ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES : I. Effects of Trypsin, Phospholipase C, and pH on the Topography of Bound Positively Charged Colloidal Particles

The effects of pH, trypsin, and phospholipase C on the topographic distribution of acidic anionic residues on human erythrocytes was investigated using colloidal iron hydroxide labeling of mounted, fixed ghost membranes. After glutaraldehyde fixation at pH 6.5–7.5, the positively charged colloidal particles were bound to the membranes in small randomly distributed clusters. The clusters of anionic sites were reversibly aggregated by incubation at pH 5.5 before fixation at the same pH. These results correlate with the distribution of intramembranous particles found by Pinto da Silva (J. Cell Biol. 53:777), with the exception that the distribution of anionic sites on a majority of the fixed ghosts at pH 4.5 was aggregated instead of dispersed. The randomly distributed clusters could be nonreversibly aggregated by trypsin or phospholipase C treatment of intact ghosts before glutaraldehyde fixation. Previous glutaraldehyde fixation prevented trypsin and pH induced aggregation of the colloidal iron sites. Evidence that N-acetylneuraminic acid groups are the principal acidic residues binding colloidal iron was the elimination of greater than 85% of the colloidal iron labeling to neuraminidase-treated cell membranes. Colloidal iron binding N-acetylneuraminic acid residues may reside on membrane molecules such as glycophorin, a sialoglycoprotein which contains the majority of the N-acetylneuraminic acid found on the human erythrocyte membrane.     

Schistosomula of Schistosoma mansoni use lysophosphatidylcholine to lyse adherent human red blood cells and immobilize red cell membrane components

Human red blood cells (RBCs) adhere to and are lysed by schistosomula of Schistosoma mansoni. We have investigated the mechanism of RBC lysis by comparing the dynamic properties of transmembrane protein and lipid probes in adherent ghost membranes with those in control RBCs and in RBCs treated with various membrane perturbants. Fluorescence photobleaching recovery was used to measure the lateral mobility of two integral membrane proteins, glycophorin and band 3, and two lipid analogues, fluorescein phosphatidylethanolamine (Fl-PE) and carbocyanine dyes, in RBCs and ghosts adherent to schistosomula. Adherent ghosts manifested 95-100% immobilization of both membrane proteins and 45-55% immobilization of both lipid probes. In separate experiments, diamide-induced cross-linking of RBC cytoskeletal proteins slowed transmembrane protein diffusion by 30-40%, without affecting either transmembrane protein fractional mobility or lipid probe lateral mobility. Wheat germ agglutinin- and polylysine-induced cross-linking of glycophorin at the extracellular surface caused 80-95% immobilization of the transmembrane proteins, without affecting the fractional mobility of the lipid probe. Egg lysophosphatidylcholine (lysoPC) induced both lysis of RBCs and a concentration-dependent decrease in the lateral mobility of glycophorin, band 3, and Fl-PE in ghost membranes. At a concentration of 8.4 micrograms/ml, lysoPC caused a pattern of protein and lipid immobilization in RBC ghosts identical to that in ghosts adherent to schistosomula. Schistosomula incubated with labeled palmitate released lysoPC into the culture medium at a rate of 1.5 fmol/h per 10(3) organisms. These data suggest that lysoPC is transferred from schistosomula to adherent RBCs, causing their lysis.     

IMMUNO-ELECTRON MICROSCOPE ANALYSIS OF THE SURFACE LAYERS OF THE UNFERTILISED SEA URCHIN EGG : I. Effects of the Antisera on the Cell Ultrastructure

