Transmembrane mobility of phospholipids in sickle erythrocytes: effect of deoxygenation on diffusion and asymmetry

We studied the effect of sickling on the transmembrane reorientation and distribution of phospholipids in the red blood cells of patients homozygous for sickle cell anemia (SS). To this purpose, we followed the redistribution kinetics of trace amounts of spin-labeled analogues of natural phospholipids first introduced in the membrane outer leaflet of normal or sickle erythrocytes exposed to air or nitrogen. Deoxygenation had no effect on the lipid redistribution kinetics in normal (AA) cell membranes. At atmospheric pO2, unfractionated SS cells were not different from normal cells. However, on deoxygenation inducing sickling, phosphatidylcholine passive diffusion was accelerated and the rate of the adenosine triphosphate-dependent transport of aminophospholipids was reduced, especially for phosphatidylserine. The stationary distribution of the aminophospholipids between the two leaflets was slightly less asymmetric, a phenomenon more pronounced with phosphatidylethanolamine. These changes were rapidly reversible on reoxygenation. When SS cells were separated by density, both dense and light cells exhibited the properties cited above. However, dense cells exposed to air possessed a lower aminophospholipid transport rate. These data favor the relationship between aminophospholipid translocase activity and phospholipid transmembrane asymmetry. Sickle cell disease is the first case of aminophospholipid translocase pathology.     

ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.

Spin-labeled analogs of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine have been used to study phospholipid transverse diffusion and asymmetry in the human erythrocyte membrane. Ascorbate reduction was used to assess the transbilayer distribution of the labels. All three spin-labeled phospholipids initially incorporated into the outer leaflet of the membrane. On fresh erythrocytes at 5 degrees C, the phosphatidylcholine label remained mainly in the outer leaflet. In contrast, the phosphatidylserine and phosphatidylethanolamine labels underwent rapid transverse diffusion that led to their asymmetric distribution in favor of the inner leaflet. The latter effect was reversibly inhibited after ATP depletion of the erythrocytes and could be reproduced on resealed erythrocyte ghosts only if hydrolyzable Mg-ATP was included in the internal medium. It is suggested that an ATP-driven transport of amino phospholipids toward the inner leaflet could be the major cause of the phospholipid asymmetry in the erythrocyte membrane. It is also proposed that the same mechanism could explain the ATP requirement of the maintenance of the erythrocyte membrane discoid shape.     

The distribution of erythrocyte phospholipids in hereditary spherocytosis demonstrates a minimal role for erythrocyte spectrin on phospholipid diffusion and asymmetry

In the human erythrocyte membrane phosphatidylcholine and sphingomyelin reside mainly in the outer leaflet, whereas the aminophospholipids, phosphatidylethanolamine and phosphatidylserine, are mainly found in the inner leaflet. Maintenance of phospholipid asymmetry has been assumed to involve interactions between the aminophospholipids and the membrane skeleton, in particular spectrin. To investigate whether spectrin contributes to maintaining the phospholipid transbilayer distribution and kinetics of redistribution, we studied erythrocytes from hereditary spherocytosis patients whose spectrin levels ranged from 34% to 82% of normal. The phospholipid composition and the accessibility of membrane phospholipids to hydrolysis by phospholipases were in the normal range. Spin-labeled phosphatidylserine and phosphatidylethanolamine analogues that had been introduced into the outer leaflet were rapidly transported at 37 degrees C to the inner leaflet, whereas the redistribution of spin-labeled phosphatidylcholine was slower. The kinetics of transbilayer movement of these spin-labeled phospholipid in all samples was in the normal range and was not affected by the level of spectrin. Although these erythrocyte membranes contained as little as 34% of the normal level of spectrin and were characterized by several physical abnormalities, the composition, distribution, and transbilayer kinetics of the phospholipids were found to be normal. We therefore conclude that spectrin plays, at best, only a minor role in maintaining the distribution of erythrocyte membrane phospholipid.     

Enzymatic synthesis and rapid translocation of phosphatidylcholine by two methyltransferases in erythrocyte membranes.

