Inhibitory effects of monovalent cations on the Ca2+-induced aggregation of porcine intestinal brush border membranes

The Ca2+-induced aggregation of porcine intestinal brush border membranes could be inhibited by addition of monovalent cations to the medium or by increasing the ionic strength of the medium, as measured by the change in optical density of the membrane suspension. The relative effectiveness of monovalent cations at 100 mM in the inhibition was in the order, (Na+ approximately equal to NH4+) greater than (K+ approximately equal to Rb+ approximately equal to Li+) greater than choline+. The Ca2+ concentration dependence profile of the membrane aggregation showed that the Ca2+ threshold at which the aggregation began was distinctly shifted to a higher concentration by the addition of KCl. In addition, the results of fluorometric studies with 1-anilino-8-naphthalene sulfonate suggested that the inhibition of the membrane aggregation by extravesicular KCl is due to a decrease of the binding affinity of Ca2+ for the membranes as a result of neutralization of the surface charges. On the other hand, measurements of the incorporation of 1,6-diphenyl-1, 3,5-hexatriene (DPH) into the membrane vesicles and of the anisotropy of DPH-labeled membranes suggested that the imposition of a salt gradient across the membrane vesicles (out greater than in) causes an increase of lipid fluidity of the membranes. Based on these results, a possible contribution of membrane surface charges and/or membrane fluidity to the Ca2+-induced aggregation of the membranes is discussed.     

Transport studies with brush border membrane vesicles: choice of experimental parameters

1. We have studied different parameters, in their effects on a transport system chosen as a model: the Na+-phosphate symporter of the renal brush border membrane. 2. Ionic strength was found to be a critical factor in the retention capacity of the filter. 3. When high ionic strength solutions containing 150 mM NaCl or KCl were used, less than 8% of the membrane proteins were lost through filtration. 4. Lowering the ionic strength by replacing NaCl or KCl by 300 mM mannitol, however, caused a 52% loss of protein. 5. Addition of 15 mM NaCl to this low ionic strength solution was sufficient to restore full retention of the vesicles by the filter. 6. The presence of arsenate, a competitive inhibitor, in the stop solution did not improve the retention of phosphate by the vesicles in high ionic strength media, but caused a pronounced temperature dependent loss of the vesicle content, as a function of time of incubation in low ionic strength solutions. 7. Addition of 5 mM phosphate in the stop solution caused a 31 and 37% loss for KCl and NaCl stop solutions, respectively, while no effect was observed for the mannitol stop solution. 8. The presence of HgCl2 gave a 32% stimulation for the mannitol solution and a 35 or 22% inhibition for the KCl or NaCl solutions. 9. Addition of NaCl in the stop solution caused an overaccumulation of 75%, after 60 sec of incubation at 25 degrees C. 10. Phosphate transport by renal vesicles is thus highly affected by the composition of the stop solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disappearance of calcium-induced phase separation in phosphatidylserine-phosphatidylcholine membranes caused by protonation and by electric current.

Disappearance of Ca2+-induced phase separation in phosphatidylserine-phosphatidylcholine membrane has been studied under several conditions by monitoring electron spin resonance spectrum of spin-labeled phosphatidylcholine. The membranes were prepared in Millipore filters. Electron micrographs of the pre parations showed formation of multilayered structures lined on the pore surface. The phase separation was disappeared when the membrane was soaked in non-buffered salt solution (100 ml KCl, pH 5.5). It was markedly contrasting that when the bathing salt solution was buffered no disappearance was observed. Disappearance of the phase separation was also observed when the Ca2+-treated membrane was transferred to acidic salt solutions (less than or equal to pH 2.5) or to low ionic strength media (less than or equal to mM) buffered at pH 5.5, and then to the buffered salt solution (100 mM KCl, pH 5.5). These are due to replacement of Ca2+ by proton, proton-induced separation, followed by disappearance of the phase separation in the buffered salt solution. Biological significance of the competition between Ca2+ and proton for the phase separation or domain formation in the membranes was emphasized.     