The response of unfertilised Paracentrotus lividus eggs to γ-globulin fractions of antisera against isolated homologous jelly coat substance or homologous homogenates of jellyless eggs has been studied at the ultrastructural level. The antijelly γ-globulin caused precipitation of the jelly layer, the density of precipitation varying between different eggs and being proportional to the γ-globulin concentration. Agglutination of the jelly substance of adjacent eggs, which is species specific, occurred frequently with higher γ-globulin concentrations. Antiegg γ-globulins (from antiserum against total homogenates of jelly-free eggs or the heat-stable fraction thereof) did not produce these effects. Instead, these γ-globulins caused various structural alterations mostly representing stages in parthenogenetic activation. This species-specific activation was induced by the reaction of antibodies with some heat-stable egg antigens different from those involved in jelly precipitation. Surface alterations included the formation of small papillae, membrane blisters, hyaline layer, and activation membrane, the release of material from the cell surface, and the breakdown of cortical granules. These alterations were dependent on both γ-globulin concentration and the variable reactivity among different females. Aster formation, found intracellularly, verified that the surface responses represented real parthenogenetic activation and were not the result of immune lysis. No such alterations appeared in the controls.     

Transient holes in the erythrocyte membrane during hypotonic hemolysis and stable holes in the membrane after lysis by saponin and lysolecithin

Ferritin and colloidal gold were found to permeate human erythrocytes during rapid or gradual hypotonic hemolysis. Only hemolysed cells contained these particles; adjacent intact cells did not contain the tracers. Ferritin or gold added 3 min after the onset of hypotonic hemolysis did not permeate the ghost cells which had, therefore, become transiently permeable. By adding ferritin at various times after the onset of hemolysis, it was determined that for the majority of the cells the permeable state (or interval between the time of development and closure of membrane holes) existed only from about 15 to 25 sec after the onset of hemolysis. It was possible to fix the transient "holes" in the open position by adding glutaraldehyde only between 10 and 20 sec after the onset of hemolysis. The existence of such fixed holes was shown by the cell entry of ferritin and gold which were added to these prefixed cells. Membrane defects or discontinuities (of the order of 200–500 A wide) were observed only in prefixed cells which were permeated by ferritin subsequently added. Adjacent prefixed cells which did not become permeated by added ferritin did not reveal any membrane discontinuities. Glutaraldehyde does not per se induce or create such membrane defects since cells which had been fixed by glutaraldehyde before the 10-sec time point or after the 180-sec time point were never permeable to added ferritin, and the cell membranes never contained any defects. It was also observed that early in hemolysis (7–12 sec) a small bulge in one zone of the membrane often occurred. Ghost cells produced by holothurin A (a saponin) and fixed by glutaraldehyde became permeated by ferritin subsequently added, but no membrane discontinuities were seen. Ghosts produced by lysolecithin and fixed by glutaraldehyde also became permeated by subsequently added ferritin, and many membrane defects were seen here (about 300 A wide).     

Factors controlling the resealing of the membrane of human erythrocyte ghosts after hypotonic hemolysis

Summary In accordance with former observations of Hoffman (1962a), ghost populations obtained by hypotonic hemolysis and subsequent restoration of isotonicity by the addition of alkali salts, were found to be composed of 3 types of ghosts. For our purposes it was useful to distinguish between: (1) ghosts which reseal immediately after hemolysis (type I); these ghosts are incapable of incorporating alkali ions which are added after hemolysis; (2) ghosts which reseal after the addition of alkali ions (type II); salt added to the hemolysate becomes trapped inside these ghosts in the course of the resealing process at temperatures above 0°C; and (3) ghosts which remain leaky regardless of the experimental condition (type III). The discrimination between the various types of ghosts was partly achieved by a kinetic method first devised by Hoffman (1962a), and partly by sucrose density gradient centrifugation.The relative sizes of the 3 fractions depend on the temperature at which hemolysis took place and on the time interval which elapsed between hemolysis and the addition of salt. At 37°C the resealing process is fast. Many of the ghosts reseal before salt can be added to the hemolysate. Hence, the fraction of type I ghosts is high after hemolysis at that temperature. At 0°C resealing is extremely slow. Hence, salt which has been added to the hemolysate at that temperature will enter the ghosts and become trapped during subsequent incubation at 37°C. There are no ghosts of type I and many ghosts of type II (about 60%). Regardless of the temperature at hemolysis, there are always ghosts which do not reseal even after prolonged incubation at 37°C. A method has been designed which permits the preparation of homogeneous populations of type II ghosts.Complexing agents (ATP, EDTA, 2,3-DPG) may prevent the resealing of the ghost membrane. However, they exert this effect only at elevated temperatures and when present in the medium at the instant of hemolysis. At 0°C, the presence of complexing agents in the medium at the instant of hemolysis has no effect on the subsequent resealing at 37°C. The recovery of the ghost membrane takes place in spite of the continued presence of the agents and eventually leads to trapping of these agents inside the resealed ghosts.The experiments support the contention that the complexing agents interact with a membrane constituent which is neither accessible from the inner nor from the outer surface of the cell membrane but becomes exposed during the hemolytic event when the complexing agents penetrate across the membrane. Apparently, at low tempertrures membrane ligands are more successful in competing with the added complexing agents for this constituent than at higher temperatures.Extending former observations of Hoffman, we found that not only Mg⁺⁺ but also Ca⁺⁺ facilitates the resealing process. Perhaps one or the other of the two alkaline earth ions is the membrane constituent which normally participates in the maintenance of the integrity of the red blood cell membrane.     