The synthesis of phosphatidylcholine from phosphatidylethanolamine is carried out by two methyltransferases in erythrocyte membranes. The first enzyme uses phosphatidylethanolamine as a substrate, requires Mg2+, and has a high affinity for methyl donor, S-adenosyl-L-methionine. The second enzyme methylates phosphatidyl-N-monomethylethanolamine to phosphatidylcholine and has a low affinity for S-adenosyl-L-methionine. The first enzyme is localized on the cytoplasmic side of the membrane and the second enzyme faces the external surface. This asymmetric arrangement of the two enzymes across the membrane makes possible the stepwide methylation of phosphatidylethanolamine localized on the cytoplasmic side and facilitates the rapid transmembrane transfer of the final product, phosphatidylcholine, to the external surface of the membrane. A mechanism for an enzyme-mediated flip-flop of phospholipids from the cytoplasmic to the outer surface of erythrocyte membranes is described.     

Measurement of outward translocation of phospholipids across human erythrocyte membrane.

Spin-labeled phospholipids have been used to study the outside----inside and inside----outside transport of phospholipids across the human erythrocyte membrane at 37 degrees C. As already shown, inward transport is much faster for aminophospholipids than for phosphatidylcholine. In addition, we show here that outward transport of the phosphatidylserine and phosphatidylethanolamine analogues is three to four times faster than that of phosphatidylcholine. Magnesium depletion of the erythrocytes considerably decreases the outward rate of both aminophospholipids to values close to that of phosphatidylcholine. These results suggest that the outward aminophospholipid translocation is, at least partly, protein mediated. The protein involved could be identical to the inward Mg-ATP-dependent aminophospholipid carrier.     

Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function

Aminophospholipid translocases (APLTs) are defined primarily by their ability to flip fluorescent or spin-labeled derivatives of phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external leaflet of a membrane bilayer to the cytosolic leaflet and are thought to establish phospholipid asymmetry in biological membranes. The identities of APLTs remain unknown, although candidate proteins include the Drs2p/ATPase II subfamily of P-type ATPases. Drs2p from budding yeast localizes to the trans-Golgi network (TGN), and here we show that this membrane contains an ATP-dependent APLT that flips 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) PS and PE derivatives from the luminal to the cytosolic leaflet. To assess the contribution of Drs2p to this activity, TGN membranes were prepared from strains harboring WT or temperature-sensitive alleles of DRS2 and null alleles of three other potential APLT genes (DNF1, DNF2, and DNF3). Assay of these membranes indicated that Drs2p was required for the ATP-dependent translocation of NBD-PS, whereas no active translocation of NBD-PE or NBD-phosphatidylcholine was detected. The specificity of Drs2p for NBD-PS suggested that translocation of PS would be required for the function of Drs2p in protein transport from the TGN. However, cho1 yeast strains that are unable to synthesize PS do not phenocopy drs2 but instead transport proteins normally via the secretory pathway. In addition, a drs2 cho1 double mutant retains drs2 transport defects. Therefore, whereas NBD-PS is a preferred substrate for Drs2p in vitro, endogenous PS is not an obligatory substrate in vivo for the role Drs2p plays in protein transport.     

Reconstitution of phospholipid translocase activity with purified Drs2p,a type-IV P-type ATPase from budding yeast

Type-IV P-type ATPases (P4-ATPases) are putative phospholipid translocases, or flippases, that translocate specific phospholipid substrates from the exofacial to the cytosolic leaflet of membranes to generate phospholipid asymmetry. In addition, the activity of Drs2p, a P4-ATPase from Saccharomyces cerevisiae, is required for vesicle-mediated protein transport from the Golgi and endosomes, suggesting a role for phospholipid translocation in vesicle budding. Drs2p is necessary for translocation of a fluorescent phosphatidylserine analogue across purified Golgi membranes. However, a flippase activity has not been reconstituted with purified Drs2p or any other P4-ATPase, so whether these ATPases directly pump phospholipid across the membrane bilayer is unknown. Here, we show that Drs2p can catalyze phospholipid translocation directly through purification and reconstitution of this P4-ATPase into proteoliposomes. The noncatalytic subunit, Cdc50p, also was reconstituted in the proteoliposome, although at a substoichiometric concentration relative to Drs2p. In proteoliposomes containing Drs2p, a phosphatidylserine analogue was actively flipped across the liposome bilayer to the outer leaflet in the presence of Mg²⁺-ATP, whereas no activity toward the phosphatidylcholine or sphingomyelin analogues was observed. This flippase activity was mediated by Drs2p, because protein-free liposomes or proteoliposomes reconstituted with a catalytically inactive form of Drs2p showed no translocation activity. These data demonstrate for the first time the reconstitution of a flippase activity with a purified P4-ATPase.     

Mechanisms of amphipath-induced stomatocytosis in human erythrocytes.

