Asymmetric binding of cytochrome b5 to the membrane of human erythrocyte ghosts

The intact, amphipatic form of cytochrome $\text{b}{}_5$ could bind to unsealed ghosts, but not to resealed ghosts, suggesting that the cytochrome could bind only to the inner (cytoplasmic) surface of the ghost membrane. This was further confirmed by the finding that the cytochrome could bind to closed, inside-out vesicles prepared from the ghosts. This asymmetric binding was not due to the exclusive localization of sialic acid and sugar chains on the outer surface of the ghosts membrane, because the cytochrome could not bind to ghosts even after enzymatic removal of these components. Although liposomes consisting of phosphatidylcholine or both phosphatidylcholine and sphingomyelin could effectively bind the cytochrome, this binding capacity was progressively decreased as increasing amount of cholesterol was included in the composition of phosphatidylcholine liposomes. Removal of cholesterol from resealed ghosts by incubation with egg phosphatidylcholine liposomes resulted in the binding of cytochrome $\text{b}{}_5$ to the outer surface of the treated ghosts. The possibility is discussed that the asymmetric binding is due to preferential localization of cholesterol in the outer leaflet of the lipid bilayer that constitutes the ghost membrane.     

Modulation of the organization of erythrocyte membrane phospholipids by cytoplasmic ATP. The susceptibility of isoionic human erythrocyte ghosts to attack by detergents and phospholipase C

Human erythrocyte ghosts were prepared in media of physiological ionic composition, and these “isoionic” ghosts were then lysed and resealed in media of varying Ca²⁺, Mg²⁺ and ATP concentrations. The susceptibilities of these ghosts to limited attack by various detergents and by phospholipases C were then compared with the susceptibilities of intact cells to similar attack: attack was assessed by measurements of lysis and of phospholipid hydrolysis. Ghosts were more readily attacked than cells by anionic detergents (cholate, glycocholate, dodecyl sulphate) and by phospholipases C, but Triton X-100 and cetyltrimethylammonium attacked cells and ghosts to the same extent. Mg · ATP^2? partially protected ghosts from attack by the anionic detergents and by the phospholipases C of Bacillus cereus and of Clostridium perfringens. Protection by Mg · ATP^2? occurred only if Mg · ATP^2? had access to the cytoplasmic surface of the membrane. Adenylyl(β-γ-methylene)diphosphonate, a non-hydrolysable ATP analogue, protected as effectively as did Mg · ATP^2?. Internal Mg · ATP^2? caused a marked reduction in the hydrolysis by phospholipases of phosphatidylethanolamine and sphingomyelin, but had no appreciable effect upon the simultaneous hydrolysis of phosphatidylcholine. It therefore seems that interaction of ATP with sites on the cytoplasmic surface of the erythrocyte membrane can, without ATP hydrolysis, cause changes in the organization of the outer surface of the membrane that specifically render phosphatidylethanolamine and sphingomyelin less accessible to attack by extracellular phospholipases.     

The effects of Ca2+ and guanylnucleotides on isoprenaline-stimulated cyclic AMP formation in rat reticulocyte ghosts

We have studied β-adrenergic stimulation of cyclic AMP formation in fragmented membranes and in unsealed or resealed ghosts prepared from rat reticulocytes. The maximal rate of isoprenaline-stimulated cyclic AMP formation with saturating MgATP concentrations and in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine was 5–8 nmol/min per ml ghosts are remained constant for at least 15 min. Transition from resealed ghosts to fragmented membranes was associated with a shift of the activation constant (K_a) for (±)-isoprenaline from 0.1 to 0.6 μM. The apparent dissociation constant for propranolol (0.01 μM) remained unchanged. The K_a values for isoprenaline in native reticulocytes and in resealed ghosts were identi The stimulating effect of NaF on cyclic AMP formation in resealed ghosts reached 15% of maximal β-adrenergic stimulation. Cyclic AMP formation, both in fragmented membranes and in ghosts, was half-maximally inhibited with Ca²⁺ concentrations ranging between 0.1 and 1 μM. GTP stimulated iosprenaline-dependent cyclic AMP formation in unsealed ghosts and in fragmented reticulocyte membranes by a factor of 3–5 but did not change the K_a value for isoprenaline. K_a values for the guanylnucleotides in different experiments varied between 0.3 and 2 μM. Ca²⁺ concentrations up to 4.6 μM reduced the maximal activation by GTP and Gpp(NH)p but did not affect their K_a values. Compared to GTP, maximal activation by Gpp(NH)p was higher in fragmented membranes, but much lower in ghosts. Our results suggest that the native β-receptor adenylate cyclase system of reticulocytes is more closely approximated in the ghost model than in fragmented membrane preparations. Membrane properties seem to modulate the actions of guanylnucleotides on isoprenaline-dependent cyclic AMP formation in ghosts. Some of these effects are not observed in isolated membranes.     