Cross-linkings between spectrin and band 3 in human erythrocyte membranes

A specific structural association between spectrin component 1 and band 3 in human erythrocyte membrane has been demonstrated by covalent cross-linkings, specific labeling, and the technique of two-dimensional gel electrophoresis. A complex of 330,000 daltons, representing 1 + 3, was produced in mildly oxidized membranes at physiologic pH and isotonic conditions but not at hypotonic conditions (< 10 mM KCl or NaCl). The yield of this complex decreased dramatically as the monovalent cation concentration decreased from 90 mM to 30 mM. The presence of Mg⁺⁺ or Ca⁺⁺ (2 mM) at low ionic strength promoted 1 + 3 cross-linking in an amount similar to that produced at isotonic conditions. The specific segment of band 3 involved in the cross-linking was also investigated by means of chymotrypsin digestion of band 3 in the intact red cells. The results showed the cross-links between spectrin component 1 and the 55,000-dalton fragment of band 3 at physiologic pH and isotonic conditions. This is consistent with the idea that band 3 is anchored on or contacted with the submembrane meshwork at the cytoplasmic membrane surface.     

The study of Ca2+ influx in human erythrocytes in isotonic polyethylene (glycol) 1500 (PEG-1500) and sucrose media

Effects of isotonic solutions of polyethylene (glycol) 1500 (PEG-1500) and sucrose on Ca2+ influx into ATP-depleted red blood cells were studied using the Ca2+ -sensitive fluorescent dye fura-2AM. When incubated in isotonic low ionic strength media (containing 2 mM CaCl2 in addition to sucrose and PEG-1500), the initial rate of Ca2+ influx was higher than that for the cells in physiological (normal ionic strength) medium. After 20 minutes of incubation in the PEG-1500-containing solution, a 10-fold increase of Ca2+ influx was observed, whereas in the sucrose medium the rate of Ca2+ influx decreased compared to that in physiological medium. 1H-NMR data provided no evidence of direct interaction between PEG-1500 and the erythrocyte membrane. Moreover, PEG-1500 did not affect lipid peroxidation (LPO) induction in erythrocyte membranes. We propose that a change in the hydrogen environment of Ca2+ -ATPase of the erythrocytes suspended in the PEG-1500 solution is the primary cause of altered Ca2+ homeostasis in these cells. The activation of the Ca2+ -ATP-ase in sucrose medium may result in an incomplete suppression of the Ca2+-pump activity in ATP-depleted cells, which is accelerated when calmodulin binds with the Ca2+-ATP-ase under the conditions of rapid Ca2+ accumulation.     

Enhancement of (Ca2+ + Mg2+)-ATPase activity of human erythrocyte membranes by hemolysis in isosmotic imidazole buffer. II. Dependence on calcium and a cytoplasmic activator.

1. Activity of the (Ca2+ + Mg2+)-ATPase of erythrocyte membrane may be enhanced by a cytoplasmic protein activator. The presence of Ca2+ is necessary for the ionic strength-dependent interaction between the erythrocyte membrane and the activator. This is true no matter the purity of activator (unfractionated hemolysis supernatant or partially purified activator) or the major source of ionic strength (imidazole or NaCl). 2. When the endogenous activator enhances (Ca2+ + Mg2+)-ATPase activity of the erythrocyte membrane, there is a physical association between activator and membrane. This association is not disrupted by a decrease in ionic strength to 0.005 but is reversed by exposure to 5 mM ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. 3. Activator binding necessary for enhancement of (Ca2+ + Mg2+)-ATPase activity may occur during preparation of membranes or during incubation for assay of ATPase.     