We studied stomatocytosis induced in human red blood cells (RBC) by vinblastine and chlorpromazine, monitoring the movements of spin-labeled phosphatidylcholine (PC*) and sphingomyelin (SM*) by electron spin resonance (ESR) spectroscopy. This technique allows determination of the fraction of labeled lipids, respectively, on the external leaflet, on the cytosol face, or trapped in endocytic vacuoles. Both vinblastine and chlorpromazine produce a time- and concentration-dependent stomatocytic shape change, which is paralleled by a shift of approximately 10% to 33% of outer leaflet SM* and PC* inward. Of this amount, 8% to 12% was trapped in endocytic vacuoles and 8% to 19% had flipped to the inner leaflet. Vanadate, while inhibiting the stomatocytosis, did not block the flip of either SM* or PC* to the inner leaflet. To explain the inhibiting effect of vanadate, as well as the adenosine triphosphate (ATP) requirement for drug-induced stomatocytosis, we propose the following model: (1) addition of amphipath partially scrambles the bilayer; and (2) the flop of phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the outer leaflet provides substrate for the aminophospholipid translocase (APLT), which flips back PS and PE inward faster than PC or SM can diffuse outward--thereby producing inner layer expansion or stomatocytosis. This role of APLT accounts for the vanadate inhibition of amphipath stomatocytosis. However, the vanadate effect can be overcome by increasing the amphipath concentration, which at such levels probably passively expands the inner leaflet.     

Identification of residues defining phospholipid flippase substrate specificity of type IV P-type ATPases

Type IV P-type ATPases (P4-ATPases) catalyze translocation of phospholipid across a membrane to establish an asymmetric bilayer structure with phosphatidylserine (PS) and phosphatidylethanolamine (PE) restricted to the cytosolic leaflet. The mechanism for how P4-ATPases recognize and flip phospholipid is unknown, and is described as the "giant substrate problem" because the canonical substrate binding pockets of homologous cation pumps are too small to accommodate a bulky phospholipid. Here, we identify residues that confer differences in substrate specificity between Drs2 and Dnf1, Saccharomyces cerevisiae P4-ATPases that preferentially flip PS and phosphatidylcholine (PC), respectively. Transplanting transmembrane segments 3 and 4 (TM3-4) of Drs2 into Dnf1 alters the substrate preference of Dnf1 from PC to PS. Acquisition of the PS substrate maps to a Tyr618Phe substitution in TM4 of Dnf1, representing the loss of a single hydroxyl group. The reciprocal Phe511Tyr substitution in Drs2 specifically abrogates PS recognition by this flippase causing PS exposure on the outer leaflet of the plasma membrane without disrupting PE asymmetry. TM3 and the adjoining lumenal loop contribute residues important for Dnf1 PC preference, including Phe587. Modeling of residues involved in substrate selection suggests a novel P-type ATPase transport pathway at the protein/lipid interface and a potential solution to the giant substrate problem.     

Synexin facilitates fusion of specific phospholipid membranes at divalent cation concentrations found intracellularly.

The effect of synexin (an adrenal medullary protein) on the kinetics of Ca2+- and Mg2+-mediated membrane fusion was examined. Membrane fusion was studied by monitoring intermixing of the aqueous contents of phospholipid vesicles. Synexin facilitated Ca2+-mediated, but not Mg2+-mediated, fusion of phosphatidate/phosphatidylethanolamine (1:3) and phosphatidate/phosphatidylserine/phosphatidylethanolamine/cholesterol (1:2:3:2) vesicles. The threshold concentration of Ca2+ for fusion was decreased to approximately equal to 10 microM in the presence of synexin at 6 micrograms/ml and 1.5 mM Mg2+ in vesicle suspensions containing 50 microM lipid. This effect of synexin was drastically inhibited by including 25% phosphatidylcholine (mol/mol) in the vesicle membrane. It is proposed that the Ca2+-dependent lipid-specific enhancement of membrane fusion by synexin contributes to an increase in the sensitivity of specific intracellular membranes to Ca2+ with respect to fusion.     

Asymmetric lateral mobility of phospholipids in the human erythrocyte membrane.

