Kinetics of Ca2+-induced fusion of cardiolipin-phosphatidylcholine vesicles: correlation between vesicle aggregation, bilayer destabilization, and fusion

We have investigated the kinetics of Ca2+-induced aggregation and fusion of large unilamellar vesicles composed of an equimolar mixture of bovine heart cardiolipin and dioleoylphosphatidylcholine. Mixing of bilayer lipids was monitored with an assay based on resonance energy transfer (RET) and mixing of aqueous vesicle contents with the Tb/dipicolinate assay. The results obtained with either assay were analyzed in terms of a mass action kinetic model, providing separate rate constants for vesicle aggregation and for the fusion reaction proper. At different Ca2+ concentrations, either at 25 degrees C or at 37 degrees C, aggregation rate constants derived from the data obtained with the RET assay were the same as those derived from the Tb/dipicolinate data, indicating that mixing of bilayer lipids occurred only during vesicle aggregation events that resulted in mixing of aqueous contents as well. At 25 degrees C, identical fusion rate constants were obtained with either assay, indicating that at this temperature the probability of lipid mixing and that of aqueous contents mixing, occurring after vesicle aggregation, were the same. The fusion rate constants for the RET assay increased more steeply with increasing temperature than the fusion rate constants derived from the Tb/dipicolinate data. As a result, at 37 degrees C the tendency of the vesicles, after aggregation, to mix lipids was slightly higher than their tendency to mix aqueous contents. The aggregation rate constants increased steeply with Ca2+ concentrations increasing in a narrow range (9.5-11 mM), indicating that, in addition to a Ca2+-dependent charge neutralization on the vesicle surface, structural changes in the lipid bilayer are involved in the aggregation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-induced fusion of phosphatidylserine vesicles: mass action kinetic analysis of membrane lipid mixing and aqueous contents mixing

We have investigated the initial kinetics of Ca2+-induced aggregation and fusion of phosphatidylserine large unilamellar vesicles at 3, 5 and 10 mM Ca2+ and 15, 25 and 35 degrees C, utilizing the Tb/dipicolinate (Tb/DPA) assay for mixing of aqueous vesicle contents and a resonance energy transfer (RET) assay for mixing of bilayer lipids. Separate rate constants for vesicle aggregation as well as deaggregation and for the fusion reaction itself were determined by analysis of the data in terms of a mass action kinetic model. At 15 degrees C the aggregation rate constants for either assay are the same, indicating that at this temperature all vesicle aggregation events that result in lipid mixing lead to mixing of aqueous contents as well. By contrast, at 35 degrees C the RET aggregation rate constants are higher than the Tb/DPA aggregation rate constants, indicating a significant frequency of reversible vesicle aggregation events that do result in mixing of bilayer lipids, but not in mixing of aqueous vesicle contents. In any conditions, the RET fusion rate constants are considerably higher than the Tb/DPA fusion rate constants, demonstrating the higher tendency of the vesicles, once aggregated, to mix lipids than to mix aqueous contents. This possibly reflects the formation of an intermediate fusion structure. With increasing Ca2+ concentrations the RET and the Tb/DPA fusion rate constants increase in parallel with the respective aggregation rate constants. This suggests that fusion susceptibility is conferred on the vesicles during the process of vesicle aggregation and not solely as a result of the interaction of Ca2+ with isolated vesicles. Aggregation of the vesicles in the presence of Mg2+ produces neither mixing of aqueous vesicle contents nor mixing of bilayer lipids.     

Kinetic study of the aggregation and lipid mixing produced by alpha-sarcin on phosphatidylglycerol and phosphatidylserine vesicles: stopped-flow light scattering and fluorescence energy transfer measurements.

alpha-Sarcin is a fungal cytotoxic protein that inactivates the eukaryotic ribosomes. A kinetic study of the aggregation and lipid mixing promoted by this protein on phosphatidylglycerol (PG) and phosphatidylserine (PS) vesicles has been performed. Egg yolk PG, bovine brain PS, dimyristoyl-PG (DMPG) and dimyristoyl-PS (DMPS) vesicles have been considered. The initial rates of the vesicle aggregation induced by the protein have been measured by stopped-flow 90 degrees light scattering. The formation of a vesicle dimer as the initial step of this process was deduced from the second-order dependence of the initial rates on phospholipid concentration. The highest alpha-sarcin concentration studied did not inhibit the vesicle aggregation, indicating that many protein molecules are involved in the vesicle cross-linking. These are common characteristics of the initial steps of the aggregation produced by alpha-sarcin in the four types of phospholipid vesicles considered. However, the kinetics of the scattering values revealed that more complex changes occurred in the later steps of the aggregation process of egg PG and brain PS vesicles than in those of their synthetic counterparts. alpha-Sarcin produced lipid mixing in vesicles composed of DMPG or DMPS, which was measured by fluorescence resonance energy transfer assays. A delay in the onset of the process, dependent on the protein concentration, was observed. Measurement of the rates of lipid mixing revealed that the process is first order on phospholipid concentration. Egg PG and brain PS vesicles did not show lipid mixing, although they avidly aggregated. However, alpha-sarcin was able to promote lipid mixing in heterogeneous systems composed of egg PG+DMPG or brain PS+DMPS vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insufficient mobilization of calcium by early breakdown of phosphatidylinositol 4,5-bisphosphate for aggregation of human platelets by collagen