Phosphoinositide metabolism of rat pineal gland in a low ionic strength medium

Pineal glands were incubated in the presence of 32P orthophosphate. When all NaCl in a conventional incubation medium was replaced by isotonic sucrose, i.e. when the ionic strength of the medium was decreased, there was a marked increase in 32P labelling of phosphatidylinositol (PI) and phosphatidic acid (PA). The 32P labelling of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was not affected. No net synthesis of PI was observed. The increased labelling of PI therefore represents an increase in the turnover of PI. The 32P labelling of PI was observed also in media where NaCl was replaced by fructose or mannitol, but not in media, where NaCl was replaced by choline chloride. The effect depends on the concentration of the HEPES buffer and was not found in the medium with a bicarbonate buffer. 32P labelling of PI was not blocked by alpha 1 adrenergic blockers, phentolamine and prazosin, and did not depend on the presence of Ca2+ in the incubation medium. The effect was blocked by a Ca2+ channel blocker, MnCl2. Only 32P labelling of PI and not that of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) was increased during prolonged incubation in the sucrose medium. It is suggested that a decrease in the charge distribution across the plasma membrane as a result of the absence of most monovalent cations is responsible for the increased metabolism of phosphatidylinositol.     

On the state of calcium ions in isolated rat liver mitochondria. I. Ion fluxes and volume changes upon Ca2+ uptake under various ionic conditions

As to functional consequences of Ca2+ uptake in isolated rat liver mitochondria, we simultaneously measured 3H2O and [14C]sucrose spaces, monovalent cation distribution, membrane potential and delta pH across the inner membrane, and [32P]phosphate and 45Ca2+ content in parallel incubations of different ionic composition. Without added Ca2+ and phosphate, mitochondrial matrix volume, membrane potential, and delta pH depended on the concentration and permeability of monovalent cations. Despite large differences in membrane potential, maximal Ca2+ uptake was identical under all conditions. Ca2+ uptake never provoked a volume change from which an osmotic active state of mitochondrial Ca2+ could be concluded. If matrix volume shrunk this could be totally accounted for by the loss of alkali ions exchanging for calcium ions. Even phosphate taken up in conjunction with Ca2+ was osmotically silent. Volume increases here occurring if K+ was permeabilized, solely resulted from K+ uptake, though this condition may give rise to irreversible mitochondrial damage with Ca2+ and phosphate release. As mitochondrial Ca2+ is bound, an electro-chemical equilibrium across the membrane is impossible for this ion. This has to be considered in any model describing equilibria of Ca2+ with mitochondria, though present models neglect this state of mitochondrial Ca2+.     

Preparation of membrane vesicles from isolated myelin: studies on functional and structural properties.

Myelin membranes purified from bovine brain are shown to form membrane vesicles when incubated in hypotonic buffer. Following restoration of isotonicity a resealing of the membrane occurs as judged by a significant decrease in 22Na+ permeability. Electron spin resonance measurements using stearic acid spin label I indicate a small decrease in membrane fluidity with increasing ionic strength between 50 and 80 mM NaCl. Iodination of myelin membrane vesicles by lactoperoxidase shows a four-fold increase in the amount of iodine incorporation into the myeline basic protein from 0--150 mM NaCl, while the iodination of the proteolipid protein remains essentially unaffected by the change in ionic strength. This dependence of the iodination of the myelin basic protein on the ionic strength can be explained by the electrostatic interactions of this protein with membrane lipids. In view of striking analogies with studies on model membranes correlating protein binding with membrane permeability changes, we suggest a similar structure-function relationship for the myelin basic protein.     

The interaction of magnesium ions with teichoic acid.

The binding of Mg2+ to the wall teichoic acid of Lactobacillus buchneri N.C.I.B. 8007 was measured by equilibrium dialysis at controlled ionic concentration and pH. In an aqueous solution containing 10mM-NaCl at pH 5.0 one Mg2+ ion was bound for every two phosphate groups of the teichoic acid, with an apparent association constant, Kassoc. = 2.7 x 10(3) M-1. On lowering the pH below the pKa of the phosphate groups the amount of bound Mg2+ decreased concomitantly with decreasing ionization of the phosphate groups. Both the amount of Mg2+ bound to the teichoic acid and the apparent association constants were similar in the presence of 10 mM concentrations of NaCl or KCl but decreased markedly in the presence of 10 mM-CaCl2 because of competition between Ca2+ and Mg2+ for the binding sites. A similar effect was found when the concentration of NaCl was increased from 0 to 50 mM. The results are discussed in relation to the function of teichoic acid in the walls of Gram-positive bacteria.     