Transmembrane distribution of sterol in the human erythrocyte

The transbilayer cholesterol distribution of human erythrocytes was examined by two independent techniques, quenching of dehydroergosterol fluorescence and fluorescence photobleaching of NBD-cholesterol. Dehydroergosterol in conjunction with leaflet selective quenching showed that, at equilibrium, 75% of the sterol was localized to the inner leaflet of resealed erythrocyte ghosts. NBD-cholesterol and fluorescence photobleaching displayed two diffusion values in both resealed ghosts and intact erythrocytes. The fractional contribution of the fast and slow diffusion constants of NBD-labelled cholesterol represent its inner and outer leaflet distribution. At room temperature the plasma membrane inner leaflet of erythrocyte ghosts as well as intact erythrocytes cells contained 78% of the plasma membrane sterol. The erythrocyte membrane transbilayer distribution of sterol was independent of temperature. In conclusion, dehydroergosterol and NBD-cholesterol data are consistent with an enrichment of cholesterol in the inner leaflet of the human erythrocyte.     

A flow EPR study of deformation and orientation characteristics of erythrocyte ghosts: Effects of lysing and resealing conditions

Summary The effects of various conditions in lysing and resealing the red cell membrane on the degree of ghost deformation and orientation in flow are investigated using the flow EPR and spin-label method. The relatively low deformability of the standard ghost, which is lysed and resealed, respectively, in hypotonic and isotonic NaCl-Tris buffer, is markedly enhanced by the presence of Mg-ATP, chlorpromazine, or Ca²⁺ ion during resealing. The effect is concentration dependent, and there is an optimal level for each treatment. Chlorpromazine and Ca²⁺ are also effective when added to the resealed ghosts. Mg²⁺ ion shows an opposite effect reducing the ghost deformability in flow at all concentrations. An isotonic lysis in NH₄HCO₃ solution with less osmotic stress substantially raises ghost deformability above that of the standard ghosts. These results are interpreted on the basis of a misalignment between the bilayer leaflets that is probably brought about during hypotonic lysis and its recovery to the nearly normal bilayer state by the agents used during or after resealing. The novel finding of deformability enhancing effect of calcium is assumed to be caused by the electrostatic expansion of the inner layer relative to the outer leaflet. The explanations are supported by the resealed ghost shapes observed before and after the treatments; shape recovery from the monoconcave spheroid toward biconcave discoid is observed in most cases concomitantly with improvements of flow characteristics.     

Transmembrane Ca2+ gradient is essential for high anion transport activity of human erythrocytes

The role of a transmembrane Ca²⁺ gradient in anion transport by Band 3 of human resealed erythrocyte ghosts has been studied. The results show that a transmembrane Ca²⁺ gradient is essential for the conformation of erythrocyte Band 3 with higher anion transport activity. The dissipation of the transmembrane Ca²⁺ gradient by the ionophore A23187 inhibits the anion transport activity. The extent of this inhibition approaches 90% as the Ca²⁺ concentration on both sides of the ghost membrane is increased to 1.0 mM and half-maximum inhibition is observed at 0.25 mM Ca²⁺. Addition of ATP (0.4 mM) to the resealing medium can partly reestablish the transmembrane Ca²⁺ gradient by activation of Ca²⁺-ATPase and alleviate the inhibition to some extent. N-ethylmaleimide, an inhibitor of erythrocyte Ca²⁺-ATPase, prevents such restoration. Electron micrographs reveal that numerous larger intramembranous particles can be observed on the P-faces of freeze-fractured resealed ghosts in the absence of a transmembrane Ca²⁺ gradient.Abbreviations DPA dipicolinic acid - EITC eosin 5-isothiocyanate - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - TES N-Tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid - PMSF phenylmethyl-sulfonylfluoride - NEM N-ethylamaleimide - BSA bovine serum albumin - EGTA ethyleneglycol-bis (aminoethylether)-tetra-acetic acid - EITC-Band 3 Band 3 labeled with EITC - Ca_i Ca²⁺ inside resealed ghosts - Ca_o Ca²⁺ outside resealed ghosts     

