Proteolytic activity of human erythrocyte membrane during ghosts preparation

1. Proteins in human erythrocyte membranes after red blood cells hemolysis revealed relatively high rate of self-digestion. 2. This indicates hemolysis as a critical moment for membrane proteases activation. 3. The detailed pattern of band 3 protein and spectrin degradation during ghosts preparation was more complicated and reflected both the changes in proteolytic susceptibility and extraction of some proteases. 4. Further extraction of membrane proteins by alkali stripping resulted in an increase in the self-digestion rate and decrease in the degradation rate of an exogenous substrate.     

Cyclic AMP transport in human erythrocyte ghosts

10^?5 M cyclic AMP has high permeability in human erythrocyte ghosts ($\text{p = 0.061 · 10}{}^{\mathrm{?6}}\text{cm}\text{·}\text{s}{}^{\mathrm{?1}}$). Saturation of influx and efflux occurs. $\text{K}{}_{\mathrm{<mtext>z</mtext><mtext>t</mtext>}}{}^{\mathrm{<mtext>oi</mtext>}}\text{= 4.43}\text{mM}$. $\text{V}{}_{\mathrm{zt}}{}^{\mathrm{oi}}\text{= 259.6 μ}\text{M · min}{}^{\mathrm{?1}}$. $\text{K}{}_{\mathrm{<mtext>z</mtext><mtext>t</mtext>}}{}^{\mathrm{<mtext>io</mtext>}}\text{= 0.475 μ}\text{M}$. $\text{V}{}_{\mathrm{<mtext>z</mtext><mtext>t</mtext>}}{}^{\mathrm{<mtext>io</mtext>}}\text{= 28.3 μ}\text{M · min}{}^{\mathrm{?1}}$ at 30°C. Equilibrium exchange entry of cyclic AMP has similar kinetics to zero trans influx, though the system does show counterflow. Cythochalasin B is an apparent competitive inhibitor of cyclic AMP exit. ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 3.9 · 10}{}^{\mathrm{?7}}\text{M}$).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.     

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.     

Alteration of membrane conductivity and fluidity in human erythrocyte membranes and erythrocyte ghosts following gamma-irradiation

Alterations of electrical properties of human erythrocyte membranes induced by gamma irradiation have been studied by means of conductivity measurements in the frequency range from 10 KHz to 100 MHz. The results clearly demonstrate the role played by haemoglobin in the structural modification of the membrane produced by gamma irradiation. Further support for this point of view has been derived from electron spin resonance measurements carried out on the same samples, labelled with different spin labels which probe the outer half layer of membrane at different penetration levels.     

Effects of calcium on potassium and water transport in human erythrocyte ghosts

Bulk water transport in reconstituted ghosts is statistically comparable to that in the parent red cells, and is unaffected by incorporation of Ca²⁺ over the range of 0.01 to 1 mM. Brief exposure of ghosts to p-chloromercuribenzene sulfonate results in a supression of osmotic water flow but leaves K⁺ permeability unchanged. Incorporation of p-chloromercuribenzene sulfonate provokes extremely rapid K⁺ loss which can be counteracted by simultaneous inclusion of Ca²⁺.Erythrocyte ghosts, when prepared with a small amount of Ca²⁺, demonstrate recovery of normal impermeability to choline, sucrose, Na⁺ and inulin and have an improved K⁺ retention over Ca²⁺-free preparations.The rate of passive transport of K⁺ from unwashed erythrocyte ghosts was measured during the initial few minutes of efflux. The initial rates vary in a bimodal fashion with the concentration of Ca²⁺ incorporated at the time of hemolysis. In low concentrations (0.01–0.1 mM), Ca²⁺ protects the K⁺ barrier while at higher concentrations (0.1–1.0 mM) it provokes a K⁺ leakage ranging from 7 to 50 times the normal rate of passive K⁺ loss. The Ca²⁺-induced K⁺ leak is thus a graded response rather than a discrete membrane transport state. The transition from a Ca²⁺-protected to a Ca²⁺-damaged membrane occurs upon an increase in Ca²⁺ concentration of less than 50 μmoles/l.     

