Association of (Ca + Mg)-ATPase activity with ATP-dependent Ca uptake in vesicles prepared from human erythrocytes.

Ghost membranes prepared from human erythrocytes exhibit 2 distinct (Ca + Mg)-ATPase1 activities (Quist and Roufogalis, Arch Biochem Biophys 168:240, 1975). (Ca + Mg)-ATPase activity dependent on a water soluble protein fraction is selectively lost from ghost membranes during preparation of vesicles under low ionic strength, slightly alkaline conditions. In this study, the Ca2+ dependence of the remaining membrane bound (Ca + Mg)-ATPase activity and ATP-dependent Ca uptake in vesicles were compared. The Ca2+ activation curves for (Ca + Mg)-ATPase activity and Ca uptake into vesicles were parallel over a Ca2+ range of 0.3-330 micrometer, and both curves have 2 apparent KA values for Ca2+ of 0.45 and 100 micrometer. Addition of a concentrated soluble protein fraction containing predominantly spectrin to the vesicles increased (Ca + Mg)-ATPase activity over twofold but did not affect the rate of Ca uptake. These findings suggest that the (Ca + Mg)-ATPase activity remaining in vesicles after extraction of the water soluble proteins is associated with the Ca pump whereas (Ca + Mg)-ATPase activity dependent on the soluble protein fraction is associated with some other function.     

Modulator binding protein antagonizes activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport of red blood cell membranes.

Red blood cells contain a protein that activates membrane-bound (Ca2+ + Mg2+)-ATPase and Ca2+ transport. The red blood cell activator protein is similar to a modulator protein that stimulates cyclic AMP phosphodiesterase. Wang and Desai [Journal of Biological Chemistry 252:4175--4184, 1977] described a modulator-binding protein that antagonizes the activation of cyclic AMP phosphodiesterase by modulator protein. In the present work, modulator-binding protein was shown to antagonize the activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport by red blood cell activator protein. The results further demonstrate the similarity between the activator protein from human red blood cells and the modulator protein from bovine brain.     

Divalent cation dependent ATPase activities of red blood cell membranes: influence of the oxidation of membrane thiol groups close to each other

An Mg2+-dependent low ATPase activity can be detected in erythrocyte "white membranes," in addition to that of the well known (Ca2+ + Mg2+)-ATPase. The thiol oxidizing agent diamide affects both activities. The oxidation of neighboring thiols seems to leave the mechanism of the (Ca2+ + Mg2+)-ATPase amplification system evoked by Ca2+ largely unaffected. The perturbation caused by diamide in the membranes seems to affect primarily a step of the ATP hydrolysis mechanism that is common to both ATPase activities. The effectiveness of diamide seems to be the same when either Ca2+ and Mg2+, or Mg2+ alone are present during the reagent action. Reduction of disulfide bonds by DTE after diamide treatment restores the (Ca2+ + Mg2+)-ATPase activity but is unable to take the Mg2+-ATPase activity back to the original level. The hypothesis is discussed that the redox state of one (or more than one) couple of --SH close to each other and possibly connected to the active site, may be an important factor in optimizing the efficiency of Ca action on the (Ca2+ + Mg2+)-ATPase.     

Ca2+ selectivity of the sarcoplasmic reticulum Ca2+-ATPase at the enzyme-water interface and in the Ca2+ entrance channel

The sarcoplasmic reticulum (SR) Ca(2+)-ATPase, a P-type transmembrane protein, can transport Ca(2+) from the cytoplasmic to the luminal side over other cations specifically. The proposed Ca(2+) entrance channel, composed of the main-chain carbonyl oxygen and side-chain carboxyl oxygen atoms of the amino acids, opens on the enzyme surface, just above the biphospholipid layer membrane-water interface, where Trp residues are frequently found. In this work, the physicochemical nature of Ca(2+) selectivity over Mg(2+) on the surface of the SR Ca(2+)-ATPase has been investigated using the density functional theory (DFT) method. The selection process can be regarded as the first step of the specificity of the enzyme to transport Ca(2+). Subsequently, the specificity of the entrance channel to conduct Ca(2+) over other cations has also been explored. As revealed by thermodynamic analyses, either the aromatic or the aliphatic amino acid residues distributed on the surface of Ca(2+)-ATPase have a bigger affinity to Mg(2+) than to Ca(2+), resulting in a concentration decrease of free Mg(2+) in the local region. Thus, Ca(2+) can transport into the Ca(2+)-entrance channel more easily. Whereafter, for a small quantity of Mg(2+) entering this channel accompanying the Ca(2+) current, the strong electrostatic interactions between Mg(2+) and the ligands will limit the activity of this metal ion, which facilitates the weakly bonded Ca(2+) passing through the channel at a relatively high rate, as suggested by the "sticky-pore" hypothesis. Furthermore, the corresponding theoretical investigations have demonstrated that the increase of the ligand electronegativity can enhance their discrimination between these two cations effectively.     