The limited depletion of cholesterol from erythrocyte membranes on treatment with incubated plasma

About 35% of the cholesterol of human erythrocyte membranes can be removed by the “preincubated plasma” technique (Murphy, J.R. (1962) J. Lab. Clin. Med. 60, 86–109), in which erythrocytes are extracted with plasma that has been preincubated to esterify a portion of its lipoprotein cholesterol. The limitation on the cholesterol depletion is shown not to be a result of insufficient plasma capacity to take up additional cholesterol or of changes in the plasma during the extraction.The maximum cholesterol depletion from “ghosts” was the same as that from whole cells. “Inside-out” membrane vesicles (Steck, T.L. (1974) in Methods in Membrane Biology (Korn, E., ed.), Vol. 2, pp. 245–281) were utilized to determine if the limitation to cholesterol depletion is a result of the remaining cholesterol being 1located at the membrane inner surface and therefore not accessible to the plasma. No further cholesterol depletion occurred when “inside-out” vesicles, prepared from erythrocytes which were depleted of cholesterol by the usual method, were extracted. Also, “inside-out” vesicles prepared from untreated erythrocytes gave the same cholesterol depletion as is usually attained.The maximal cholesterol depletion was unaffected by a number of modifications of the extracting preincubated plasma: addition of lysolecithin or albumin, dialysis against isotonic buffer, and variation in pH of the preincubated plasma from 6.0 to 9.0.It is concluded that the limitation on the cholesterol depletion is a result of a firm binding of the remaining cholesterol.     

THE LOCALIZATION OF Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE ON RED CELL GHOST MEMBRANES

The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO₃)₂ resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb²⁺ and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of P_i on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis P_i is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase.     

The effects of Ca and guanylnucleotides on isoprenaline-stimulated cyclic AMP formation in rat reticulocyte ghosts

We have studied β-adrenergic stimulation of cyclic AMP formation in fragmented membranes and in unsealed or resealed ghosts prepared from rat reticulocytes. The maximal rate of isoprenaline-stimulated cyclic AMP formation with saturating MgATP concentrations and in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine was 5–8 nmol/min per ml ghosts are remained constant for at least 15 min. Transition from resealed ghosts to fragmented membranes was associated with a shift of the activation constant (K_a) for (±)-isoprenaline from 0.1 to 0.6 μM. The apparent dissociation constant for propranolol (0.01 μM) remained unchanged. The K_a values for isoprenaline in native reticulocytes and in resealed ghosts were identi The stimulating effect of NaF on cyclic AMP formation in resealed ghosts reached 15% of maximal β-adrenergic stimulation. Cyclic AMP formation, both in fragmented membranes and in ghosts, was half-maximally inhibited with Ca²⁺ concentrations ranging between 0.1 and 1 μM. GTP stimulated iosprenaline-dependent cyclic AMP formation in unsealed ghosts and in fragmented reticulocyte membranes by a factor of 3–5 but did not change the K_a value for isoprenaline. K_a values for the guanylnucleotides in different experiments varied between 0.3 and 2 μM. Ca²⁺ concentrations up to 4.6 μM reduced the maximal activation by GTP and Gpp(NH)p but did not affect their K_a values. Compared to GTP, maximal activation by Gpp(NH)p was higher in fragmented membranes, but much lower in ghosts. Our results suggest that the native β-receptor adenylate cyclase system of reticulocytes is more closely approximated in the ghost model than in fragmented membrane preparations. Membrane properties seem to modulate the actions of guanylnucleotides on isoprenaline-dependent cyclic AMP formation in ghosts. Some of these effects are not observed in isolated membranes.     

Studies on carnitine palmitoyl transferase: The similar nature of CPTI (inner form) and CPTO (outer form)

