Ca2+-cardiolipin interaction in a model system Selectivity and apparent high affinity

The interaction of cardiolipin with Ca²⁺ was assessed by measuring the cardiolipin-mediated extraction of ⁴⁵Ca²⁺ from an aqueous to an organic (methylene chloride) phase. Cardiolipin binds Ca²⁺ with high affinity [$\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{(}\text{apparent}\text{)=0.70±0.17 μ}\text{M (S.D.)}$]. Cation-cardiolipin interactions are selective. Interaction of cardiolipin with Ca²⁺ is insensitive to Na⁺, but is inhibited by divalent cations with $\text{Mn}{}^{\mathrm{2+}}\text{>}\text{Zn}{}^{\mathrm{2+}}\text{>}\text{Mg}{}^{\mathrm{2+}}$. In addition La³⁺ and Ruthenium red are particularly potent inhibitors of Ca²⁺ binding by cardiolipin. Cardiolipin-mediated extraction of Ca²⁺ into an aqueous phase is also inhibited by phosphatidylcholine. Inhibition of Ca²⁺-cardiolipin interaction by phosphatidylcholine (a phospholipid known to stabilize the bilayer conformation) may implicate inverted, non-bilayer lipid structures in the binding.     

Spin-label studies on the specificity of interaction of cardiolipin with beef heart cytochrome oxidase

The selectivity of interaction of various cardiolipin analogues with beef heart cytochrome oxidase in reconstituted complexes with dimyristoylphosphatidylcholine has been studied by electron spin resonance spectroscopy, using lipids spin-labeled in the acyl chains. No difference in selectivity is observed between cardiolipin and its monolyso derivative, and similarly no selectivity is observed between phosphatidylcholine and lysophosphatidylcholine. Removal of the cardiolipin charge by methylation of the phosphate groups reduces but does not eliminate selectivity relative to phosphatidylcholine. The dependence of the lipid selectivity on head group and chain composition is in the order cardiolipin approximately equal to monolysocardiolipin greater than acylcardiolipin greater than dimethylcardiolipin greater than phosphatidylcholine approximately equal to lysophosphatidylcholine, where acylcardiolipin has the spin-label chain attached at the center -OH of the head group. The degree of association of the negatively charged cardiolipin derivatives with cytochrome oxidase decreases with increasing salt concentration, to a level comparable to that for dimethylcardiolipin. At high ionic strength there is still a marked selectivity relative to phosphatidylcholine. Li+ ions are more effective in screening the interaction than are Na+ ions, and divalent ions are more effective than monovalent ions. The selectivity for cardiolipin is only slightly reduced on titrating the protein to high pH. Alkylation of the protein with N-ethylmaleimide has little effect on the titration behavior. Covalent modification of the protein by reaction with citraconic anhydride decreases the selectivity of interaction with cardiolipin. It is concluded that cardiolipin possesses an additional specificity of interaction with cytochrome oxidase other than that of purely electrostatic origin.     

Molecular aspects of the bilayer stabilization induced by poly(L-lysines) of varying size in cardiolipin liposomes

The interaction between poly(L-lysines) of varying size with cardiolipin was investigated via binding assays, X-ray diffraction, freeze-fracture electron microscopy, and 31P- and 13C-NMR. Binding of polylysines to the lipid only occurred when three or more lysine residues were present per molecule. The strength of the binding was highly dependent on the polymerization degree, suggesting a cooperative interaction of the lysines within the polymer. Upon binding, a structural reorganization of the lipids takes place, resulting in a closely packed multilamellar system in which the polylysines are sandwiched in between subsequent bilayers. Acyl chain motion is reduced in these liquid-crystalline peptide-lipid complexes. From competition experiments with Ca2+ it could be concluded that when the affinity of the polylysine for cardiolipin was much larger than that of Ca2+, a lamellar polylysine-lipid complex was formed, irrespective of whether an excess of Ca2+ was added prior to or after the polypeptide. When the affinity of the polylysine for cardiolipin was less or of the same order as that of Ca2+, the lipid was organized in the hexagonal HII phase in the presence of Ca2+. These results are discussed in the light of the peptide specificity of bilayer (de)stabilization in cardiolipin model membranes.     