Hemoglobin-depleted human erythrocyte ghosts: Characterization of morphology and transport functions

Summary A method of preparing hemoglobin-depleted resealed ghosts with an extremely low hemoglobin content is described. The membrane morphology, the crossed immunoelectrophoresis pattern of the membrane proteins, and the transport function of these ghosts have been examined.Electron microscopic examination of the ghosts on hydrophilic as well as hydrophobic grid surfaces revealed a faint filamentous network (spectrin) associated with the membrane. The ghosts were found to have permeabilities towards small polar molecules (water and mannitol) and ions (chloride, sodium, and potassium) which are quantitatively very close to those of intact red cells.It is concluded that white ghosts prepared by the present method are well suited for simultaneous studies of morphology, membrane biochemistry, and membrane transport functions.     

A spectrin-dependent ATPase of the human erythrocyte membrane

Removal of spectrin from erythrocyte membranes results in the simultaneous loss of a calcium-stimulated, magnesium-dependent ATPase with an apparent KD for Ca2+ of 1 microM. This ATPase activity with high Ca2+ affinity is specifically reconstituted by addition of purified spectrin to spectrin-depleted membranes, and the reconstituted activity is directly proportional to the amount of spectrin that is reassociated with the membranes. Spectrin binding and activation of the high Ca2+ affinity Mg2+-ATPase are proportionally inhibited by thermal denaturation, trypsin digestion, or treatment of the membranes with thiol-reactive reagents. Binding of calmodulin to the Ca2+ pump ATPase requires that calmodulin contains bound ca2+. By contrast, spectrin binding to the erythrocyte membrane is Ca2+-independent. Direct assay of calmodulin is purified spectrin and absence of chlorpromazine inhibition of reconstitution demonstrate that activation of the high Ca2+ affinity ATPase resulting from spectrin binding is not a result of contamination of spectrin by calmodulin. Additional evidence that the spectrin-activated ATPase is an entity separate and distinct from the Ca2+ pump is provided by other characteristics of the activation phenomenon. It is suggested that spectrin constitutes part of an ATPase which may function as a component of the "cytoskeleton" controlling erythrocyte shape and membrane flexibility.     

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.     

Ca-2+-stimulated membrane phosphorylation and ATPase activity of the human erythrocyte.

1. Human erythrocyte membranes were preincubated with ethyleneglycolbis-(beta-aminoethyl)-N,N' tetraacetate (EGTA) and subsequently labelled for short periods with micromolar concentrations of [8-3-H, gamma-32-P]ATP. Under these conditions, and at temperatures smaller than or equal to 22 degrees C, both ATP hydrolysis and membrane phosphorylation were stimulated by Ca-2+. 2. The properties of the Ca-2+-stimulated ATP hydrolysis and associated phosphorylation of a 150 000 molecular weight protein component, previously described (Knauf, P. A., Proverbio, F. and Hoffman, J. F. (1974) J. Gen. Physiol. 63, 324-336), have been studied. The behavior of the phosphorylated component, ECaP, has properties consistent with its role as a phosphorylated intermediate of Ca-2+-ATPase activity, including: (1) similar dependence of the steady-state level of ECaP and Ca-2+-ATPase on ATP concentration; (2) rapid turnover apparent upon the addition of excess non-radioactive ATP; and (3) good correlation between the steady-state levels of Ca-2+-dependent phosphorylation and Ca-2+-ATPase activity in separate preparations possessing variable specific activity. Addition of excess EGTA to ECaP caused only partial dephosphorylation. Sensitivity of Ca-2+-stimulated ATP hydrolysis and associated phosphorylation to micromolar concentrations of Ca-2+ implicates this activity in the "high-affinity" Ca-2+-pump system of the human erythrocyte (Schatzmann, H. J. (1973) J. Physiol. London 235, 551-569).     

Glycosylation of the human erythrocyte glucose transporter is essential for glucose transport activity

The human erythrocyte glucose transporter is a fully integrated membrane glycoprotein having only one N-linked carbohydrate chain on the extracellular part of the molecule. Several authors have suggested the involvement of the carbohydrate moiety in glucose transport, but not definitive results have been published to date. Using transport glycoproteins reconstituted in proteoliposomes, kinetic studies of zero-trans influx were performed before and after N-glycanase treatment of the proteoliposomes: this enzymatic treatment results in a 50% decrease of the Vmax. The orientation of transport glycoproteins in the lipid bilayer of liposomes was investigated and it appears that about half of the reconstituted transporter molecules are oriented properly. Finally, it could be concluded that the release of the carbohydrate moiety from the transport glycoproteins leads to the loss of their transport activity.     

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++.