Rat liver mitochondrial carnitine palmitoyl transferase (CPT) was found to reside in two mitochondrial locations. Twenty to twenty-five percent of the total CPT activity was easily released and solubilized by digitonin. This activity appeared to be the outer form of CPT (CPT_O). The remainder of the activity or the inner CPT (CPT_I) was tightly membrane bound. Trypsin digestion of the digitonin prepared mitoplast did not affect residual CPT activity indicating that this activity probably resided on the inner side of the membrane. Following their separation by digitonin treatment, CPT_O and CPT_I were partially purified 14.7-and 16.7-fold, respectively. The purification of each enzyme involved extraction from the membrane with Tween 80, ammonium sulfate fractionation, gel filtration, and another ammonium sulfate fractionation. The partially purified CPT_O and CPT_I were found to have identical elution volumes from a G-200 column corresponding to a molecular weight of 430,000. Also they both were found to have nearly identical K_m values for palmitoyl-CoA, palmitoyl-carnitine, CoA, and carnitine suggesting they were identical enzymes. The V values could not be compared due to differences in purity, but the ratio of V in the forward direction to V in the reverse direction was identical for CPT_I and CPT_O again suggesting enzyme identity. Assay of the CPT system “in situ” by following the reduction of the acyl-CoA dehydrogenase, a flavoprotein, suggested that the activity of CPT_I was 450-fold greater than the activity of CPT_O when both were present in the intact membrane. These data suggest that “in situ” factors exist which greatly change the catalytic properties of CPT_I compared to CPT_O.     

The role of interactions between O2, H2O2, .OH,e- and O2- in free radical damage to biological systems

The occurrence of the Haber-Weiss reaction and other interactions between free radicals has been investigated in the effects of mixtures of free radicals on the permeability of resealed erythrocyte ghosts and on the activity of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. The following mixtures were found to induce damage greater than that which could be accounted for by the independent actions of the constituent free radicals: (i) · OH + H₂O₂, and (ii) · OH + H₂O₂ + O₂^?. In contrast, the following mixtures were found to induce less damage than that predicted on the basis of independent actions of constituent free radicals: (i) H₂O₂ + O₂^?, and (ii) oxidizing radicals ( · OH, H₂O₂) + reducing radicals (e^?, H · ). These results suggest a Haber-Weiss-like interaction between H₂O₂ and O₂^?and an interaction between H₂O₂ and · OH to produce a species more potent than either in causing increased permeability. The decrease in damage observed in the simultaneous presence of oxidizing and reducing radicals suggests an antagonistic effect by which each tends to moderate damage by the other. Inactivation of glyceraldehyde-3-phosphate dehydrogenase was found to be more sensitive to radiation than permeability by an order of magnitude, while permeability was more sensitive to the enhancement of damage by oxygen. Comparison of the effectiveness of free radical scavengers in inhibiting the increase in permeability caused by free radicals showed the following order of effectiveness, expressed in terms of percentage protection: formate (90%) > nitrogen (65%) > catalase (60%) > dismutase (32%), and with respect to enzymatic inactivation, nitrogen (100%) > formate (77%) > dismutase (48%) > catalase (44%). The relative rates observed anaerobically and aerobically in the presence and absence of the above scavengers suggest that (at least in the case of radiation damage to the membranes of erythrocyte ghost cells) the “oxygen effect” is due to the interaction of oxygen with e^? and H., producing O₂^? which aggravates damage under conditions which allow consequent Haber-Weiss-like reactions. The further increase in damage when oxygen concentration is raised yet higher is due to the interaction of oxygen with the sites of initial damage.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca2+/Mg2+)-ATPase,and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Carbohydrate composition of cells and plasma membranes of Distyostelium discoideum at selected stages of development

Cells of Distyostelium discoideum representing four developmental stages were atuo-analysed for constituent monosaccharides and their compositions compared. Rhamnose, ribose, fucose, glucose, mannose, galactose, glucosamine, galactosamine and an unidentified sugar were recovered after hydrolysis in 2 M HCl for 2 h at 100°C. The relative proportions of the individual sugars were found to vary as a function of development. The largest variations were in the proportions contributed by galactose (from 2% of vegetative cell carbohydrate to 12% of the carbohydrate of fruiting bodies) and galactosamine (present in measurable quantity only in fruiting bodies).Plasma membrane “ghosts” were found to have the same monosccharide constituents as whole cells, but in different proportions. Mannose contributed over 24% of the total carbohydrate recovered from aggregating cell “ghosts”, but only 13% of carbohydrate recovered from “ghosts” prepared from vegetative cells. Galactose was the most abundant sugar recovered from vegetative “ghosts”, and was second only to mannose in aggregating “ghosts”.     