The interaction of cardiolipin with rat liver carbamoyl phosphate synthetase I

A selective interaction of rat liver carbamoyl phosphate synthetase I with cardiolipin, and other anionic phospholipids, has been demonstrated. The enzymatic activity of the synthetase is inhibited by cardiolipin and, to a lesser extent, by phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine. This group of anionic phospholipids also induced a conformational change in the synthetase, yielding a species with increased exposure of the linkages between independently folded domains of the enzyme, as determined by limited proteolysis under nondenaturing conditions. The interaction of cardiolipin with carbamoyl phosphate synthetase I was a fairly slow process, with complex kinetics, and was apparently irreversible. The inclusion of Mg2+ or of MgATP in the incubation mixture prevented the cardiolipin effects. The zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine had negligible effects on the structure and activity of the synthetase. This interaction between cardiolipin and carbamoyl phosphate synthetase I potentially constitutes one of the mechanisms by which the synthetase forms its loose association with the inner mitochondrial membrane. Multiple mechanisms, including synthetase conformational changes, cardiolipin phase changes, and ATP/ADP binding site involvement, are possibly involved in the phospholipid/synthetase interaction and the resulting potential regulatory mechanism(s) for urea cycle activity.     

Translocation and binding of adenine nucleotides by rat liver mitochondria partially depleted of phospholipids.

1. Rat liver mitochondria were partially depleted of their phospholipids using phospholipase A prepared from porcine pancreas (substrate specificity, cardiolipin greater than phosphatidylethanolamine greater than phosphatidylcholine) or from Crotalus adamanteus venom (substrate specificity, phosphatidylethanolamine = phosphatidylcholine greater than cardiolipin). 2. Removal of only about 1% of the mitochondrial phospholipid with the pancreatic enzyme leads to 50% and 25% losses in ADP and ATP translocation, respectively. Concomitant with the loss in translocation is a decline in the ability of both carbonylcyanide m-chlorophenylhydrazone and Ca2+ to stimulate ATP translocation. 3. To achieve comparable losses in ADP and ATP translocation with the venom enzyme, it is necessary to remove about 8% of the total mitochondrial phospholipid. Following such treatment, carbonylcyanide m-chlorophenylhydrazone and Ca2+ are still capable of stimulating ATP translocation. 4. Control experiments involving treatment of the mitochondria with the products of phospholipase digestion indicate that the effects observed on the translocase reflect a loss of phospholipid from the membrane. 5. Binding studies indicate that the loss in adenine nucleotide translocation following phospholipase treatment cannot be accoundted for by an altered ability to bind adenine nucleotides to atractyloside-sensitive sites. 6. The data are interpreted in terms of a mechanism of adenine nucleotide translocation involving a lipoprotein carrier system, consisting of the translocator protein and phospholipids, possibly cardiolipin and phosphatidylethanolamine.     

The interaction of adriamycin with small unilamellar vesicle liposomes. A fluorescence study

The interaction of the antineoplastic agent adriamycin with sonicated liposomes composed of phosphatidylcholine alone and with small amounts (1-6%) of cardiolipin has been studied by fluorescence techniques. Equilibrium binding data show that the presence of cardiolipin increases the amount of drug bound to liposomes when the bilayer is below its phase transition temperature and when the ionic strength is relatively low (0.01 M). At higher ionic strength (0.15 M) and above the Tm (i.e. conditions which are closer to the physiological state) the binding of the drug to the two liposome types is nearly the same. Thus the differences in the interactions of adriamycin with cardiolipin-containing membranes, as opposed to those composed of phosphatidylcholine alone, are not due simply to increased binding but rather to an altered membrane structure when this lipid is present. Quenching of adriamycin fluorescence by iodide shows that bound drug is partially, but not completely, buried in the liposomal membrane. Both in the presence and absence of cardiolipin the bulk of the adriamycin is more accessible to the quencher below the Tm than above it; that is, a solid membrane tends to exclude the drug from deep penetration. Above the Tm, the presence of cardiolipin alters the nature of liposome-adriamycin interaction. Here the fluorescence quenching data suggest that the presence of small amounts of cardiolipin (3%) in a phosphatidylcholine matrix creates two types of binding environments for drug, one relatively exposed and the other more deeply buried in the membrane. The temperature dependence of the adriamycin fluorescence and the liposome light scattering reveal that cardiolipin alters the thermal properties of the bilayer as well as its interaction with adriamycin. At low ionic strength lateral phase separations may occur with both pure phosphatidylcholine and when 3% cardiolipin is present; under these conditions the bound adriamycin exists in two kinds of environment. It is notable that only adriamycin fluorescence reveals this phenomenon; thebulk property of liposome light scattering reports only on the overall membrane phase change. These data suggest that under certain conditions the drug binding sites in the membranes are decoupled from the bulk of the lipid bilayer.     