The fluorescent phospholipid 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminododecanoyl]phosphatidylcholine (NBD-phosphatidylcholine) and the corresponding aminophospholipid derivatives (NBD-phosphatidylethanolamine and NBD-phosphatidylserine) were introduced in the human erythrocyte membrane by a nonspecific phospholipid exchange protein purified from corn. The lateral mobility of the fluorescent phospholipids was measured by using an extension of the classical photobleaching recovery technique that takes advantage of a modulated fringe pattern and provides a high sensitivity. In intact erythrocytes and in ghosts resealed in the presence of ATP, the fluorescence-contrast curves after photobleaching decayed biexponentially corresponding to two lateral diffusion constants. With NBD-phosphatidylcholine, the majority of the signal corresponded to a "slow" component (1.08 X 10(-9) cm2/sec at 20 degrees C), whereas with the amino derivatives the majority of the signal corresponded to a "fast" component (5.14 X 10(-9) cm2/sec at 20 degrees C). If the ghosts were resealed without ATP, the fast component of the aminophospholipids disappeared. We interpret these results as follows: (i) Provided the cells or the ghosts contain ATP, the three fluorescent phospholipids distribute spontaneously between inner and outer leaflets as endogenous phospholipids, namely NBD-phosphatidylcholine is located in the outer leaflet, while both aminophospholipids are preferentially located in the inner leaflet. (ii) The viscosity of the inner leaflet of human erythrocyte membranes is lower than that of the outer leaflet.     

Platelet and erythrocyte Mg2+, Ca2+, Na+, K+ and cell membrane adenosine triphosphatase activity in essential hypertension in blacks.

OBJECTIVE: To assess the relationship between intracellular Mg2+, Ca2+, Na+ and K+ and cell membrane adenosine triphosphatase (ATPase) activity in normotensive and hypertensive blacks. DESIGN: Intracellular cations and cell membrane ATPase activity were studied in black patients with untreated essential hypertension and age-, weight- and height-matched normotensive controls. Platelet, erythrocyte and serum Mg2+, Ca2+, Na+ and K+ levels as well as platelet and erythrocyte membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase activities were measured in all subjects. METHODS: Intracellular Na+ and K+ were measured by flame photometry and Mg+ and Ca+ by atomic absorption spectrophotometry. Cell membrane ATPase activity was determined by a colorimetric method. RESULTS: The hypertensive group consistently demonstrated depressed activity of each ATPase studied, with significantly lower serum Mg2+, serum K+, erythrocyte Mg2+ and platelet Mg2+ levels compared with the normotensive group. Platelet Na+ and Ca2+ and erythrocyte Ca2+ were significantly elevated in the hypertensive group. In the hypertensive group, mean arterial pressure (MAP) was inversely correlated with platelet and erythrocyte membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase. Serum Mg2+, serum Ca2+ and platelet Mg2+ were negatively correlated with MAP in the hypertensive group whilst erythrocyte and platelet Ca2+ were positively correlated. In the normotensive group, platelet Mg2+ and MAP were negatively, and erythrocyte Ca2+ and MAP, positively correlated. CONCLUSIONS: Black patients with essential hypertension have widespread depression of cell membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase activities with serum and intracellular Mg2+ depletion and cytosolic Na+ and Ca2+ overload, which may reflect an underlying membrane abnormality in essential hypertension. These cellular abnormalities may be related to the defective transport mechanisms that in turn may be aggravated by Mg2+ depletion.     

Isolation of a Chinese hamster ovary cell mutant defective in intramitochondrial transport of phosphatidylserine.

A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09-0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-[N-[6(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to approximately 40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.     

Demonstration and partial characterisation of phospholipid methyltransferase activity in bile canalicular membrane from hamster liver

BACKGROUND/AIMS: Methylation of phosphatidylethanolamine to phosphatidylcholine predominantly takes place in mitochondrial-associated membrane and the endoplasmic reticulum of the liver. The transport of the phospholipids from endoplasmic reticulum to the bile canalicular membrane is via vesicular and protein transporters. In the bile canalicular membrane a flippase enzyme helps to transport phosphatidylcholine specifically to the biliary leaflet. The phosphatidylcholine then enters the bile where it accounts for about 95% of the phospholipids. We postulated that the increased proportion of phosphatidylcholine in the bile canalicular membrane and the bile compared to the transport vesicles may be due to a methyltransferase activity in the bile canalicular membrane which, using s-adenosyl methionine as the substrate, converts phosphatidylethanolamine on the cytoplasmic leaflet to phosphatidylcholine, which is transported to the biliary leaflet. The aim of our study was to demonstrate and partially characterise methyltransferase activity in the bile canalicular membrane. METHODS: Organelles were obtained from hamster liver by homogenisation and separation by sucrose gradient ultracentrifugation. These, along with phosphatidylethanolamine, were incubated with radiolabelled s-adenosyl methionine. Phospholipids were separated by thin-layer chromatography and radioactivity was counted by scintigraphy. RESULTS: We demonstrated methyltransferase activity (nmol of SAMe converted/mg of protein/h at 37 degrees C) in the bile canalicular membrane of 0.442 (SEM 0.077, n=8), which is more than twice that found in the microsomes at 0.195 (SEM 0.013, n=8). The Km and pH optimum for the methyltransferase in the bile canalicular membrane and the microsomes were similar (Km 25 and 28 microM, respectively, pH 9.9 for both). The Vmax was different at 0.358 and 0.168 nmol of SAMe converted/mg of protein/h for the bile canalicular membrane and the microsomes, respectively. CONCLUSION: The presence of the methyltransferase activity in the bile canalicular membrane may be amenable to therapeutic manipulation.     