Interaction between cytoplasmic (Ca2+--Mg2+) ATPase activator and the erythrocyte membrane.

Human red blood cells (RBC) contain a cytoplasmic, nonhemoglobin protein which activates the (Ca2+-Mg2+)ATPase of isolated RBC membranes. Results presented in this paper confirm that activation of (Ca2+-Mg2+)ATPase is associated with binding of the cytoplasmic activator to the membrane. Binding of the cytoplasmic activator is reversible and dependent on ionic strength and Ca2+. Cytoplasmic activator is sensitive to trypsin but is not degraded when intact RBC are exposed to trypsin. Cytoplasmic activator does not modify the (Ca2+-Mg2+)-ATPase of membranes from RBC exposed to activator prior to hemolysis. Thus, the activator is located in the cell and appears to act by binding to the inner membrane surface.     

Binding of Ca2+, Mg2+, and heparin by human serum amyloid P component in affinity capillary electrophoresis

Human serum amyloid P component (SAP) is a glycoprotein circulating in the blood and found in association with all types of amyloid (malfolded potein aggregates) examined so far. Despite uncertainties regarding the precise function of SAP in vivo, the lectin-like properties of this Ca(2+)-activated protein with affinity for anionic saccharides and malfolded proteins are well known. The propensity to form homomeric penta- or decamers in solution and the selfaggregation in the presence of Ca(2+) as well as the tendency of SAP to attach to uncoated fused silica have precluded the analysis of SAP by microelectrophoretic methods. We now work out conditions to characterize the binding of Ca(2+) and Mg(2+) and the binding of heparin to SAP in the presence of divalent metal ions by ACE. The results show a strong binding of heparin (sub-muM apparent dissociation constants) even in the abscence of Ca(2+) at low ionic strength, pH 8.2. Also, a selective interaction with Ca(2+) compared with Mg(2+) is demonstrated. The approach will further the use of microelectrophoretic methods to examine the interactions of SAP with ligands of putative pathophysiological relevance such as lipopolysaccharides and misfolded proteins.     

Active calcium treatment transport via coupling between the enzymatic and the ionophoric sites of Ca2+ + Mg2+-ATPase.

The 20K dalton fragment of Ca2+ + Mg2+-ATPase obtained from th tryptically digested sarcoplasmic reticulum has been further purified using Bio-Gel P-100. This removed low-molecular-weight UV-absorbing and positive Lowry-reacting contaminants. The ionophoric activity of the 20K fragment in both oxidized cholesterol and phosphatidylcholine:cholesterol membranes is unaltered by this further purification. The 20K selectivity sequence in phosphatidylcholine:cholesterol membrane is Ba2+ greater than Ca2+ greater than Sr2+ greater than Mn2+ Mg2+. Digestion of intact sarcoplasmic reticulum vesicles with trypsin, which results in the dissection of the hydrolytic site (30K) from the ionophoric site (20K), is shown to disrupt energy transduction between ATP hydrolysis and calcium transport. This further implicates the 20K dalton fragment as a calcium transport site. These data and previous evidence are discussed in terms of a proposed model for the ATPase molecular structure and the mechanisms of cation transport in sarcoplasmic reticulum.     