Phase transition characteristics of diphosphatidyl-glycerol (cardiolipin) and stereoisomeric phosphatidyldiacylglycerol bilayers. Mono- and divalent metal ion effects

Synthesis and phase transition chaaracteristics of aqueous dispersions of the homologous (12 : 0, 14 : 0, 16 : 0) diphosphatidylglycerols (cardiolipins) and phosphatidyldiacylglycerols are reported. Electron microscopy of the negatively stained aqueous dispersions reveals a characteristic lamellar structure suggesting that these phospholipid molecules are organized as bilayers in the aqueous dispersions. The phase transition temperature (Tm) and the enthalpy of transition (delta H) increase monotonically with chain length in the cardiolipin and phosphatidyldiacylglycerol series; Tm for phosphatidyldiacylglycerol is higher than that for cardiolipin of the same chain-length. The transition temperatures for the enantiomeric sn-3,3- and sn-1,1-phosphatidyldiacylglycerol and for the diastereomeric, meso-sn-1,3-phosphatidyldiacylglycerol are approximately the same. The molar enthalpy for the transition of cardiolipin-NH+4 bilayers is approximately twice the value for the phosphatidylcholines of the same chain length, i.e., the molar enthalpy per acyl chain is approximately the same in the two systems. The transition temperatures for metal ion salts of C16-cardiolipin exhibit a biphasic dependence upon the unhydrated ionic radii, i.e., the highest Tm is observed for Ca2+-cardiolipin and decreases for the salts of ions with smaller and larger ionic radii than that of Ca2+. The lowest Tm is observed for Rb+-cardiolipin. Monovalent metal salts of cardiolipin exhibit two phase transitions. This effect may result from different conformational packing of the four acyl chains due to differences in metal-phosphate binding.     

Cytochrome c specifically induces non-bilayer structures in cardiolipin-containing model membranes

(1) The effect of cytochrome c addition on the phospholipid structure of liposomes composed of cardiolipin, phosphatidylserine, phosphatidylglycerol, phosphatidylcholine or phosphatidylethanolamine in a pure form or in mixtures was investigated by 31P-NMR and freeze-fracture techniques. (2) Cytochrome c specifically induces the hexagonal Hii phase and possibly an inverted micellar structure of part of the phospholipids in cardiolipin-containing model membranes. (3) These results are compared with the effect of Ca2+ on cardiolipin and are discussed in relation to the structure and function of the inner mitochondrial membrane.     

Phosphoinositide-protein interactions of the plasma-membrane Ca2(+)-transport ATPase as revealed by fluorescence energy transfer.