Inhibition of the binding of cytochrome b5 to phosphatidylcholine vesicles by cholesterol

The binding of cytochrome b₅ to single-walled liposomes of egg phosphatidylcholine was inhibited by the presence of cholesterol in the lipid bilayer under conditions where a limited amount of liposomes was incubated with the cytochrome. Since similar conditions seem to apply for the binding of cytochrome b₅ to erythrocyte ghosts, this observation supports the conclusion of Enomoto and Sato (Enomoto, K. and Sato, R. (1977) Biochim. Biophys. Acta 466, 136–147) that the localization of cholesterol on the outer surface of the ghost membrane prevents the binding of cytochrome b₅ to this surface. The finding reported by Roseman et al. (Roseman, M.A., Holloway, P.W. and Calabro, M.A. (1978) Biochim. Biophys. Acta 507, 552–556) that cholesterol did not prevent the cytochrome binding to phosphatidylcholine liposomes in the presence of a large excess of liposomes could be confirmed in the present study, but this does not contradict the abovementioned conclusion.     

Cytochrome P-450 from mitochondria of bovine adrenal cortex Comparison of cholesterol side-chain cleavage P-450 with steroid 11β-hydroxylation P-450 and immunochemical cross-reactivity between adrenal mitochondrial and liver microsomal cytochromes P-450

Adrenocortical mitochondrial cytochrome P?450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11β-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0°C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11β-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H₂O₂, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents.Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450_LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450_LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450.     

Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase

Cytochrome c oxidase is a multisubunit membrane-bound enzyme, which catalyzes oxidation of four molecules of cytochrome c²⁺ and reduction of molecular oxygen to water. The electrons are taken from one side of the membrane while the protons are taken from the other side. This topographical arrangement results in a charge separation that is equivalent to moving one positive charge across the membrane for each electron transferred to O₂. In this reaction part of the free energy available from O₂ reduction is conserved in the form of an electrochemical proton gradient. In addition, part of the free energy is used to pump on average one proton across the membrane per electron transferred to O₂. Our understanding of the molecular design of the machinery that couples O₂ reduction to proton pumping in oxidases has greatly benefited from studies of so called “uncoupled” structural variants of the oxidases. In these uncoupled oxidases the catalytic O₂-reduction reaction may display the same rates as in the wild-type CytcO, yet the electron/proton transfer to O₂ is not linked to proton pumping. One striking feature of all uncoupled variants studied to date is that the (apparent) pK_a of a Glu residue, located deeply within a proton pathway, is either increased or decreased (from 9.4 in the wild-type oxidase). The altered pK_a presumably reflects changes in the local structural environment of the residue and because the Glu residue is found near the catalytic site as well as near a putative exit pathway for pumped protons these changes are presumably important for controlling the rates and trajectories of the proton transfer. In this paper we summarize data obtained from studies of uncoupled structural oxidase variants and present a hypothesis that in quantitative terms offers a link between structural changes, modulation of the apparent pK_a and uncoupling of proton pumping from O₂ reduction.     

TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP

Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (M_f). The apparent diffusion coefficient (D_app) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a “membrane fence” model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more “fluid” and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of G_q/G₁₁ family.In conclusion: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R–G_q/G₁₁α protein coupling.     

A comparison of intact human red blood cells and resealed and leaky ghosts with respect to their interactions with surface labelling agents and proteolytic enzymes

Resealed ghosts and intact red blood cells were directly compared with respect to their interactions with surface probes and to digestion by pronase. The amount and pattern of labelling of surface proteins by 4.4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) and by pyridoxal phosphate-borohydride (as seen after sodium dodecylsulfate/acrylamide gel electrophoresis) was substantially the same in cells and resealed ghosts under conditions in which a relatively small change would be apparent. In each membrane system, DIDS labels a protein component of apparent molecular weight 95 000 and pyridoxal phosphate labels the same protein plus three glycoprotein components. The sensitivity of surface proteins and of DIDS and pyridoxal phosphate-labelled sites to pronase was also similar in the cells and resealed ghosts. The glycoproteins were digested, in each case, and the 95 000 (molecular weight) protein was largely split into two portions of apparent molecular weights 65 000 and 35 000, with both portions containing DIDS and pyridoxal phosphate binding sites. The pattern of labelling of “leaky” ghosts by pyridoxal phosphate in the presence of hemoglobin was similar to the labelling of intact cells, provided that the pyridoxal phosphate was present on both the outside and inside of the cells. Virtually all of the major protein components visible by staining on acrylamide gels were labelled. It is concluded that none of the probes could detect any substatial differences in reactivity of proteins of the outer surface of the membrane of the ghosts as compared to the cells and that no irreversible changes in membrane protein conformation or arrangement occur as a consequence of lysis and resealing of ghosts, that are detectable by the reported procedures.