Activation of hepatic microsomal Ca2+-adenosine triphosphatase by calcium-binding protein regucalcin

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on Ca2+-adenosine triphosphatase (ATPase) activity in hepatic microsomes was investigated. Mg2+-ATPase activity was clearly increased by the presence of 50 microM Ca2+. Regucalcin (1.0-4.0 microM) caused a remarkable elevation (about 3-fold) of Ca2+-ATPase activity. Also, Mg2+-ATPase activity was increased (about 1.6-fold) by the presence of regucalcin (2.0 and 4.0 microM). Guanosine-5'-O-(3-thiotriphosphate) (GTPrs; 10(-5) and 10(-4) M) and nicotinamide adenine dinucleotide phosphate oxidized form (NADP+; 10(-5) to 10(-3) M) or reduced form (NADPH; 10(-4) and 10(-3) M) significantly increased Ca2+-ATPase activity. These increases were not enhanced by the presence of regucalcin (2.0 microM). Of various metal ions, a comparatively low concentration of V5+ (10(-5) M) or Cd2+ (10(-6) M) significantly increased Ca2+-ATPase activity, while Hg2+, Zn2+, Cu2+ and Mn2+ did not have such an effect. Regucalcin (2.0 microM) did not enhance the effect of V5+ and Cd2+ on Ca2+-ATPase activity. The present finding, that regucalcin activates hepatic microsomal Ca2+-ATPase, suggests a cell physiological role of regucalcin as an activator in the microsomal Ca2+-pump activity. This action of regucalcin may not be influenced by other regulators.     

Effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) and water coordination on the structure of glycine and zwitterionic glycine

Interactions between metal ions and amino acids are common both in solution and in the gas phase. Here, the effect of metal ions and water on the structure of glycine is examined. The effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) and water on structures of Gly.Mn+(H2O)m and GlyZwitt.Mn+(H2O)m (m = 0, 2, 5) complexes have been determined theoretically by employing the hybrid B3LYP exchange-correlation functional and using extended basis sets. Selected calculations were carried out also by means of CBS-QB3 model chemistry. The interaction enthalpies, entropies, and Gibbs energies of eight complexes Gly.Mn+ (Mn+ = Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) were determined at the B3LYP density functional level of theory. The computed Gibbs energies DeltaG degrees are negative and span a rather broad energy interval (from -90 to -1100 kJ mol(-1)), meaning that the ions studied form strong complexes. The largest interaction Gibbs energy (-1076 kJ mol(-1)) was computed for the NiGly2+ complex. Calculations of the molecular structure and relative stability of the Gly.Mn+(H2O)m and GlyZwitt.Mn+(H2O)m (Mn+ = Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+; m = 0, 2, and 5) systems indicate that in the complexes with monovalent metal cations the most stable species are the NO coordinated metal cations in non-zwitterionic glycine. Divalent cations Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+ prefer coordination via the OO bifurcated bonds of the zwitterionic glycine. Stepwise addition of two and five water molecules leads to considerable changes in the relative stability of the hydrated species. Addition of two water molecules at the metal ion in both Gly.Mn+ and GlyZwitt.Mn+ complexes reduces the relative stability of metallic complexes of glycine. For Mn+ = Li+ or Na+, the addition of five water molecules does not change the relative order of stability. In the Gly.K+ complex, the solvation shell of water molecules around K+ ion has, because of the larger size of the potassium cation, a different structure with a reduced number of hydrogen-bonded contacts. This results in a net preference (by 10.3 kJ mol(-1)) of the GlyZwitt.K+H2O5 system. Addition of five water molecules to the glycine complexes containing divalent cations Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+ results in a net preference for non-zwitterionic glycine species. The computed relative Gibbs energies are quite high (-10 to -38 kJ mol(-1)), and the NO coordination is preferred in the Gly.Mn+(H2O)5 (Mn+ = Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) complexes over the OO coordination.     

Ionophoric properties of atropine: complexation and transport of Na+, K+, Mg2+ and Ca2+ ions across a liquid membrane

The activity of atropine on the complexation and transport of Na(+), K(+), Mg(2+) and Ca(2+) ions across a liquid membrane was investigated using a spectrophotometric method. Atropine is a natural drug that blocks muscarinic receptors. It is a competitive antagonist of the action of acetylcholine and other muscarinic agonists. Atropine is shown to extract Na(+), K(+), Mg(2+) and Ca(2+) ions from an aqueous phase into an organic one with a preference for Ca(2+) ions. According to a kinetic study, divalent cations (Mg(2+) and Ca(2+)) are more rapidly transported than monovalent ones (Na(+) and K(+)). In both complexation and transport, the flux of the ions increases with the increase of atropine concentration. Atropine might act on the membrane permeability; its complexation and ionophoric properties shed new lights on its therapeutic properties.     

