Cyclic AMP transport in human erythrocyte ghosts.

10(-5) M cyclic AMP has high permeability in human erythrocyte ghosts (p = 0.061-10(-6) cm.s-1). Saturation of influx and efflux occurs. Koizt = 4.43 mM. Voizt = 259.6 micron.min-1-Kiozt = 0.475 micron. Viozt = 28.3 micron.min-1 at 30 degrees C. Equilibrium exchange entry of cyclic AMP has similar kinetics to zero trans influx, though the system does show counterflow. Cytochalasin B is an apparent competitive inhibitor of cyclic AMP exit. (Ki = 3.9.10(-7) M). Control experiments indicated that cyclic AMP remains intact during incubation with red blood cell ghosts and is contained within the intravesicular space during the transport experiments.     

Fluidity of human erythrocyte ghosts.

The fluidity, defined by its two components, the order parameter, S, and the rotation correlation time, tau c, was studied on healthy human erythrocytes ghosts. We also measured ghost protein, cholesterol and phospholipid contents as well as acetylcholinesterase activities. No statistically significant difference was evidenced between erythrocyte ghosts from men and women. Whereas tau c values did not significantly vary among sample elements, variations of ghost order parameters about the mean were explained at 61% by changes in cholesterol contents and, to a lesser extent, in protein contents. No relationship was evidenced between ghost order parameter values and those of corresponding acetylcholinesterase activities. Liposomes prepared from ghost lipid extracts had much lower order parameter values than did corresponding ghosts. A few experiments were performed in the same way on ghosts from sickle blood. This disease appeared to decrease the bilayer lipid motionnal freedom as an increase of the order parameter values was evidenced.     

Phospholipid asymmetry in human erythrocyte ghosts

Using phospholipase digestion and the fluorescent probe merocyanine 540 the maintenance of phospholipid asymmetry in the plasma membrane of human erythrocyte ghosts was investigated. Digestion with phospholipase A2 indicated that ghosts prepared in the presence of Mg++ as the only divalent cation retained the normal phospholipid asymmetry characteristic of intact erythrocytes. These ghosts, like normal erythrocytes, also failed to stain with merocyanine 540. However, the presence of as little as 5-10 microM Ca++ during ghost preparation resulted in ghosts in which lipid asymmetry had been abolished, as indicated by phospholipase digestion. Moreover, these ghosts stained with merocyanine 540. In contrast to ghosts, intact erythrocytes treated with ionophore required millimolar levels of Ca++ ions to disrupt membrane lipid asymmetry. To discover the reason for this difference in behavior between ghosts and intact cells, ghosts were prepared from preswollen cells using only small volumes of buffer for lysis. These experiments demonstrated that as the cellular contents of erythrocytes are diluted, the asymmetric arrangement of phospholipids becomes more sensitive to disruption by Ca++.     

Calcium efflux from human erythrocyte ghosts

Summary The passive Ca efflux from human red cell ghosts was studied in media of differing ion compositions and compared to the ATP-dependent Ca efflux. Cells were loaded with⁴⁵Ca during reversible hemolysis, and the loss of radioactivity into the non-radioactive incubation medium was measured, usually for 3 hr at 37°C. Analysis of the efflux curves revealed that⁴⁵Ca efflux followed the kinetics of a simple two-compartment system. In the concentration range between 0 and 1mm Ca in the external solution ([Ca⁺⁺] _o ), the rate constant of passive Ca efflux (k min^–1, fraction of⁴⁵Ca lost per minute into the medium) increased from 0.00732 to 0.0150 min^–1. There was no further increase at higher [Ca⁺⁺] _o . The relation between the rate constant of Ca efflux and [Ca⁺⁺] _o is thus characterized by saturation kinetics. The passive transfer system for Ca could also be activated by Sr. The alkali metal ions Na, K and Li did not seem to have any significant influence on passive Ca transfer. The passive Ca efflux was slightly inhibited by Mg and strongly inhibited by Pb. Under most experimental conditions, a fraction of 15 to 50% of the intracellular Ca seemed to be inexchangeable. The inexchangeable fraction decreased with increasing [Ca⁺⁺] _o and increased with increasing [Ca⁺⁺] _i . It was not influenced by alkali metal ions, CN or Pb, but it could be completely removed from the cells by the addition of 0.1mm Mersalyl to the incubation medium or by hemolysis with addition of a detergent. The active ATP-dependent Ca transport differed characteristically from passive transfer; the rate constant decreased with increasing [Ca⁺⁺] _o , and the inexchangeable Ca fraction increased with increasing [Ca⁺⁺] _o . The experimental results suggest that there exists a carrier-mediated Ca–Ca exchange diffusion in the erythrocyte membrane and that only a fraction of the ghost cell population participates in the Ca exchange diffusion.     