When human platelets (5 X 10(8)/ml) were stimulated by a threshold concentration of collagen (2 micrograms/ml), a lag period of about 60 s was seen before the initiation of release reaction and aggregation. Breakdown of [32P]phosphatidylinositol 4,5-bisphosphate was seen within 10 s after the addition of collagen. The concentration of intracellular free Ca2+ (monitored by Quin II) rose from 80 nM to 145 nM within 10 s after stimulation by collagen. However, a lag period of about 50 s remained. The rise was not blocked by indomethacin. It was supposed that the initial Ca2+ mobilization by myo-inositol 1,4,5-trisphosphate was too small to cause aggregation. Thromboxane A2 was gradually accumulated during the lag period and then abruptly increased in parallel with aggregation. These events were completely inhibited by 10 microM indomethacin. Thus, aggregation appeared to be dependent on the generation of thromboxane A2. Addition of 25 nM A23187 at 10 s after stimulation by collagen shortened the lag period before initiation of the abrupt thromboxane A2 generation, secretion and aggregation, whereas 25 nM A23187 could not cause these reactions in the absence of collagen. Accordingly, the lag period is assumed to be required for accumulation of free Ca2+ to the threshold for aggregation of platelets. It is considered that thromboxane A2 plays a central role in Ca2+ mobilization during stimulation of human platelets by collagen.     

Calcium-induced dissociation of human plasma factor XIII and the appearance of catalytic activity

1. The Ca(2+) dependence of the activity of plasma Factor XIII(a) was studied by using the continuous assay based on the incorporation of dansylcadaverine into dephosphorylated acetylated beta-casein (beta-substrate). The K(m) for Ca(2+) is about 0.170mm. 2. At low concentrations of Ca(2+) there was a lag in attaining the steady-state rate. The size of the lag was decreased and eventually abolished if the enzyme was preincubated with a high concentration of Ca(2+) before assay. The concentration of Ca(2+) required to decrease the lag phase by 50% in 10min depended on the protein concentration: at 0.87mg of protein/ml it required 17mm-Ca(2+) and at 0.44mg/ml it needed 10mm-Ca(2+). 3. The concentrations of Ca(2+) required either to abolish the lag phase in the appearance of enzyme activity or to activate the essential thiol for reaction with 5,5'-dithiobis-(2-nitrobenzoate) in 10min incubation were similar at the same protein concentration. This indicated that Ca(2+) induces a conformation change that is responsible for both phenomena. A model is proposed that links this conformation change to the dissociation of the tetrameric enzyme. 4. This was supported by the observation that the addition of excess of b chains to the Factor XIII(a) (a'(2)b(2)) increased the concentration of Ca(2+) required to expose the reactive thiol, and inhibited the Ca(2+)-dependent aggregation of a' chains. 5. Platelet Factor XIII(a) (a'(2)) was inhibited by 5,5'-dithiobis-(2-nitrobenzoate) in the absence of Ca(2+), and no lag phases were observed in attaining the steady-state rate at low Ca(2+) concentrations, thus confirming the model for the activation of the plasma enzyme. 6. The Ca(2+) dependence of platelet Factor XIII(a) indicated that Ca(2+) has an additional role in the enzyme mechanism of the plasma enzyme, perhaps being involved in substrate binding. 7. The dependence of the stability of plasma Factor XIII(a) on Ca(2+) and protein concentration indicates that the decay in activity is related to the tetramer dissociation. 8. beta-Substrate decreased the Ca(2+) concentration required for (1) abolition of the lag phase and (2) enzyme inhibition by thiol reagents. The effect on the former is greater than on the latter. 9. The role of the b chains of the plasma Factor and the evolutionary significance of the plasma and platelet Factors are considered.     