Fluorescence energy transfer has been used to study the interaction of various phospholipids with the erythrocyte (Ca2+ + Mg2+)-ATPase. The fluorescence energy transfer between tryptophan residues of the (Ca2+ + Mg2+)-ATPase purified from erythrocytes and pyrene-labelled analogues of phosphatidylcholine (Pyr-PC), phosphatidylinositol (Pyr-PI), phosphatidylinositol 4-phosphate (Pyr-PIP), phosphatidylinositol 4,5-bisphosphate (Pyr-PIP2), phosphatidylglycerol (Pyr-PG) and phosphatidic acid (Pyr-PA) was measured. A positive correlation was found between the number of negative charges on the phospholipids (PIP2 greater than PIP greater than PA greater than PI = PG greater than PC) and the potency of their pyrene-labelled analogues to act as quantum acceptors in fluorescence energy transfer from the tryptophan residues of the (Ca2+ + Mg2+)-ATPase. This is the first time that a physical interaction between PIP/PIP2 and an intrinsic membrane protein has been demonstrated. The dependence of the energy transfer on the number of negative charges of the phospholipids closely resembles the previously demonstrated charge dependence of the enzymatic activity of the (Ca2+ + Mg2+)-ATPase (Missiaen, L., Raeymaekers, L., Wuytack, F., Vrolix, M., Desmet, H. and Casteels, R. (1989) Biochem. J. 263, 687-694). It is concluded that the stimulation of the (Ca2+ + Mg2+)-ATPase activity by negatively charged phospholipids is based on a binding of these lipids to the (Ca2+ + Mg2+)-ATPase and that the negative charges are a major modulatory factor for this interaction.     

Calcium ion binding between lipid bilayers: the four-component system of phosphatidylserine, phosphatidylcholine, calcium chloride, and water

Ca2+ binding between lamellae of phosphatidylserine (PS) and phosphatidylcholine (PC) gives rise to a rigid phase of Ca(PS)2. When aqueous Ca2+, hydrated PS/PC, and Ca(PS)2 coexist at equilibrium, the aqueous Ca2+ concentration is invariant and is characteristic of the PS/PC ratio. This characteristic Ca2+ concentration is 0.040 microM for palmitoyloleoylphosphatidylserine without PC and increases as the inverse square of the PS mole fraction at high PS concentration (Raoult's law) and as the inverse square of the PS mole fraction multiplied by a constant at low PS concentration (Henry's law). For example, for palmitoyloleoylphosphatidylserine/palmitoyloleoylphosphatidylcholi ne = 0.6/0.4 or 0.2/0.8, this characteristic Ca2+ concentration is about 0.1 or about 6 microM, respectively. These observations at constant temperature are summarized in a quaternary phase diagram for the four-component system CaCl2/PS/PC/water.     

A fluorescent calmodulin that reports the binding of hydrophobic inhibitory ligands.

Ca2+ binding to calmodulin in the pCa range 5.5-7.0 exposes hydrophobic sites that bind hydrophobic inhibitory ligands, including calmodulin antagonists, some Ca2+-antagonists and calmodulin-binding proteins. The binding of these hydrophobic ligands to calmodulin can be followed by the approx. 80% fluorescence increase they produce in dansylated (5-dimethylaminonaphthalene-1-sulphonylated) calmodulin (CDRDANS). In the presence of Ca2+, calmodulin binds the calmodulin inhibitor, R24571, with an affinity of approx. 2-3 nM and hydrophobic ligands, including trifluoperazine (TFP), W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide], fendiline, felodipine and prenylamine, with affinities in the micromolar range. This binding is strongly Ca2+-dependent and Mg2+-independent. Calmodulin shows a reasonably high degree of specificity in its binding of these ligands over other ligands tested. CDRDANS, therefore, provides a convenient and simple means of monitoring the interaction of a variety of hydrophobic ligands with the Ca2+-dependent regulatory protein, calmodulin. CDRDANS binds to phospholipid vesicles made of (dimyristoyl)phosphatidylcholine (DMPC) or (dipalmitoyl)phosphatidylcholine (DPPC) and produces fluorescence increases only in the presence of Ca2+ and at temperatures above their gel-to-liquid crystalline phase transition. Although the fluorescence changes in CDRDANS accurately report phase transitions in these liposomes, its binding to these vesicles is weak. Calmodulin probably requires a high-affinity lipid-bound receptor protein for its high-affinity binding to natural membranes.     