A unique phospholipid organization in bovine erythrocyte membranes

Ruminant erythrocytes are remarkable for their choline-phospholipid anomalies; namely, low or absent phosphatidylcholine (PC) along with high sphingomyelin levels. Here, we report another anomaly in bovine erythrocytes that affects aminophospholipids: phosphatidylethanolamine (PE) shows an extreme asymmetry, with only 2% of the total present in the outer leaflet. Furthermore, we found that phospholipase A(2), an enzyme located on the external surface of the erythrocytes, shows higher activity against PC than against PE. In addition, we observed that acylation of PE is by far the most important biosynthetic event in this system. We propose that deacylation of PE and PC by phospholipase A(2) to generate lysocompounds, followed by selective reacylation of lyso-PE in the inner leaflet, can account for the compositional and architectural peculiarities of bovine erythrocyte membranes.     

Sendai virus-induced hemolysis: reduction in heterogeneity of erythrocyte lipid bilayer fluidity.

Hemolysis of human or chicken erythrocytes by Sendai virus causes a change in the structure of the erythrocyte membrane lipid bilayer that can be detected by spin label electron spin resonance. In the intact erythrocyte, the phosphatidylcholine derivative spin label exists in a more rigid environment than the corresponding phosphatidylethanolamine label. Virus-induced hemolysis tends to abolish this difference in fluidity, i.e., the region of the phosphatidylcholine spin label becomes more fluid and that of the phosphatidylethanolamine spin label becomes more rigid. Fatty acid derivative spin labels, which may detect some "average" environment, show no change in fluidity. The fluidity change is detected at several different positions in the fatty acyl chain of the phosphatidylcholine spin label. Sendai virions grown in Madin-Darby bovine kidney (MDBK) cells or grown in eggs and harvested early, which lack hemolytic activity, cause no significant change in bilayer structure. Hemolytic activity and the ability to alter erythrocyte bilayer fluidity can be activated in MDBK-grown Sendai virions by trypsin treatment in vitro and in early-harvest egg-grown Sendai virions by freezing and thawing. Erythrocyte ghosts prepared by osmotic hemolysis and resealed by treatment with Mg2+ or elevated ionic strength exhibit a difference in fluidity between phosphatidylcholine and phosphatidylethanolamine spin labels, although less than that observed in whole cells. Incubation of resealed ghosts with Sendai virus abolishes the difference in fluidity. Unsealed ghosts that have been extensively washed show no heterogeneity in membrane bilayer fluidity, and incubation with Sendai virus causes no further fluidity change. Virus-induced hemolysis as measured by hemoglobin release is more sensitive to inhibition by Ca2+ than is the associated fluidity change in the bilayer.     

Erythrocyte membrane ATPase activity of G6PD-deficient individuals and the effect of primaquine metabolite(s) on membrane ATPase enzymes

Erythrocyte membrane Na+/K+, Ca2+/Mg2+ and Mg2+ ATPase activities in addition to the calmodulin-activated Ca2+/Mg2+ ATPase enzyme were measured in both G6PD-deficient and normal individuals. Although all three membrane ATPase activities were somewhat higher in the G6PD-deficient erythrocytes, only activated Ca2+/Mg2+ ATPase activity was significantly increased. The effect of primaquine on the membrane ATPases was also compared with other ATPase inhibitors. Primaquine was ineffective on erythrocyte membrane ATPase in-vitro. However, sera containing primaquine metabolite(s) were inhibitory to Ca2+/Mg2+ and Mg2+ ATPase systems of only G6PD-deficient erythrocytes. Other ATPase inhibitors showed a similar inhibitory effect in G6PD-deficient and normal erythrocytes, indicating a specific influence of primaquine on ATPase system in G6PD deficiency. It is suggested that this effect of primaquine may be an additional factor for haemolysis observed in the people with GdB- type of G6PD deficiency among the Mediterranean populations.     