Membrane contact, fusion, and hexagonal (HII) transitions in phosphatidylethanolamine liposomes

The behavior of phosphatidylethanolamine (PE) liposomes has been studied as a function of temperature, pH, ionic strength, lipid concentration, liposome size, and divalent cation concentration by differential scanning calorimetry (DSC), by light scattering, by assays measuring liposomal lipid mixing, contents mixing, and contents leakage, and by a new fluorometric assay for hexagonal (HII) transitions. Liposomes were either small or large unilamellar, or multilamellar. Stable (impermeable, nonaggregating) liposomes of egg PE (EPE) could be formed in isotonic saline (NaCl) only at high pH (greater than 8) or at lower pH in the presence of low ionic strength saline (less than 50 mOsm). Bilayer to hexagonal (HII) phase transitions and gel to liquid-crystalline transitions of centrifuged multilamellar liposomes were both detectable by DSC only at pH 7.4 and below. The HII transition temperature increased, and the transition enthalpy decreased, as the pH was raised above 7.4, and it disappeared above pH 8.3 where PE is sufficiently negatively charged. HII transitions could be detected at high pH following the addition of Ca2+ or Mg2+. No changes in light scattering and no lipid mixing, mixing of contents, or leakage of contents were noted for EPE liposomes under nonaggregating conditions (pH 9.2 and 100 mM Na+ or pH 7.4 and 5 mM Na+) as the temperature was raised through the HII transition region. However, when aggregation of the liposomes was induced by addition of Ca2+ or Mg2+, or by increasing [Na+], it produced sharp increases in light scattering and in leakage of contents and also changes in fluorescent probe behavior in the region of the HII transition temperature (TH). Lipid mixing and contents mixing were also observed below TH under conditions where liposomes were induced to aggregate, but without any appreciable leakage of contents. We conclude that HII transitions do not occur in liposomes under conditions where intermembrane contacts do not take place. Moreover, fusion of PE liposomes at a temperature below TH can be triggered by H+, Na+, Ca2+, or Mg2+ or by centrifugation under conditions that induce membrane contact. There was no evidence for the participation of HII transitions in these fusion events.     

Kinetics of activation of L-lactate dehydrogenase from Streptococcus faecalis by fructose 1,6-bisphosphate and by metal ions

The lactate dehydrogenase from Streptococcus faecalis is activated either by fructose 1,6-bisphosphate or by divalent cations such as Mn2+ or Co2+. With both types of activator, a lag is observed before attainment of the steady state rate of pyruvate reduction if the activator is added to the enzyme at the same time as the substrates. This lag can be largely abolished by preincubation of enzyme with activator before mixing with substrates. For fructose 1,6-bisphosphate (Fru(1,6)P2) as the activator, the rate constant for the lag phase showed a linear dependence on activator concentration but was independent of enzyme concentration. This suggests that binding of fructose 1,6-bisphosphate induces a conformational change in the enzyme which leads to increased activity, without association of enzyme subunits or dimers. With Co2+ as activator, the rate constant for the lag phase showed a hyperbolic dependence on Co2+ concentration and was also dependent on enzyme concentration. This suggests that activation by Co2+, in contrast to that by Fru(1,6)P2, involves association of enzyme dimers, followed by ligand binding.     

Effects of prostaglandins, cAMP, and changes in cytosolic calcium on platelet aggregation induced by a thromboxane A2 mimic (U46619).

The effects of U46619, a thromboxane mimic, on cytosolic Ca2+ concentration and platelet aggregation were determined in human platelets. Cytosolic Ca2+ concentration was determined by Quin-2 fluorescence and platelet aggregation quantitated with an aggregometer. Addition of U46619 (1 x 10(-7) M) to the platelet suspension produced a rapid increase in cytosolic Ca2+ and platelet aggregation. Pretreatment of platelets with EGTA (3 x 10(-3) M), verapamil (5 x 10(-4) M), a calcium entry blocker, or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (1 x 10(-3) M), an inhibitor of intracellular Ca2+ release, either blunted or markedly delayed the rate, but not the magnitude, of increase in cytosolic Ca2+ and prevented platelet aggregation by U46619. Pretreatment of platelets with prostaglandin I2 (PGI2) (5 x 10(-8) M), PGD2 (5 x 10(-8) M), PGE1 (5 x 10(-8) M), PGF2 alpha (1 x 10(-5) M), dibutyryl cAMP (5 x 10(-3) M), or forskolin (1 x 10(-6) M) prevented both the increase in cytosolic Ca2+ and the associated platelet aggregation induced by U46619. These data suggest that U46619 may induce platelet aggregation through an increase in cytosolic Ca2+, and that both Ca2+ entry and its release from intracellular storage sites probably contribute to the increase in cytosolic Ca2+. Furthermore, the rate of the increase in cytosolic Ca2+ concentration, as well as the magnitude of the increase, appear to be critical for platelet aggregation induced by U46619. Our data are consistent with the hypothesis that PGs inhibit U46619-induced platelet aggregation by preventing the increase in cytosolic Ca2+, and that these effects may be mediated via an increase in cAMP, since they were induced by PGs and cAMP.     