The effect of adriamycin and duramycin on calcium translocation in liposome systems modeled on the inner mitochondrial membrane

Adriamycin (doxorubicin, AdM) is a potent antineoplastic agent which binds specifically and with high affinity to the acidic phospholipid cardiolipin (CL) [Goormaghtigh et al. (1980) Biochim. Biophys. Acta 597, 1]. Duramycin (DM), a polypeptide antibiotic, has been reported to interact selectively with phosphatidylethanolamine (PE) and monogalactosyldiacylglycerol [Navarro et al. (1985) Biochemistry 24, 4645]. The selectivity of DM-PE interaction was confirmed. AdM and DM were then used to explore the roles of CL and PE in Ca2+ translocation in a phosphatidylcholine (PC)/PE/CL liposome system modeled on the inner mitochondrial membrane with the following results: (i) AdM (100-400 microM) altered Ca2+ uptake by PC/PE/CL (4/4/1, mol/mol) liposomes in a concentration-dependent fashion which varied with temperature, external Ca2+ concentration, and liposome PE content. (ii) Addition of AdM was qualitatively equivalent to increasing temperature, Ca2+ concentration, or liposome PE content, and cooperative interactions among these parameters were observed. An increase in any one factor generally enhanced Ca2+ uptake; simultaneous increases in several factors inhibited uptake. (iii) Inhibition of Ca2+ uptake was correlated with efflux of Arsenazo III. (iv) Ca2+ uptake by PC/PE/CL liposomes is biphasic [Kester and Sokolove (1989) Biochim. Biophys. Acta 980, 127]. DM suppressed the PE-dependent slow phase and stimulated the PE-independent initial phase. Ca2+ uptake by PC/PE/CL liposomes in the presence of DM resembled uptake by PC/CL liposomes. These data confirm the ability of PE to enhance the slow, highly temperature-dependent component of CL-mediated Ca2+ translocation and suggest that this process is sensitive to lipid phase behavior.     

Interaction of peroxidized cardiolipin with rat-heart mitochondrial membranes: induction of permeability transition and cytochrome c release

Cardiolipin peroxidation plays a critical role in mitochondrial cytochrome c release and subsequent apoptotic process. Mitochondrial pore transition (MPT) is considered as an important step in this process. In this work, the effect of peroxidized cardiolipin on MPT induction and cytochrome c release in rat heart mitochondria was investigated. Treatment of mitochondria with micromolar concentrations of cardiolipin hydroperoxide (CLOOH) resulted in a dose-dependent matrix swelling, DeltaPsi collapse, release of preaccumulated Ca2+ and release of cytochrome c. All these events were inhibited by cyclosporin A and bongkrekic acid, indicating that peroxidized cardiolipin behaves as an inducer of MPT. Ca2+ accumulation by mitochondria was required for this effect. ANT (ADP/ATP translocator) appears to be involved in the CLOOH-dependent MPT induction, as suggested by the modulation by ligands and inhibitors of adenine nucleotide translocator (ANT). Together, these results indicate that peroxidized cardiolipin lowers the threshold of Ca2+ for MPT induction and cytochrome c release. This synergistic effect of Ca2+ and peroxidized cardiolipin on MPT induction and cytochrome c release in mitochondria, might be important in regulating the initial phase of apoptosis and also may have important implications in those physiopathological situations, characterized by both Ca2+ and peroxidized cardiolipin accumulation in mitochondria, such as aging, ischemia/reperfusion and other degenerative diseases.     

The effect of holotoxin A1 on transport of calcium ions across the lipid models of biological membranes

Planar bilayer lipid membranes formed from trepang phospholipids possess an intrinsic Ca2(+)-permeability. These phospholipids dissolved in a non-polar solvent can extract 45Ca2+ from the aqueous to the organic phase. The triterpenic glycoside holotoxin A isolated from the trepang Stichopus japonicus inhibits the Ca2+ flux of lipid bilayers from trepang phospholipids as well as the Ca2+ flux induced in phosphatidylcholine bilayers by the calcium ionophore X-537A. Toxin inhibits the Ca2+ ionophore A23187 induced Ca2+ efflux from phosphatidylcholine liposomes and 45Ca2+ transition from the aqueous to the organic phase. Holotoxin A does not inhibit the 45Ca2+ transfer to the non-polar phase induced by holoturia phospholipids and does not affect the phosphatidylcholine hydroperoxide-induced Ca2+ flux of lipid bilayers. Using the fluorescent probe pyrene, it was demonstrated that toxin increases the microviscosity of liposomal membranes and trepang oocyte "ghosts".     

