearance of injured or apoptotic cells. We recently identified a human erythrocyte membrane protein that induces rapid transbilayer movement of PL at elevated Ca2+. We also presented evidence that this PL scramblase is expressed in a variety of other cells and tissues where transbilayer movement of plasma membrane PL is promoted by intracellular Ca2+ [Zhou, Q., et al. (1997) J. Biol. Chem. 272, 18240-18244]. We have now cloned murine PL scramblase for comparison with the human polypeptide. Both human and murine PL scramblase are acidic proteins (pI = 4.9) with a predicted inside-outside (type 2) transmembrane segment at the carboxyl-terminus. Whereas human PL scramblase (318 AA) terminates in a short exoplasmic tail, murine PL scramblase (307 AA) terminates in the predicted membrane-inserted segment. The aligned polypeptide sequences reveal 65% overall identity, including near identity through 12 residues of an apparent Ca2+ binding motif (D[A/S]DNFGIQFPLD) spanning codons 273-284 (human) and 271-282 (murine), respectively. This conserved sequence in the cytoplasmic domain of PL scramblase shows similarity to Ca2+-binding loop motifs previously identified in known EF hand structures. Recombinant murine and human PL scramblase were each expressed in Escherichia coli and incorporated into proteoliposomes. Measurement of transbilayer movement of NBD-labeled PL confirmed that both proteins catalyzed Ca2+-dependent PL flip-flop similar to that observed for the action of Ca2+ at the cytoplasmic face of plasma membranes. Mutation of residues within the putative EF hand loop of human PL scramblase resulted in loss of its PL mobilizing function, suggesting that these residues directly participate in the Ca2+-induced active conformation of the polypeptide.
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molecular cloning of human plasma membrane phospholipid scramblase a protein mediating transbilayer movement of plasma membrane Phospholipids
Journal of Biological Chemistry, 1997Co-Authors: Quansheng Zhou, James G Stout, Ji Zhao, Robert A Luhm, Therese WiedmerAbstract:Abstract 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 (Basse, 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.
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molecular cloning of human plasma membrane phospholipid scramblase a protein mediating transbilayer movement of plasma membrane Phospholipids
Journal of Biological Chemistry, 1997Co-Authors: Quansheng Zhou, James G Stout, Therese Wiedmer, Ji Zhao, Robert A Luhm, Peter J SimsAbstract: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 approximately 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 (Basse, 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 approximately 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 ( approximately 10(4) molecules/cell) than in erythrocyte ( approximately 10(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.
Mohammed Jemal - One of the best experts on this subject based on the ideXlab platform.
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Phospholipids in liquid chromatography mass spectrometry bioanalysis comparison of three tandem mass spectrometric techniques for monitoring plasma Phospholipids the effect of mobile phase composition on Phospholipids elution and the association of Phospholipids with matrix effects
Rapid Communications in Mass Spectrometry, 2009Co-Authors: Yuanqing Xia, Mohammed JemalAbstract:Because plasma Phospholipids may cause matrix effects in bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods, it is important to establish optimal mass spectrometric techniques to monitor the fate of Phospholipids during method development and application. We evaluated three MS/MS techniques to monitor Phospholipids using positive and negative electrospray ionization (ESI). The first technique is based on using positive precursor ion scan of m/z 184, positive neutral loss scan of 141 Da and negative precursor ion scan of m/z 153. The second technique is based on using class-specific positive and negative selected reaction monitoring (SRM) transitions to monitor class-representative Phospholipids. The third technique, previously reported, utilizes in-source collision-induced dissociation (CID)-based positive SRM of m/z 184 --> 184. We recommend the all-inclusive technique 1 for use in qualitative assessment of all classes of Phospholipids and technique 2 for use in quantitative assessment of class-representative Phospholipids. Secondly, we evaluated the elution behaviors of the plasma Phospholipids under different reversed-phase mobile phase conditions. The phospholipid-eluting strength of a mobile phase was mainly dependent on the type and amount (%) of the organic eluent and the strength increased in the order of methanol, acetonitrile and isopropyl alcohol. Under the commonly used gradient and isocratic elution schemes in LC/MS/MS bioanalysis, not all the Phospholipids are eluted off the column. Thirdly, we investigated the association between Phospholipids and matrix effects in positive and negative ESI using basic, acidic and neutral analytes. While the Phospholipids caused matrix effects in both positive and negative ESI, the extent of ionization suppression was analyte-dependent and was inversely related to the retention factor and broadness of the Phospholipids peaks. The lysoPhospholipids which normally elute earlier in reversed-phase chromatography are more likely to cause matrix effects compared to the later-eluting Phospholipids in spite of the larger concentrations of the latter in plasma.