Effect of phloretin on monosaccharide transport in erythrocyte ghosts

Summary ³H-labelled phloretin was shown to be bound reversibly by human erythrocyte and ghost membranes but not to penetrate across them in either direction. Kinetic parameters ofd-xylose andd-galactose transport in intact cells and in ghosts, as well as the inhibition by phloretin of these transports were found to be in fair agreement. By enclosing phloretin in ghosts, its inhibition of monosaccharide transport was found to be symmetrical and thus an equivalence of the outer and the inner membrane sides of the human erythrocyte was demonstrated.     

Ultrastructure of hemoglobin-depleted human erythrocyte resealed ghosts

Both negative-stain and freeze-fracture electron microscopic techniques revealed that the ultrastructure of resealed white ghosts prepared at high dilution during the hemolysis step is very different from that of resealed ghosts prepared at low or moderate dilution (pink ghosts). The negative-stained resealed white ghosts showed light halo substructures on membrane surfaces and protrusions at the edge of the ghosts. Freeze-fracturing of these ghosts showed that membrane blebbing had occurred and that fragments of the membranes resealed to form small right-side-out vesicles ranging from 0.1 to 0.3 μm in diameter.     

Spin probe clustering in human erythrocyte ghosts

Summary A model has been developed for 5-nitroxide stearate, I(12,3), distribution in human erythrocyte ghosts which accurately predicts ESR spectral alterations observed with increased probe/total lipid (P/L) at 37°C. This spin probe occupies a class of high-affinity, noninteracting sites at low loading. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membranebound clusters of variable size. No low probe remains at high P/L where all I(12,3) clusters in a concentrated phase. This model allows determination of the dilute/clustered probe ratio, and shows that I(12,3) segregates in erythrocytes at what might otherwise be considered low P/L (e.g., 1/359). These findings validate the earlier use of empirical parameters to estimate probe sequestration in biological membranes.     

Spectrin phosphorylation and shape change of human erythrocyte ghosts

Human erthrocyte membranes in isotonic medium change shape from crenated spheres to biconcave disks and cup-forms when incubated at 37 degrees C in the presence of MgATP (M. P. Sheetz and S. J. Singer, 1977, J. Cell Biol. 73:638-646). The postulated relationship between spectrin phosphorylation and shape change (W. Birchmeier and S. J. Singer, 1977, J. Cell Biol. 73:647-659) is examined in this report. Salt extraction of white ghosts reduced spectrin phosphorylation during shape changes by 85-95%. Salt extraction did not alter crenation, rate of MgATP-dependent shape change, or the fraction (greater than 80%) ultimately converted to disks and cup-forms after 1 h. Spectrin was partially dephosphorylated in intact cells by subjection to metabolic depletion in vitro. Membranes from depleted cells exhibited normal shape-change behavior. Shape-change behavior was influenced by the hemolysis buffer and temperature and by the time required for membrane preparation. Tris and phosphate ghosts lost the capacity to change shape after standing for 1-2 h at 0 degrees C. Hemolysis in HEPES or N- tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid yielded ghosts that were converted rapidly to disks in the absence of ATP and did not undergo further conversion to cup-forms. These effects could not be attributed to differential dephsphorylation of spectrin, because dephosphorylation during ghost preparation and incubation was negligible. These results suggest that spectrin phosphorylation is not required for MgATP-dependent shape change. It is proposed that other biochemical events induce membrane curvature changes and that the role of spectrin is passive.     