Plasma Membrane-Associated Actin in Bright Yellow 2 Tobacco Cells : Evidence for Interaction with Microtubules

Plasma membrane ghosts form when plant protoplasts attached to a substrate are lysed to leave a small patch of plasma membrane. We have identified several factors, including the use of a mildly acidic actin stabilization buffer and the inclusion of glutaraldehyde in the fixative, that allow immunofluorescent visualization of extensive cortical actin arrays retained on membrane ghosts made from tobacco (Nicotiana tabacum L.) suspension-cultured cells (line Bright Yellow 2). Normal microtubule arrays were also retained using these conditions. Membrane-associated actin is random; it exhibits only limited coalignment with the microtubules, and microtubule depolymerization in whole cells before wall digestion and ghost formation has little effect on actin retention. Actin and microtubules also exhibit different sensitivities to the pH and K⁺ and Ca²⁺ concentrations of the lysis buffer. There is, however, strong evidence for interactions between actin and the microtubules at or near the plasma membrane, because both ghosts and protoplasts prepared from taxol-pretreated cells have microtubules arranged in parallel arrays and an increased amount of actin coaligned with the microtubules. These experiments suggest that the organization of the cortical actin arrays may be dependent on the localization and organization of the microtubules.     

THE LOCALIZATION OF SPECTRIN ON THE INNER SURFACE OF HUMAN RED BLOOD CELL MEMBRANES BY FERRITIN-CONJUGATED ANTIBODIES

Spectrin, a major protein constituent of mammalian red blood cell membrane preparations, has been localized on the inner surface of human red blood cell membranes by techniques that utilized specific ferritin-conjugated antibodies and fixation of membranes shortly after hemolysis so as to allow penetration of the ferritin-antibody labels. The labeling of spectrin was shown to be specific by the following criteria. (a) Nonhomologous ferritin-conjugated antibodies did not specifically bind to either membrane surface. (b) Blocking the membrane-bound spectrin with excess unconjugated antispectrin antibodies prevented ferritin-antibody labeling. (c) Removal of spectrin by treating the membrane preparation with a low ionic strength buffer containing ethylenediaminetetraacetate and β-mercaptoethanol prevented labeling by specific ferritin-conjugated antibodies.     

STRUCTURE OF MEMBRANE HOLES IN OSMOTIC AND SAPONIN HEMOLYSIS

Serial section electron microscopy of hemolysing erythrocytes (fixed at 12 s after the onset of osmotic hemolysis) revealed long slits and holes in the membrane, extending to around 1 µm in length. Many but not all of the slits and holes (about 100–1000 Å wide) were confluent with one another. Ferritin and colloidal gold (added after fixation) only permeated those cells containing membrane defects. No such large holes or slits were seen in saponin-treated erythrocytes, and the membrane was highly invaginated, giving the ghost a scalloped outline. Freeze-etch electron microscopy of saponin-treated membranes revealed 40–50 Å-wide pits in the extracellular surface of the membrane. If these pits represent regions from which cholesterol was extracted, then cholesterol is uniformly distributed over the entire erythrocyte membrane.     