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Phospholipids in liquid chromatography mass spectrometry bioanalysis comparison of three tandem mass spectrometric techniques for monitoring plasma Phospholipids the effect of mobile phase composition on Phospholipids elution and the association of Phospholipids with matrix effects
Rapid Communications in Mass Spectrometry, 2009Co-Authors: Mohammed JemalAbstract:Because plasma Phospholipids may cause matrix effects in bioanalytical liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods, it is important to establish optimal mass spectrometric techniques to monitor the fate of Phospholipids during method development and application. We evaluated three MS/MS techniques to monitor Phospholipids using positive and negative electrospray ionization (ESI). The first technique is based on using positive precursor ion scan of m/z 184, positive neutral loss scan of 141 Da and negative precursor ion scan of m/z 153. The second technique is based on using class-specific positive and negative selected reaction monitoring (SRM) transitions to monitor class-representative Phospholipids. The third technique, previously reported, utilizes in-source collision-induced dissociation (CID)-based positive SRM of m/z 184 → 184. We recommend the all-inclusive technique 1 for use in qualitative assessment of all classes of Phospholipids and technique 2 for use in quantitative assessment of class-representative Phospholipids. Secondly, we evaluated the elution behaviors of the plasma Phospholipids under different reversed-phase mobile phase conditions. The phospholipid-eluting strength of a mobile phase was mainly dependent on the type and amount (%) of the organic eluent and the strength increased in the order of methanol, acetonitrile and isopropyl alcohol. Under the commonly used gradient and isocratic elution schemes in LC/MS/MS bioanalysis, not all the Phospholipids are eluted off the column. Thirdly, we investigated the association between Phospholipids and matrix effects in positive and negative ESI using basic, acidic and neutral analytes. While the Phospholipids caused matrix effects in both positive and negative ESI, the extent of ionization suppression was analyte-dependent and was inversely related to the retention factor and broadness of the Phospholipids peaks. The lysoPhospholipids which normally elute earlier in reversed-phase chromatography are more likely to cause matrix effects compared to the later-eluting Phospholipids in spite of the larger concentrations of the latter in plasma. Copyright © 2009 John Wiley & Sons, Ltd.
Peter J Sims - One of the best experts on this subject based on the ideXlab platform.
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palmitoylation of phospholipid scramblase is required for normal function in promoting ca2 activated transbilayer movement of membrane Phospholipids
Biochemistry, 1998Co-Authors: Ji Zhao, Therese Wiedmer, Quansheng Zhou, Peter J SimsAbstract:Accelerated transbilayer movement of plasma membrane Phospholipids (PL) plays a central role in the initiation of plasma clotting and in phagocytic clearance of injured or apoptotic cells. We recen...
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molecular cloning of human plasma membrane phospholipid scramblase a protein mediating transbilayer movement of plasma membrane Phospholipids
Journal of Biological Chemistry, 1997Co-Authors: Quansheng Zhou, James G Stout, Therese Wiedmer, Ji Zhao, Robert A Luhm, Peter J SimsAbstract: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 approximately 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 (Basse, 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 approximately 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 ( approximately 10(4) molecules/cell) than in erythrocyte ( approximately 10(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.
Sathyanarayana N Gummadi - One of the best experts on this subject based on the ideXlab platform.
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Are cysteine residues of human phospholipid scramblase 1 essential for Pb^2+ and Hg^2+ binding-induced scrambling of Phospholipids?
European Biophysics Journal, 2021Co-Authors: Ashok Kumar Shettihalli, Santosh Kumar Palanirajan, Sathyanarayana N GummadiAbstract:Lead and mercury being common environmental pollutants are often associated with erythrocytes, where phosphatidylserine (PS) exposure-mediated procoagulant activation is induced. Human phospholipid scramblase 1 (hPLSCR1) identified in the erythrocyte membrane is a type II transmembrane protein involved in Ca^2+-dependent bidirectional scrambling of Phospholipids (PL) during blood coagulation, cell activation, and apoptosis. The prominent role of hPLSCR1 in Pb^2+ and Hg^2+ poisoning was demonstrated by a biochemical assay, where recombinant hPLSCR1 induced PL scrambling across bilayer with a higher binding affinity ( K _d) towards Hg^2+ (4.1 µM) and Pb^2+ (5.8 µM) than Ca^2+ (25.6 mM). The increased affinity could be the outcome of heavy metals interacting at auxiliary sites other than the calcium-binding motif of hPLSCR1. Similar to other metal-binding proteins, cysteine-based metal-binding motifs could be the potential additional binding sites in hPLSCR1. To explore the hypothesis, the cysteines were chemically modified, which significantly reduced only the Hg^2+- and Pb^2+-induced scrambling activity leaving Ca^2+-induced activity unaltered. Recombinant constructs with deletion of prominent cysteine residues and point mutation in the calcium-binding motif including Δ100-hPLSCR1, Δ160-hPLSCR1, and D275A-hPLSCR1 were generated, purified, and assayed for scramblase activity. The cysteine-deleted constructs of hPLSCR1 showed reduced binding affinity ( K _d) for Hg^2+ and Pb^2+ without altering the Ca^2+-binding affinity whereas the point mutant had completely lost its affinity for Ca^2+ and reduced affinities for Hg^2+ and Pb^2+. The results accentuated the significance of cysteine residues as additional binding sites for heavy metal ions in hPLSCR1.