ATPase activity and transport by a cGMP transporter in human erythrocyte ghosts and proteoliposome-reconstituted membrane extracts

We previously described the [(3)H]cGMP-binding characteristics of a CHAPS-solubilized protein that we proposed to be a cGMP transporter. We now report the ATPase activity of the membrane-bound, solubilized and reconstituted form of a cGMP transporter. The membrane-bound protein of unsealed ghosts had a linear ATPase activity over a 120 min incubation period with optimal activity of about 400 pmol/mg/min. The apparent K(m) and V(max) for ATP were about 0.5 mM and 300 pmol/mg/min, respectively. When solubilized with CHAPS the specific activity of the protein was reduced to about 70 pmol/mg/min. Reconstitution of the CHAPS preparation into phospholipid bilayer using rapid detergent removal by Extracti-gel column resulted in proteoliposomes which had ATPase activity similar to that found in the erythrocyte membranes. The proteoliposomes displayed a linear ATP-dependent uptake of [(3)H]cGMP with an apparent K(m) value of 1. 0 microM. This low K(m)-uptake of [(3)H]cGMP in proteoliposomes was not affected by 10 microM of AMP, cAMP and GMP, but was completely abolished in the presence of the non-hydrolyzable ATP analogue, ATP-gamma-S. Some ATPase activation was also observed in the presence of 2 microM cAMP, but it is unclear whether this activity was coupled to the cGMP transporter. Our results show that the membrane protein responsible for cGMP transport has an ATPase activity and transports the cyclic nucleotide in the presence of ATP.     

The effect of the strongly bound protein fraction on sugar transport in human erythrocyte ghosts

The protein fraction released from human erythrocyte membranes with 90% acetic acid enhanced the transport of several sugar species when enclosed in erythrocyte ghosts. Both the influx and the efflux of d-glucose were increased so that permeation rather than sugar binding was involved. The permeation increase was selective, being found with d-glucose, d-galactose and d-xylose but not with l-glucose or lactose. The protein-dependent sugar transport was saturable and the incorporation of proteins into the ghost membrane brought J_max to the level corresponding to intact erythrocytes, leaving K_m unchanged.     

Anion transport in single erythrocyte ghosts measured by fluorescence microphotolysis

Fluorescence microphotolysis was used to measure in single resealed human erythrocyte ghosts the band 3-mediated transport of the fluorescent anion N-(7-nitrobenzofurazan-4-yl)-taurine (NBD-taurine). Transport was reduced to less than 5% of the control by the specific inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). The accuracy of the determination of the rate constant for NBD-taurine influx was approximately +/- 15% as calculated from repetitive measurements in individual ghosts. The sample population distribution of the rate constant was slightly skewed towards values larger than the mean. The rate of NBD-taurine transport showed an optimum near pH 7. The Arrhenius plot was linear in the range from 28.5 degrees C to 51 degrees C with an apparent activation enthalpy of 21.4 kcal/mol.     

Effect of hemoglobin A and S on human erythrocyte ghosts

The interaction of erythrocyte ghosts and vesicles with chromatographed hemoglobin (Hb) A and Hb S was studied under various conditions. Although no binding of either Hb A or Hb S to inside-out vesicles was detected, under conditions of physiological ionic strength and pH, several properties of white membrane ghosts were effected by the presence of Hb. Addition of Hb A and Hb S (2 g/dl) to membrane ghosts in 6 mM MgATP, 150 mM NaCl, 10 mM Tris-HCl buffer, pH 7.4, was found to effect the echinocyte-discocyte transition, the extent of endocytosis, the volume, and the sealing of ghosts. Our observations suggest that the structure of membrane ghosts is influenced by cytosol proteins and that the environment of the red cell membrane plays an important role in the definition and the control of the membrane structure and function.