Spin label studies on the selectivity of lipid-protein interaction of cardiolipin analogues with the Na+/K+-ATPase

The selectivity of lipid-protein interaction for various spin-labelled cardiolipin analogues in Na+/K+-ATPase membranes from Squalus acanthias has been investigated by ESR spectroscopy. Cardiolipin derivatives with different numbers of acyl chains, or in which the headgroup charge has been removed by methylation of the phosphate groups, all show a pronounced selectivity relative to phosphatidylcholine. Maximally three times more of the cardiolipin analogue is associated with the protein, than is phosphatidylcholine. The selectivity pattern in the absence of salt is in the order: cardiolipin approximately monolysocardiolipin greater than or equal to acylcardiolipin greater than dimethylcardiolipin much greater than phosphatidylcholine, where acylcardiolipin has the spin label chain attached to the centre-OH group of the headgroup. The degree of association of the negatively charged cardiolipins with the protein is reduced by salt, corresponding to the lower selectivity for dimethylcardiolipin. It is concluded that the selectivity of the Na+/K+-ATPase for cardiolipin is not solely of electrostatic origin, nor is it likely to originate in the larger number of fatty acid chains relative to diacyl phospholipids.     

Protection against membrane damage: a 1H-NMR investigation of the effect of Zn2+ and Ca2+ on the permeability of phospholipid vesicles

1H-NMR spectroscopy of small, unilamellar dipalmitoyl phosphatidylcholine (DPPC) vesicles in conjunction with the lanthanide shift reagent Pr3+ was used to study the effect of Zn2+ and Ca2+ ions on the permeability induced at the lipid phase transition temperature (Tc) of the vesicles and by the bee venom polypeptide melittin. In addition, the effects of Zn2+ and Ca2+ were studied on Triton X-100 stabilized channels at Tc and in the presence of n-alcohols. The results show that the presence of 10 mM Zn2+ and Ca2+ inhibited most of the forms of vesicular permeability investigated. The results are discussed in terms of the nature of the binding of the metal ions to the vesicles and support the proposal that one biological function of Zn2+ and Ca2+ is protection against membrane damage.     

Fusion of erythrocyte ghosts induced by calcium phosphate. Kinetic characteristics and the role of Ca2+, phosphate and calcium-phosphate complexes

Using an assay which allows continuous monitoring of the mixing of aqueous contents during membrane fusion, we have investigated the kinetics of calcium-phosphate-induced fusion of erythrocyte ghosts. In the presence of 10 mM phosphate, the threshold concentration for Ca2+-induced fusion was 1.25 mM, while the optimal concentration was approx. 1.75 mM Ca2+. Further enhancement of the cation concentration (greater than or equal to 2 mM) inhibited fusion of the ghosts. Initiation of fusion required the addition of phosphate prior to the addition of Ca2+, indicating that the combined interaction of Ca2+ and phosphate in or at the plane of the bilayer was a prerequisite for the induction of fusion. Furthermore, fusion was greatly facilitated upon transformation of calcium phosphate in the bulk medium from an amorphous to a solid, crystalline phase. It is suggested that membrane aggregation, and hence fusion, is facilitated by the formation of crystalline calcium phosphate nucleating on the ghost membrane. La3+, Mg2+ and Mn2+ did not trigger the fusion process, although aggregation of the ghosts did occur. Under conditions where calcium phosphate precipitation was inhibited, lanthanum phosphate precipitates facilitated fusion after prior treatment of ghosts with phosphate and Ca2+. These results indicated that fusion-prone conditions were induced prior to calcium phosphate precipitation. It is proposed that prior to calcium phosphate precipitation membrane changes are induced by separate interaction of Ca2+ and phosphate with the ghost membrane. Such an interaction could then render the ghosts susceptible to fusion and as soon as conditions are provided allowing close contact between adjacent membranes, fusion will be observed.     

