Elevation of intracellular Ca2؉ in erythrocytes, platelets, and other cells initiates rapid redistribution of plasma membrane phospholipids (PL) between inner and outer leaflets, collapsing the normal asymmetric distribution. Consequently, phosphatidylserine and other lipids normally sequestered to the inner leaflet become exposed at the cell surface. This Ca 2؉-induced mobilization of phosphatidylserine to the surface of activated, injured, or apoptotic cells confers a procoagulant property to the plasma membrane, which promotes fibrin clotting and provides a signal for cell removal by the reticuloendothelial system. To identify the constituent of the membrane that mediates this Ca 2؉-dependent "PL scramblase" activity, we undertook purification and reconstitution of membrane component(s) with this activity from detergent extracts of erythrocyte ghosts depleted of cytoskeleton. Active fractions were identified by their capacity to mediate the Ca 2؉ -dependent redistribution of 7-nitrobenz-2-oxa-1,3-diazol-4-yllabeled PL between leaflets of reconstituted proteoliposomes. This PL scramblase activity co-eluted through multiple chromatographic steps with a single polypeptide of ϳ37 kDa, which was purified to apparent homogeneity as resolved by silver staining. The activity associated with this protein band was inactivated by trypsin. The isolated protein reconstituted in proteoliposomes mediated nonselective, bidirectional transport of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-PL between membrane leaflets, with half-maximal activation between 20 and 60 M Ca 2؉ (saturation >100 M), mimicking the Ca 2؉
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.
The extracellular ATPase (ecto-ATPase) is a divalent cation-dependent nucleoside triphosphatase with an unusually high specific activity. Monoclonal antibodies, described previously [Stout, J. G., Strobel, R. S., & Kirley, T. L. (1995) J. Biol. Chem. 270, 11845-11850], and newly generated polyclonal antibodies, both raised against the chicken gizzard ecto-ATPase, were evaluated for their ability to cross-react with mammalian ecto-ATPases and were used as specific immunochemical probes to identify non-cross-linked and cross-linked ecto-ATPase. Unlike previous results obtained with the rabbit skeletal muscle ecto-ATPase enzyme, cross-linking the chicken gizzard smooth muscle ecto-ATPase with 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) and dithiobis(succinimidylpropionate) (DSP) increased the activity of the enzyme which corresponded to an increase in a approximately 130 kDa immunoreactive band, proposed to be a ecto-ATPase homodimer, and a concomitant decrease in a approximately 66 kDa immunoreactive band, the ecto-ATPase monomer. Ecto-ATPase was immunochemically identified in chicken, rat, mouse, rabbit, and pig. Interestingly, under nonreducing conditions, the ecto-ATPase activity in rat and pig (unlike chicken and rabbit) was evident on Western blots as an immunoreactive band at approximately 200 kDa, proposed to be an intermolecularly disulfide-linked ecto-ATPase homotrimer. Nonreducing Western blot analysis of various rat tissues with three different monoclonal antibodies that recognize the 66 kDa chicken gizzard ecto-ATPase monomer strengthened the hypothesis that this 200 kDa band indeed represents the trimeric ecto-ATPase. After reduction, ecto-ATPase monomers were found to be approximately 66 kDa in all species examined. The differences in ecto-ATPase quaternary structure stability may account for the observed species differences in ecto-ATPase enzymatic properties. Intermolecular disulfide bonds appear to be one of the species-specific ways to stabilize the native, active ecto-ATPase quaternary structure (the homotrimer). Based on the data obtained, as well as previous data from this and other laboratories, a hypothesis was developed to explain the modulation of ecto-ATPase activity by a variety of agents, including detergents, chemical cross-linkers, lectins, antibodies, and small molecule inhibitors. It is proposed that agents and conditions stabilizing ecto-ATPase oligomers stimulate enzyme activity, whereas agents and conditions destabilizing ecto-ATPase homooligomers would inhibit the ecto-ATPase.
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