Cation-induced aggregation and fusion of N-acyl-N-methyl-phosphatidylethanolamine vesicles.

Aggregation and fusion of unilamellar vesicles consisting of N-acyl-N-methylphosphatidylethanolamine were studied as a function of mono- and divalent cation concentrations. The aggregation reactions were irreversible processes, as demonstrated by changes in monovalent ion concentrations and by the addition of ethylenediaminetetraacetic acid (EDTA) to chelate divalent cations, suggesting the possibility of some cation-induced vesicle fusion. An increase in the NaCl ionic strength of the vesicle suspension solutions diminishes the threshold concentration for Li+ and K+ and increases that corresponding to Mn2+, Mg2+ and Ca2+. However NaCl concentrations above 300 mM yield smaller threshold values for the divalent cation-induced processes, probably due to the increased size of phospholipid vesicles as the ionic strength of the medium increases.     

Ion-binding to phospholipids. Interaction of calcium with phosphatidylserine.

The binding of Ca2+ to monolayers and bilayers of phosphatidylserine has been investigated as a function of pH, ionic strength (NaCl concentration) and Ca2+ concentration using surface and colloid chemical techniques. The molar ratio of lipid to bound calcium decreases to 2 as the Ca2+ concentration is increased to about 0.1 mM. At [Ca2+] greater than 0.1 mM a 1:1 complex is formed. The apparent binding constant Ka ranges from about approximately 10(6) - 10(4) l/mol depending on the Ca2+ concentration. After allowing for electrostatic effects and neighbour group interactions, the intrinsic binding constant Ki of the phosphorylserine polar group at pH 7 (I = 0.01 M), where it carries a net negative charge of one, is approximately 10(4) l/mol; consistent values for Ki were obtained using several independent approaches. Ka for Ca2+ binding decreases with increasing NaCl concentration because the monovalent cations compete with Ca2+ for the same binding site. Na+ and K+ are equally effective in displacing 45Ca2+ adsorbed to monolayers of phosphatidylserine, both with respect to the kinetics and the equilibrium of the displacement. Ka for the reaction between phosphatidylserine and monovalent cations is about 10(3)-fold smaller than that of Ca2+. An investigation of the binding of Mn2+ to phosphatidylserine by both surface chemical and nuclear magnetic resonance methods shows that this cation has a similar binding constant to that of Ca2+. The Ca2+-binding capabilities of monolayers containing only carboxyl groups (i.e. arachidic acid) and phosphodiester groups (i.e. dicetyl phosphate) have also been determined; the apparent pK for the - COOH group in monolayers is larger than or equal to 9 and that for the phosphodiester group is less than 4. Since these groups do not retain the same pK values when they are in close proximity in the phosphorylserine group, the relative contributions of the two groups to the binding of Ca2+ to phosphatidylserine is not obvious.     

Calcium ion as a probe of the monovalent cation center of sodium, potassium ATPase