Al3+, Ca2+, Mg2+, and Li+ in aqueous solution: calculated first-shell anharmonic OH vibrations at 300 K

The anharmonic OH stretching vibrational frequencies, ν(OH), for the first-shell water molecules around the Li(+), Ca(2+), Mg(2+), and Al(3+) ions in dilute aqueous solutions have been calculated based on classical molecular dynamics (MD) simulations and quantum-mechanical (QM) calculations. For Li(+)(aq), Ca(2+)(aq), Mg(2+)(aq), and Al(3+)(aq), our calculated IR frequency shifts, Δν(OH), with respect to the gas-phase water frequency, are about -300, -350, -450, and -750?cm(-1), compared to -290, -290, -420, and -830?cm(-1) from experimental infrared (IR) studies. The agreement is thus quite good, except for the order between Li(+) and Ca(2+). Given that the polarizing field from the Ca(2+) ion ought to be larger than that from Li(+)(aq), our calculated result seems reasonable. Also the absolute OH frequencies agree well with experiment. The method we used is a sequential four-step procedure: QM(electronic) to make a force field+MD simulation+QM(electronic) for point-charge-embedded M(n+) (H(2)O)(y) (second?shell) (H(2)O)(z) (third?shell) clusters+QM(vibrational) to yield the OH spectrum. The many-body Ca(2+)-water force-field presented in this paper is new. IR intensity-weighting of the density-of-states frequency distributions was carried out by means of the squared dipole moment derivatives.     

Effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+, and Zn2+) and water coordination on the structure and properties of L-arginine and zwitterionic L-arginine

Interactions between metal ions and amino acids are common both in solution and in the gas phase. The effect of metal ions and water on the structure of L-arginine is examined. The effects of metal ions (Li(+), Na(+), K(+), Mg(2+), Ca(2+), Ni(2+), Cu(2+), and Zn(2+)) and water on structures of Arg x M(H2O)m , m = 0, 1 complexes have been determined theoretically by employing the density functional theories (DFT) and using extended basis sets. Of the three stable complexes investigated, the relative stability of the gas-phase complexes computed with DFT methods (with the exception of K(+) systems) suggests metallic complexes of the neutral L-arginine to be the most stable species. The calculations of monohydrated systems show that even one water molecule has a profound effect on the relative stability of individual complexes. Proton dissociation enthalpies and Gibbs energies of arginine in the presence of the metal cations Li(+), Na(+), K(+), Mg(2+), Ca(2+), Ni(2+), Cu(2+), and Zn(2+) were also computed. Its gas-phase acidity considerably increases upon chelation. Of the Lewis acids investigated, the strongest affinity to arginine is exhibited by the Cu(2+) cation. The computed Gibbs energies DeltaG(o) are negative, span a rather broad energy interval (from -150 to -1500 kJ/mol), and are appreciably lowered upon hydration.     

Properties of plasma membranes from granulation tissue with reference to extracellular matrix.

Treatment with neuraminidase decreased the activity of Na+,K+-activated Mg2+-adenosine triphosphatase in plasma membranes isolated from experimental granulation tissue but not that of 5'-nucleotidase or leucine-beta-naphthylamidase. A temporary lowering of the pH of the plasma membrane suspension to 2-3 inactivated all three enzymes, which remained inactive after the pH had been readjusted to 7.4. Addition of dextran preparations to the membrane suspension decreased the activity of adenosine triphosphatase. Ethanol (0.4%) had a similar effect. These marker enzymes of plasma membranes were not affected by additions of hyaluronate, chondroitin sulfate, protein polysaccharide or soluble collagen. Serotonin stimulated the adenosine triphosphatase activity slightly. About 10-20% of the protein in the plasma membrane preparation was extracted with EDTA. This "fuzzy coat" fraction yielded a distinct gel-electrophoretic protein pattern. Hyaluronidase was not helpful in cleaving this surface layer from the plasma membranes.     

ATP synthesis in the disk membranes of rod outer segments of bovine retina

ATP is synthesized on the disk membrane isolated from rod outer segments of the bovine retina. Together with a slow component which accounted for a constant rate of about 22 nmol ATP/min/mg of protein and which was due to the adenylate kinase activity, a fast component with a maximal activity of about 58 nmol ATP/min/mg of protein was measured at physiological calcium concentrations. This fast activity disappeared in the presence of the Ca(2+) ionophore A23187, was inhibited by vanadate or thapsigargin but not by oligomycin, suggesting that this ATP synthesis is due to the reversal functioning of the Ca(2+)-ATPase previously found on the disk membranes.     