Phosphorylation in membranes of intact human erythrocytes.

Studies of phosphorylation in membranes of intact human erythrocytes were performed by incubating erythrocytes in inorganic [32P]phosphate. Analysis of membrane proteins by polyacrylamide gel electrophoresis showed a pattern of phosphorylation similar to that observed when ghost membranes were incubated with [gamma-32P]ATP. Membrane lipid phosphorylation was also similar in intact cells and ghosts. The most heavily phosphorylated lipid, polyphosphoinositide, was closely associated with glycophorin A, the major erythrocyte membrane sialoglycoprotein obtained when the sialoglycoprotein fraction was isolated by the lithium diiodosalicylate-phenol partition procedure. Only 1 molecule of glycophorin A out of every 100 was found to be phosphorylated, and the phosphate exchange occurred specifically in the COOH-terminal intracellular portion of glycophorin A. These studies show that the human erythrocyte can be used as a model for membrane phosphorylation in an intact cell system.     

ISOLATION OF PLASMA MEMBRANE FRAGMENTS FROM HELA CELLS

A method for isolating plasma membrane fragments from HeLa cells is described. The procedure starts with the preparation of cell membrane "ghosts," obtained by gentle rupture of hypotonically swollen cells, evacuation of most of the cell contents by repeated washing, and isolation of the ghosts on a discontinuous sucrose density gradient. The ghosts are then treated by minimal sonication (5 sec) at pH 8.6, which causes the ghost membranes to pinch off into small vesicles but leaves any remaining larger intracellular particulates intact and separable by differential centrifugation. The ghost membrane vesicles are then subjected to isopycnic centrifugation on a 20–50% w/w continuous sucrose gradient in tris-magnesium buffer, pH 8.6. A band of morphologically homogeneous smooth vesicles, derived principally from plasma membrane, is recovered at 30–33% (peak density = 1.137). The plasma membrane fraction contained a Na-K-activated ATPase activity of 1.5 µmole Pi/hr per mg, 3% RNA, and 13.8% of the NADH-cytochrome c reductase activity of a heavier fraction from the same gradient which contained mitochondria and rough endoplasmic vesicles. The plasma membranes of viable HeLa cells were marked with ¹²⁵I-labeled horse antibody and followed through the isolation procedure. The specific antibody binding of the plasma membrane vesicle fraction was increased 49-fold over that of the original whole cells.     

TRANSLATIONAL MOBILITY OF THE MEMBRANE INTERCALATED PARTICLES OF HUMAN ERYTHROCYTE GHOSTS : pH-Dependent, Reversible Aggregation

This paper demonstrates the translational movement along the plane of the human erythrocyte ghost of the membrane particles exposed by freeze-fracture. The membrane particles can be aggregated by incubation of the ghosts in media with a pH in the vicinity of 5 5 or 3 5. The particles are disaggregated in neutral and alkaline media (pH 9 5) and also at pH 4.5 Aggregation of the particles at pH 5.5 is reversible, prevented by prefixation in glutaraldehyde and by media of high ionic strength. Particle aggregation occurs within 2–4 min. These results are consistent with the concept that the erythrocyte ghost membrane is a planar fluid domain formed by a bilayer membrane continuum which is interrupted by localized, yet mobile, proteic intercalations.     

Membrane Damage by an α-Helical Pore-forming Protein,Equinatoxin II,Proceeds through a Succession of Ordered Steps

Actinoporin equinatoxin II (EqtII) is an archetypal example of α-helical pore-forming toxins that porate cellular membranes by the use of α-helices. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal α-helix into the lipid bilayer. We studied these separate steps with an EqtII triple cysteine mutant. The mutant was engineered to monitor the insertion of the N terminus into the lipid bilayer by labeling Cys-18 with a fluorescence probe and at the same time to control the flexibility of the N-terminal region by the disulfide bond formed between cysteines introduced at positions 8 and 69. The insertion of the N terminus into the membrane proceeded shortly after the toxin binding and was followed by oligomerization. The oxidized, non-lytic, form of the mutant was still able to bind to membranes and oligomerize at the same level as the wild-type or the reduced form. However, the kinetics of the N-terminal helix insertion, the release of calcein from erythrocyte ghosts, and hemolysis of erythrocytes was much slower when membrane-bound oxidized mutant was reduced by the addition of the reductant. Results show that the N-terminal region needs to be inserted in the lipid membrane before the oligomerization into the final pore and imply that there is no need for a stable prepore formation. This is different from β-pore-forming toxins that often form β-barrel pores via a stable prepore complex.     