Sodium and potassium ion-transport adenosine triphosphatase from dog kidney was incubated with 0.4-2 mM Ca2+ at 23 degrees C for more than 2 min in the absence of monovalent inorganic cations, cooled to 0 degrees C, and phosphorylated from 1 mM Pi with 2.4 mM MgCl2. The resultant phosphoenzyme resembled that obtained by incubating the enzyme with K+ in place of Ca2+ in six respects. It was concluded that Ca2+ can occupy the monovalent cation-binding center for K+. The rate constant for release of Ca2+ from the dephosphoenzyme at 0 degrees C was 0.17 s-1. The rate of release from the phosphoenzyme was at least 7-fold slower. Phosphorylation stabilized the binding of Ca2+ to the enzyme in contrast to its destabilization of the corresponding K X enzyme complex. K-sensitive phosphoenzyme did not respond to free Ca2+. Thus Ca2+ was not easily accepted by nor released from the phosphoenzyme and would not be an effective substrate for transport. A selective barrier against Ca2+ between the monovalent cation binding center and the extracellular solution is proposed. Release of calcium from the dephosphoenzyme yielded a conformation that was not phosphorylated from Pi. The enzyme changed the conformation of its center for phosphorylation before or at the same time that it changed the conformation of its center for ion transport.     

Membrane damage by Cerebratulus lacteus cytolysin A-III. Effects of monovalent and divalent cations on A-III hemolytic activity

The effects of monovalent and divalent cations on the hemolytic activity of Cerebratulus lacteus toxin A-III were studied. The activity of cytolysin A-III is remarkably increased in isotonic, low ionic strength buffer, the HC50 (the toxin concentration yielding 50% lysis of a 1% suspension of erythrocytes after 45 min at 37 degrees C) being shifted from 2 micrograms per ml in Tris or phosphate-buffered saline to 20-30 ng per ml in sucrose or mannitol buffered with Hepes, corresponding to a 50-100-fold increase in potency. On the contrary, hemolytic activity decreases progressively as the monovalent cation concentration in the medium increases for Na+, K+, or choline salts. The divalent cations Ca2+ and Zn2+ likewise inhibit the cytolysin A-III activity, but more strongly than do the monovalent cations specified above. Zn2+ at a concentration of 0.3 mM totally abolishes both toxin A-III-dependent hemolysis of human erythrocytes and toxin-induced leakage from liposomes. The observation of similar effects in both natural membranes and artificial bilayers suggests an effect of Zn2+ on the toxin A-III-induced membrane lesion, especially since Zn2+ does not alter binding of the cytolysin. The dose-response curve for toxin A-III exhibits positive cooperativity, with a Hill coefficient of 2 to 3. However, analysis of toxin molecular weight by analytical ultracentrifugation reveals no tendency to aggregate at protein concentrations up to 2 mg per ml. These data are consistent with a post-binding aggregational step which may be affected by the ionic strength of the medium.     

Dependence of Na+-Ca2+ exchange and Ca2+-Ca2+ exchange on monovalent cations

Two mechanisms of passive Ca2+ transport, Na+-Ca2+ exchange and Ca2+-Ca2+ exchange, were studied using highly-purified dog heart sarcolemmal vesicles. About 80% of the Ca2+ accumulated by Na+-Ca2+ exchange or Ca2+-Ca2+ exchange could be released as free Ca2+, while up to 20% was probably bound. Na+-Ca2+ exchange was simultaneous, coupled countertransport of Na+ and Ca2+. The movement of anions during Na+-Ca2+ exchange did not limit the initial rate of Na+-Ca2+ exchange. Na+-Ca2+ exchange was electrogenic, with a reversal potential of about -105 mV. The apparent flux ratio of Na+-Ca2+ exchange was 4 Na+:1 Ca2+. Coupled cation countertransport by the Na+-Ca2+ exchange mechanism required a monovalent cation gradient with the following sequence of ion activation: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+. In contrast to Na+-Ca2+ exchange, Ca2+-Ca2+ exchange did not require a monovalent cation gradient, but required the presence of Ca2+ plus a monovalent cation on both sides of the vesicle membrane. The sequence of ion activation of Ca2+-Ca2+ exchange was: K+ much greater than Rb+ greater than Na+ greater than Li+ greater than Cs+. Na+ inhibited Ca2+-Ca2+ exchange when Ca2+-Ca2+ exchange was supported by another monovalent cation. Both Na+-Ca2+ exchange and Ca2+-Ca2+ exchange were inhibited, but with different sensitivities, by external MgCl2, quinidine, or verapamil.     