Reconstitution of purified P-glycoprotein into liposomes

To study the molecular function of the multidrug-resistance gene product P-glycoprotein, we purified and reconstituted it into liposomes. Twelve detergents were examined in an attempt to solubilize and reconstitute the transport activity of K562/ADM membrane proteins containing P-glycoprotein. We found that transport activity was effectively reconstituted after solubilization with cholate, glycocholate and taurocholate. Other detergents, such as CHAPS, Triton X-100 and deoxycholate, diminished the transport activity. The K562/ADM membrane was solubilized by 1% glycocholate, and P-glycoprotein was purified by MRK-16 immunoaffinity column chromatography to a homogeneous single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified P-glycoprotein was reconstituted by detergent dialysis into liposomes composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The reconstituted P-glycoprotein specifically bound [³H]azidopine and had an ATPase activity that was slightlystimulated when vincristine was added. Furthermore, though its activity was reduced, the reconstituted P-glycoprotein was shown to be an ATP-dependent transporter of vincristine.     

Glutathione disulfide-stimulated Mg2+-ATPase of human erythrocyte membranes.

Inside-out erythrocyte membranes attached to polycationic beads manifested glutathione disulfide (GSSG)-stimulated ATPase activity. A Lineweaver-Burk plot of the ATPase activity as a function of GSSG concentration was biphasic and gave apparent Km values of 0.13 mM and 2.0 mM. These kinetics are similar to those reported for the ATP-requiring GSSG-transport systems in human erythrocytes and for the GSSG-stimulated ATPase activity in the plasma membranes of rat hepatocytes. Erythrocyte membranes that were depleted of extrinsic proteins were solubilized in 0.5% Triton X-100. Affinity chromatography on S-hexylglutathione-Sepharose 6B, with elution by a linear gradient of S-hexyl-glutathione, resulted in the resolution of two peaks of enzyme activity. One enzyme, which was eluted at approximately 0.5 mM S-hexylglutathione, had a high affinity for GSSG (apparent Km of 150 microM) and for ATP (80 microM). The other enzyme, which was eluted at approximately 1 mM S-hexylglutathione, had a low affinity for GSSG (apparent Km of 2.0 mM) and ATP (140 microM). GSSG-independent Mg2+-ATPase, Ca2+-dependent Mg2+-ATPase and Na+, K+-dependent Mg2+-ATPase were undetectable in the fractions. Addition of Ca2+, ouabain, or vanadate neither activated nor inhibited the activities, further indicating that the enzymes are distinguishable from ion-pumping ATPases. The enzymes required GSSG for activation; reduced glutathione (GSH) was ineffective. The ATPase activity of the high-Km enzyme was inhibited by addition of p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide and was activated by treatment with dithiothreitol, whereas the ATPase activity of the low-Km enzyme was not modified by these thiol reagents. The properties of the enzymes are similar to those of ATP-dependent GSSG-transport systems in human erythrocytes, suggesting that these ATPases may function in the active transport of GSSG.     

Phosphatidylserine transport in Rhnull erythrocytes

Phosphatidylserine transport in normal and Rhnull red blood cells was determined by measuring characteristic morphologic changes induced by synthetic phospholipids. Treating normal A+ cells with commercial anti-A antisera, anti-Rho(D) antisera, or with saturating concentrations of purified Rho(D) antibodies had no effect on phosphatidylserine transport. Normal B- cells treated with purified anti-B antibodies transported phosphatidylserine at rates equal to those of cells not treated with antibody. Rhnull cells, deficient in the protein bearing the Rho(D) antigen, incorporated dimyristoylphosphatidylcholine and dimyristoylphosphatidylserine at rates and to extents similar to normal cells. Furthermore, incorporated phosphatidylserine, but not phosphatidylcholine, was rapidly transported across the membrane bilayer. Energy depletion or treatment with sulfhydryl reagents inhibited phosphatidylserine transport equally in normal and Rhnull cells. These results indicate that, although Rhnull cells have numerous membrane defects, they are capable of adenosine triphosphate-dependent transport of exogenously added dimyristoylphosphatidylserine. Normal phosphatidylserine transport in the presence of anti-Rho(D) antibodies or in cells deficient in the Rho(D) polypeptide indicates that this protein is not the aminophospholipid transporter.