Cation [M = H+, Li+, Na+, K+, Ca2+, Mg2+, NH4+, and NMe4+] interactions with the aromatic motifs of naturally occurring amino acids: a theoretical study

Ab initio (HF, MP2, and CCSD(T)) and DFT (B3LYP) calculations were done in modeling the cation (H(+), Li(+), Na(+), K(+), Ca(2+), Mg(2+), NH(4)(+), and NMe(4)(+)) interaction with aromatic side chain motifs of four amino acids (viz., phenylalanine, tyrosine, tryptophan and histidine). As the metal ion approaches the pi-framework of the model systems, they form strongly bound cation-pi complexes, where the metal ion is symmetrically disposed with respect to all ring atoms. In contrast, proton prefers to bind covalently to one of the ring carbons. The NH(4)(+) and NMe(4)(+) ions have shown N-H...pi interaction and C-H...pi interaction with the aromatic motifs. The interaction energies of N-H...pi and C-H...pi complexes are higher than hydrogen bonding interactions; thus, the orientation of aromatic side chains in protein is effected in the presence of ammonium ions. However, the regioselectivity of metal ion complexation is controlled by the affinity of the site of attack. In the imidazole unit of histidine the ring nitrogen has much higher metal ion (as well as proton) affinity as compared to the pi-face, facilitating the in-plane complexation of the metal ions. The interaction energies increase in the order of 1-M < 2-M < 3-M < 4-M < 5-M for all the metal ion considered. Similarly, the complexation energies with the model systems decrease in the following order: Mg(2+) > Ca(2+) > Li(+) > Na(+) > K(+) congruent with NH(4)(+) > NMe(4)(+). The variation of the bond lengths and the extent of charge transfer upon complexation correlate well with the computed interaction energies.     

Three types of membranous ATPase on rat liver lysosomes.

At least three types of vanadate-insensitive membranous ATPase were identified on rat liver lysosomes: bafilomycin A1-sensitive Mg(2+)-ATPase (H(+)-ATPase), N-ethylmaleimide (NEM)-sensitive but bafilomycin A1-insensitive Mg(2+)-ATPase (ATPase I), and NEM-insensitive Ca2+/Mg(2+)-ATPase (ATPase II). They showed different sensitivity to chemicals and ions with apparent molecular masses of 700-800, 500-650, and 360 kDa, respectively. Of these membranous ATPases, H(+)-ATPase seemed to constitute only one tenth of the ATPase activity on rat liver lysosomes and to be the only ATPase that exposed its active site to the cytoplasmic side of the lysosomal membranes.     

Phosphorprotein intermediate in the Ca2+-dependent ATPase reaction of macrophage plasma membrane.

ATPase activity and phosphorylation by [gamma-32P] ATP of isolated plasma membrane of alveolar macorphages are stimulated in a parallel fashion by physiologic concentrations of Ca2+, with half-maximal activating effect of this ion at (3--7) X 10(-7) M. For various membrane preparations, a direct proportionality exists between Ca2+-dependent ATPase activity and amount of 32P incorporated. Labeling of membrane attains the steady-state level by 10 sec at 0 degrees C, and is rapidly reversed by adenosine diphosphate (ADP), K+ decreases the amount of membrane-bound 32P, mainly by enhancing the rate of dephosphorylation of the 32P-intermediate. Hydroxylamine causes a release of about 90% of 32P bound to the membrane, thus indicating that the 32P-intermediate contains an acyl-phosphate bond. When the labeled plasma membrane is solubilized and electrophoresed on acrylamide gels in the presence of sodium dodecyl sulphate, the radioactivity appears to be largely associated with a single protein fraction of 132,000 +/- 2,000 aarent molecular weight. These features of the macrophage Ca2+-ATPase suggest that the enzyme activity might be part of a surface-localized Ca1+-extrusion system, participating in the regulation of Ca2+-dependent activities of the macrophage.     