An electron microscopic investigation of the surface coat of the electrocyte of Electrophorus electricus

Summary The surface coat of the electrocyte of the main electric organ of Electrophorus electricus was studied using cytochemical methods (periodic acid-silver methenamine, periodic acid-chromic acid-silver methenamine, periodic acid-thiosemicarbazide-silver proteinate, Concanavalin A — horseradish peroxidase, ruthenium red, Alcian-blue lanthanum nitrate, colloidal iron hydroxide and cationized ferritin). The surface of the electrocyte presents perpendicularly oriented tubular invaginations of the cell membrane. The fibrous coat 50–100 nm thick, penetrates into the lumen of the invaginations. It is also observed in the synaptic clefts existent in the posterior face of the electrocyte. The coating of the surface membrane gives a positive reaction with all techniques used. Binding of colloidal iron hydroxide particles was observed only in the outer layer of the coat. With the Alcian-blue lanthanum nitrate technique, microtubules were observed in the cytoplasm of the electrocyte.The results indicate that the surface coat of the electrocyte contains mucopolysaccharides, glycoproteins, acid mucopolysaccharides and anionic sites detected at low (colloidal iron hydroxyde) and neutral (cationized ferritin) pH.This work has been supported by Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Conselho de Ensino e Pesquisa da UFRJ (CEPG) and Banco Nacional de Desenvolvimento Econômico     

Limited proteolysis of the erythrocyte membrane skeleton by calcium-dependent proteinases

The action of purified calcium-dependent proteinases on human erythrocyte membrane skeleton proteins has been examined. Preferential cleavage of proteins 4.1 a and b and band 3 and limited cleavage of alpha- and beta-spectrin occur when either calcium-dependent proteinase I or calcium-dependent proteinase II has access to the cytoplasmic side of the ghost membrane skeleton in the presence of calcium. Thus, when these proteinases are incubated with sealed ghosts they do not cleave these proteins. Leupeptin, mersalyl, the specific cellular protein inhibitor of these enzymes, and calcium chelators can inhibit proteolysis of the red cell ghost proteins by Ca2+-dependent proteinases. Each proteinase has also been loaded into erythrocyte ghosts in the absence of calcium at low ionic strength and subsequently trapped inside by resealing the ghosts. The proteinases were activated by incubating these ghosts in the presence of the calcium ionophore A23187 and calcium. Examination of the ghost proteins by electrophoresis demonstrated calcium-dependent proteolysis of Bands 4.1 and 3 and limited cleavage of alpha- and beta-spectrin similar to that observed on proteolysis of the open, leaky ghosts. In the presence of calcium each calcium-dependent proteinase appears to associate with the erythrocyte ghost membrane.     

Protein Components of Bacteriophages λ and λ Virulent

Parallel studies have been made of the protein coats of the temperate bacteriophage λ and of a deletion mutant, λ virulent. A new method for preparing ghosts of both phages by the action of Cu⁺⁺ is described. Protein ghosts of both phages can be dissolved in citrate at pH values below 3, more rapidly in the presence of 8 m urea. Both phages yielded three apparently identical protein components which can be separated by thin-layer gel filtration and thin-layer gel electrophoresis. The protein of molecular weight 47,000 ± 1,500 represents about 55% of the protein of the ghosts and is therefore likely to be the subunit of the head. The other proteins of molecular weight 30,000 ± 1,500 and 16,000 ± 1,500 represent approximately 25% and 20% of the protein, respectively. Amino acid analyses of the ghosts from the two phages have been carried out and show no significant differences. The buoyant density of phage λ virulent is 0.016 g/ml less than that of λ. Since no differences have been found in the protein components of the two phages, this indicates that the virulent mutant contains approximately 16% less deoxyribonucleic acid than the temperate phage.     