Permeation of neutral molecules through calcium channel in sarcoplasmic reticulum vesicles.

Permeation of neutral molecules as well as Ca2+ through the Ca2+ channel in sarcoplasmic reticulum vesicles has been studied by the tracer and/or by the light scattering methods. In the absence of KCl, the Ca2+ channel was found not to be able to pass neutral molecules such as glucose, xylose, and glycine under the condition that the channel was open, although the channel could pass Ca2+. On the other hand, submolar concentrations of KCl made the channel become permeable to neutral molecules as well as Ca2+. Since the effect of KCl could be replaced by NaCl and KNO3, but not by sucrose and glucose, this effect of KCl is considered to be due to ionic strength and not to osmotic pressure. These results suggest that low ionic strength transforms the Ca2+ channel protein in such a manner as to block the permeation of neutral molecules without modifying the gating mechanism of the channel.     

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca2+ concentration was increased to a threshold of 3--5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 micrometer diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2+-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca2+ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca2+ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca2+ concentration.     

Interaction of myosin with thin filaments during contraction and relaxation: effect of ionic strength

The influence of ionic strength on the isometric tension, stiffness, shortening velocity and ATPase activity of glycerol-treated rabbit psoas muscle fiber in the presence and the absence of Ca2+ has been studied. When the ionic strength of an activating solution (containing Mg2+-ATP and Ca2+) was decreased by varying the KCl concentration from 120 to 5 mM at 20 degrees C, the isometric tension and stiffness increased by 30% and 50%, respectively. The ATPase activity increased 3-fold, while the shortening velocity decreased to one-fourth. At 6 degrees C, similar results were obtained. These results suggest that at low ionic strengths ATP is hydrolyzed predominantly without dissociation of myosin cross-bridges from F-actin. In the absence of Ca2+, with decreasing KCl concentration the isometric tension and stiffness developed remarkably at 20 degrees C. However, the ATPase activity and shortening velocity were very low. At low ionic strength, even in the absence of Ca2+ myosin heads are bound to thin filaments. The development of the tension and stiffness were greatly reduced at 6 degrees C or at physiological ionic strength.     

Cadmium as a tool for studying calcium-dependent cation permeability of the human red blood cell membrane.

Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes.     

Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations

Fibrinogen binding to platelet plasma membranes, which is a prerequisite for platelet aggregation, was determined by incubating 125I-labeled fibrinogen with isolated membranes and measuring the amount of radioactivity sedimenting with the membranes through 15% sucrose. Fibrinogen binding was optimal at 10(-3) M Ca2+. Scatchard analyses of the fibrinogen binding showed that the membrane capacity for fibrinogen was 1.6 X 10(-12) mol/mg of membrane protein, with a dissociation constant (Kd) = 1.2 X 10(-8) M. When Ca2+ levels were manipulated by the addition of varying amounts of EGTA at a fixed Mg2+ concentration of 3 X 10(-3) M, specific binding of fibrinogen to platelet membranes occurred only at Ca2+ concentrations greater than or equal to 10(-6) M. Membranes isolated from platelets of an individual with Glanzmann's thrombasthenia bound only 12% as much fibrinogen as control platelets. The data in the present study suggest that there are two divalent cation binding sites that must be occupied for fibrinogen to bind: one site is specific for calcium and is saturated at 10(-6) M Ca2+; the other site is less specific and is saturated at a 10(-3) M concentration of either Ca2+ or Mg2+. Fibrinogen binding to intact platelets and, consequently, platelet aggregation only required 10(-3) M extracellular divalent cation and was not specific for Ca2+. These data indicate that the cytoplasm is a potential source for the requirement of 10(-6) M Ca2+, and that changes in the intracellular concentration of Ca2+ may cause the expression of fibrinogen receptors during ADP-induced platelet activation.