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heavy metals mediated Phospholipids scrambling by human phospholipid scramblase 3 a probable role in mitochondrial apoptosis
Chemical Research in Toxicology, 2020Co-Authors: Santosh Kumar Palanirajan, Sathyanarayana N GummadiAbstract:Human phospholipid scramblases are a family of four homologous transmembrane proteins (hPLSCR1-4) mediating Phospholipids (PLs) translocation in plasma membrane upon Ca2+ activation. hPLSCR3, the only homologue localized to mitochondria, plays a vital role in mitochondrial structure, function, maintenance, and apoptosis. Upon Ca2+ activation, hPLSCR3 mediates PL translocation at the mitochondrial membrane enhancing t-bid-induced cytochrome c release and apoptosis. Mitochondria are important target organelles for heavy-metals-induced apoptotic signaling cascade and are the central executioner of apoptosis to trigger. Pb2+ and Hg2+ toxicity mediates apoptosis by increased reactive oxygen species (ROS) and cytochrome c release from mitochondria. To discover the role of hPLSCR3 in heavy metal toxicity, hPLSCR3 was overexpressed as a recombinant protein in Escherichia coli Rosetta (DE3) and purified by affinity chromatography. The biochemical assay using synthetic proteoliposomes demonstrated that hPLSCR3 translocated aminoPhospholipids in the presence of micromolar concentrations of Pb2+ and Hg2+. A point mutation in the Ca2+-binding motif (F258V) led to a ∼60% loss in the functional activity and decreased binding affinities for Pb2+ and Hg2+ implying that the divalent heavy metal ions bind to the Ca2+-binding motif. This was further affirmed by the characteristic spectra observed with stains-all dye. The conformational changes upon heavy metal binding were monitored by circular dichroism, intrinsic tryptophan fluorescence, and light-scattering studies. Our results revealed that Pb2+ and Hg2+ bind to hPLSCR3 with higher affinity than Ca2+ thus mediating scramblase activity. To summarize, this is the first biochemical evidence for heavy metals binding to the mitochondrial membrane protein leading to bidirectional translocation of PLs specifically toward phosphatidylethanolamine.
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biochemical evidence for lead and mercury induced transbilayer movement of Phospholipids mediated by human phospholipid scramblase 1
Chemical Research in Toxicology, 2013Co-Authors: Ashok Kumar Shettihalli, Sathyanarayana N GummadiAbstract:Human phospholipid scramblase 1(hPLSCR1) is a transmembrane protein involved in bidirectional scrambling of plasma membrane Phospholipids during cell activation, blood coagulation, and apoptosis in response to elevated intracellular Ca(2+) levels. Pb(2+) and Hg(2+) are known to cause procoagulant activation via phosphatidylserine exposure to the external surface in erythrocytes, resulting in blood coagulation. To explore its role in lead and mercury poisoning, hPLSCR1 was overexpressed in Escherichia coli BL21 (DE3) and purified using affinity chromatography. The biochemical assay showed rapid scrambling of Phospholipids in the presence of Hg(2+) and Pb(2+). The binding constant (Ka) was calculated and found to be 250 nM(-1) and 170 nM(-1) for Hg(2+) and Pb(2+), respectively. The intrinsic tryptophan fluorescence and far ultraviolet circular dichroism studies revealed that Hg(2+) and Pb(2+) bind to hPLSCR1 and induce conformational changes. hPLSCR1 treated with protein modifying reagent N-ethylmaleimide before functional reconstitution showed 40% and 24% inhibition in the presence of Hg(2+) and Pb(2+), respectively. This is the first biochemical evidence to prove the above hypothesis that hPLSCR1 is activated in heavy metal poisoning, which leads to bidirectional transbilayer movement of Phospholipids.