Stacking of safranine in liposomes during valinomycin-induced efflux of potassium ions.

Liposomes were prepared from phosphatidylcholine and cardiolipin in a KCl medium and suspended in a choline chloride medium with safranine. When efflux of K+ was induced by valinomycin, spectral shifts characteristic of stacking were observed. Ca2+ inhibited the rate of stacking in a competitive manner with a Ki of about 200 muM, while La3+ was about 10 times more potent. When liposomes were prepared from phospholipids with a higher ratio of cardiolipin to phosphatidylcholine the inhibition was more potent. No effect on the stacking phenomena was seen when CA2+ was added after the stacking was completed. When CA2+ or an organic cation with four charges, spermine was trapped in the intraliposomal compartment, no significant change in the rate of stacking was seen. However, the extent of stacking was decreased. It is suggested that safranine is driven by a diffusion potential to a site that is inaccessible to CA2+ in the medium, presumably to the inner boundaries of the liposomal membranes.     

Characterization of calcium binding to brush-border membranes from rat duodenum.

The Ca2+-binding properties of isolated brush-border membranes at physiological ionic strength and pH were examined by rapid Millipore filtration. A comprehensive analysis of the binding data suggested the presence of two types of Ca2+-binding sites. The high-affinity sites, Ka = (6.3 +/- 3.3) X 10(5) M-1 (mean +/- S.E.M.), bound 0.8 +/- 0.1 nmol of Ca2+/mg of protein and the low-affinity sites, Ka = (2.8 +/- 0.3) X 10(2) M-1, bound 33 +/- 3.5 nmol of Ca2+/mg of protein. The high-affinity site exhibited a selectivity for Ca2+, since high concentrations of competing bivalent cations were required to inhibit Ca2+ binding. The relative effectiveness of the competing cations (1 and 10 mM) for the high-affinity site was Mn2+ approximately equal to Sr2+ greater than Ba2+ greater than Mg2+. Data from the pH studies, treatment of the membranes with carbodi-imide and extraction of phospholipids with aqueous acetone and NH3 provided evidence that the low-affinity sites were primarily phospholipids and the high-affinity sites were either phosphoprotein or protein with associated phospholipid. Two possible roles for the high-affinity binding sites are suggested. Either high-affinity Ca2+ binding is involved with specific enzyme activities or Ca2+ transport across the luminal membrane occurs via a Ca2+ channel which contains a high-affinity Ca2+-specific binding site that may regulate the intracellular Ca2+ concentration and gating of the channel.     

The protein-mediated transfer of phosphatidylcholine between membranes. The effect of membrane lipid composition and ionic composition of the medium.

The phosphatidylcholine exchange protein from bovine liver catalyzes the transfer of phosphatidylcholine between rat liver mitochondria and sonicated liposomes. The effect of changes in the liposomal lipid composition and ionic composition of the medium on the transfer have been determined. In addition, it has been determined how these changes affected the electrophoretic mobility i.e. the surface charge of the membrane particles involved. Transfer was inhibited by the incorporation of negatively charged phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol into the phosphatidylcholine-containing vesicles; zwitterionic phosphatidyl-ethanolamine had much less of an inhibitory effect while positively charged stearylamine stimulated. The cation Mg2+ and, to a lesser extent, K+ overcame the inhibitory effect exerted by phosphatidic acid, in that concentration range where these ions neutralized the negative surface charge most effectively. Under conditions where Mg2+ and K+ affected the membrane surface charge relatively little inhibition was observed. In measuring the protein-mediated transfer between a monolayer and vesicles consisting of only phosphatidylcholine, cations inhibited the transfer in the order La3+ greater than Mg2+ larger than or equal to Ca2+ greater than K+ = Na+. Inhibition was not related to the ionic strength, and very likely reflects an interference of these cations with an electrostatic interaction between the exchange protein and the polar head group of phosphatidylcholine.