Role of the bilayer in the shape of the isolated erythrocyte membrane

Summary The determinants of cell shape were explored in a study of the crenation (spiculation) of the isolated erythrocyte membrane. Standard ghosts prepared in 5mm NaP_i (pH 8) were plump, dimpled disks even when prepared from echinocytic (spiculated) red cells. These ghosts became crenated in the presence of isotonic saline, millimolar levels of divalent cations, 1mm 2,4-dinitrophenol or 0.1mm lysolecithin. Crenation was suppressed in ghosts generated under conditions of minimal osmotic stress, in ghosts from red cells partially depleted of cholesterol, and, paradoxically, in ghosts from red cells crenated by lysolecithin. The susceptibility of ghosts to crenation was lost with time; this process was potentiated by elevated temperature, low ionic strength, and traces of detergents or chlorpromazine.In that ghost shape was influenced by a variety of amphipaths, our results favor the premise that the bilayer and not the subjacent protein reticulum drives ghost crenation. The data also suggest that vigorous osmotic hemolysis induces a redistribution of lipids between the two leaflets of the bilayer which affects membrane contour through a bilayer couple mechanism. Subsequent relaxation of that metastable distribution could account for the observed loss of crenatability.     

Proteolytic degradation of human erythrocyte band 3 by membrane-associated protease activity

Summary Antisera directed against the cytoplasmic portion of human erythrocyte Band 3 were used to follow the degradation of the band 3 molecule. Small amounts of Band 3 were degraded when well-washed red cell membrane ghosts were incubated in the cold; this process was greatly accelerated by incubating ghosts at 37°C. Band 3 labeled with pyridoxal-phosphate was digested at comparable rates. Band 3 digestion also took place when alkali-extracted ghost membranes were incubated at 37° for prolonged periods. These results suggest that human erythrocytes contain tightly bound, membrane-associated proteolytic activity.     

Monopalmitoylphosphatidylcholine incorporation into human erythrocyte ghost membranes causes protein and lipid immobilization and cholesterol depletion

The effects of lysophosphatidylcholine (lysoPC) on human erythrocyte (RBC) ghost morphology, transmembrane protein and lipid lateral mobilities, and membrane lipid composition were studied in order to elucidate mechanisms by which lysoPC immobilizes ghost membrane components [Golan, D. E., Brown, C. S., Cianci, C. M. L., Furlong, S. T., & Caulfield, J. P. (1986) J. Cell Biol. 103, 819-828]. Under standardized conditions 1.0-1.5 micrograms/mL egg lysoPC lysed 50% of RBCs and induced, in some ghosts, the formation of large patches of wrinkled membrane. Patches exhibited complete immobilization of glycophorin and band 3 and partial immobilization of the phospholipid analogue fluorescein phosphatidylethanolamine (Fl-PE), whereas adjacent smooth membrane areas manifested only partial immobilization of proteins and no immobilization of Fl-PE. Supralytic concentrations of lysoPC induced both progressive, homogeneous wrinkling of RBC ghost membranes and concentration-dependent decreases in the lateral mobilities of glycophorin, band 3, and Fl-PE. Complete immobilization of glycophorin and band 3 occurred at 8.4 micrograms/mL lysoPC and of Fl-PE at 16.8 micrograms/mL lysoPC. Monopalmitoylphosphatidylcholine (MPPC), the major component of egg lysoPC, induced both membrane wrinkling and a concentration-dependent decrease in Fl-PE mobility, with complete immobilization at 10 micrograms/mL. Other synthetic lysoPCs did not completely immobilize Fl-PE, although some caused membrane wrinkling. MPPC was incorporated into ghost membranes with a linear dependence (r = 0.97) on MPPC concentration. Relative to total membrane lipid, the lysoPC mole fraction increased from 0.2 +/- 0.1% at 0 micrograms/mL MPPC to 25 +/- 2% at 16 micrograms/mL MPPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.