Direct AFM measurements of adhesion forces between calcium oxalate monohydrate and kidney epithelial cells in the presence of Ca2+ and Mg2+ ions

Adhesion forces between the calcium oxalate monohydrate (COM, whewellite) crystal and the layer of the epithelial kidney cells have been directly measured under buffer solutions by using atomic force microscope (AFM). Two renal epithelial lines, MDCK (a collecting duct line) and LLC-PK1 (a proximal tubular line), were used. All experiments were conducted in buffer solutions containing additional Ca(2+) and Mg(2+) ions in the various concentrations. For MDCK-cells, the obtained values of the adhesion force were in the range 0.12-0.51 nN and 0.12-0.20 nN for Ca(2+) and Mg(2+), respectively. No adhesion force (larger than 0.05 nN) has been found for LLC-PK1 cells. The "critical" concentrations of ions, near which the adhesion force (for MDCK-cells) was maximal, were found to be 100 mM. The "critical" concentration of ions and the tendency of the adhesion forces with the changing ions concentration, confirm earlier results of Lieske et al. [J.C. Lieske, G. Farell, S. Deganello, Urol. Res. 32 (2004) 117-123], in which the affinity (rather than the adhesion force) between the COM micro-crystals and the layer of the MDCK-cells were measured, calculating the radioactive signal of radioactive (14)C COM-crystals stuck to the cells. We believe that the aggregation of the COM crystals does not occur in the bulk urine due to short travel time through the nephron. If so, the kidney stone formation is determined by COM-seeding on the tubules walls. The further growth of the stone on the seed can take practically unlimited time because the COM crystal is practically is not soluble in water or urine solutions. The value of the adhesion force can be useful for evaluation of the adhesion energy or probability of the COM-aggregates to stick to the kidney epithelium under the urine flow. This probability is calculated taking into account the adhesion force, F(ad), and hydrodynamic driving force of the flow. This probability reflects the opportunity of the small aggregates to grow and form the kidney stones.     

Single molecular multianalyte (Ca2+, Mg2+) fluorescent probe and applications to bioimaging

Intracellular signal transduction relies on spatial and temporal signal transmitter dynamics. To clarify the correlations of these transmitter molecules, multicolor-imaging has been widely used. However, in the case of applying multiple indicators in a cell, spectral overlap of the indicators prevents accurate quantitative analysis. Moreover, the invasive (toxic) effect, the localization, the metabolism, as well as photobleaching of these indicators complicate the situation. Here, we show that single-molecular multifluorescent probes can overcome these problems. While intracellular calcium plays a critical role as a signal transmitter and magnesium acts as a cofactor in many situations, the correlations between the two cations are now the main issue. We designed and synthesized a Ca2+-Mg2+ responsive multifluorescent probe, KCM-1. KCM-1 shows a spectral blue shift upon complexation to Ca2+ and a red shift to the presence of Mg2+. With data analyzed at different excitation wavelengths, the concentrations of Ca2+ and Mg2+ are simultaneously quantified. Furthermore, by using the AM-ester method, intracellular Ca2+ and Mg2+ concentrations are simultaneously imaged. Such a type of intracellular multiple analyte imaging by a single-molecular multifluorescent probe is successfully demonstrated for the first time.     

Coagulation of bitumen with kaolinite in aqueous solutions containing Ca2+, Mg2+ and Fe3+: effect of citric acid

Heterocoagulation experiments of kaolinite with solvent-diluted-bitumen were carried out to investigate the effect of hydrolyzable metal cations and citric acid on the liberation of bitumen from kaolinite. The adsorption of Ca(2+) and Mg(2+) on kaolinite, and zeta potentials of kaolinite and bitumen droplets in solutions containing 10(-3)mol/L of Ca(2+), Mg(2+) and Fe(3+) with or without citric acid were also measured. It was found that the heterocoagulation of bitumen with kaolinite was enhanced in the presence of the metal cations from pH 7 to pH 10.5, accompanied by a decrease in the magnitude of the zeta potentials and an increase in the adsorption of the metal cations on kaolinite and possibly on bitumen droplets. The addition of 5 x 10(-4)mol/L citric acid reduced the degree of coagulation from 90% to less than 40% in the presence of 10(-3)mol/L Ca(2+) and Mg(2+) cations at pH approximately 10, and at pH approximately 8 for Fe(3+). It was found that hydrolyzable metal cations enhanced bitumen-kaolinite interactions through electrical double layer compression and specific adsorption of the metal hydrolysis species on the surface of kaolinite. The effect of metal cations was removed by citric acid through formation of metal-citrate complexes and/or the adsorption of citrate anions, which restored the zeta potentials of both kaolinite and bitumen. Therefore, electrostatic attraction or repulsion was responsible for the coagulation or dispersion of kaolinite particles from bitumen droplets in the tested system.