Free fatty acid enhancement of cation-induced fusion of liposomes: synergism with synexin and other promoters of vesicle aggregation

The effect of free fatty acids on the cation-induced fusion of large unilamellar vesicles (liposomes) was investigated by using fluorescent assays which monitor the mixing of aqueous contents of liposomes. Overall fusion was modeled as a two-step process involving aggregation of vesicles followed by actual fusion. Different experimental conditions were used which favored either aggregation or fusion as the rate-limiting step in the overall process. When phosphatidylserine liposomes were induced to fuse by 4 mM Ca2+ plus 5 mM Mg2+, preincubation with arachidonic acid showed a dramatically increased overall rate of fusion compared to the same liposomes not treated with fatty acid. When fusion was induced by 3 mM Ca2+, arachidonic acid had little effect. These results were interpreted in terms of the action of arachidonic acid only at the fusion step per se and not the aggregation step. Therefore, the enhancement of the overall fusion rate would be observed solely under conditions where the actual fusion of liposomes was rate limiting (Ca/Mg) rather than the aggregation of liposomes (Ca alone). When other liposome systems were tested, the effect of arachidonic acid was observed only under fusion rate-limiting conditions. Arachidonic acid was found to act synergistically with promoters of liposomal aggregation, such as Mg2+, spermine, and synexin, to enhance the overall rate of liposome fusion, as would be expected from action at separate kinetic steps. The dependence of the fusion rates on arachidonic acid concentration demonstrated an apparently cooperative effect. The structure of the fatty acid is of critical importance in determining its effects, as shown by the fact that 16-doxylstearic acid always increased the rate of fusion while 5-doxylstearic acid always decreased the rate of fusion under all conditions tested. A number of different fatty acids, including oleic acid, elaidic acid, 16-doxylstearic acid, myristic acid, and stearic acid, were effective at increasing the fusion rate to varying extents. In general, unsaturated fatty acids were more effective than saturated ones, either due to partitioning into the membrane or because of structural requirements for promotion of fusion.     

Ca2+-induced phosphatidylcholine vesicle aggregation in the presence of ferricyanide

The titration of sonicated vesicles of egg phosphatidylcholine with ferricyanide in the presence of Ca2+ results in the formation of aggregates. The turbidity increase caused by these aggregates cannot be reversed by EDTA treatment. In addition, no rearrangement of the bilayer structure has been found in this process, either measuring leakage of vesicle content or exchange of lipids among the bilayers themselves. The aggregation is dependent on the Ca2+ content of the vesicles, the outer Ca2+ and Fe(CN)3-(6) concentration and the order of addition of Ca2+ and ferricyanide. The results can be explained by a specific adsorption of Fe(CN)3-(6) to bilayers of sonicated vesicles, in contrast to other multivalent anions. In contrast to the stability found with sonicated vesicles, the aggregation causes a leakage of the internal solution when multilamellar liposomes are titrated with Fe(CN)3-(6).     

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.     

Evidence for the activation of two different Ca2+ channels during the egg jelly-induced acrosome reaction of sea urchin sperm

The influx of Ca2+ and its subsequent intracellular increase are required for the acrosome reaction of sea urchin sperm to occur. Spermatozoa must undergo this reaction, which is triggered by the egg jelly, in order to fertilize the egg. Here, the egg jelly-induced Ca2+ influx mechanisms have been studied in sperm loaded with FURA-2 using Mn2+ under the assumption that this divalent ion is an indicator of Ca2+ influx through Ca2+ channels. Egg jelly induced the immediate entry of Ca2+ (mixing time 1 s), however; we found that the influx of Mn2+ increased after a lag time of 5 s. Nisol-dipine (a Ca2+ channel blocker) did not block the Mn2+ influx which was inhibited by 40 mM of external [K+], low Na+, and 5 mM of tetraethylammonium (a K+ channel blocker). These conditions also inhibited the alkalinization and the acrosome reaction. The inhibition of the Mn2+ influx could be overcome by increasing internal pH (pHi) with ammonium (10 mM). On the contrary the influx of Ca2+ during the first 5 s was not inhibited by any of the conditions indicated before, except by nisoldipine. These data could be explained by the activation of two different Ca2+ channels by egg jelly. The first one being a receptor-operator Ca2+ channel that opens when the receptor for egg jelly is occupied independently of the ionic conditions. The other one could be considered as a second messenger-operated Ca2+ channel that requires at least an increase in pHi to open.     

Binding of monovalent cations to phosphatidylserine and modulation of Ca2+- and Mg2+-induced vesicle fusion

The effect of several monovalent cations on the Ca2+-induced aggregation and fusion of sonicated phosphatidylserine (PS) vesicles is studied by monitoring the mixing of internal compartments of the fusing vesicles using the Tb/dipicolinic acid assay. The dissociation of the fluorescent Tb-dipicolinate complex which accompanies Ca2+-induced vesicle fusion is determined directly and is due to leakage of contents and entry of medium into vesicles. PS vesicles do not fuse when the medium contains only monovalent cations (at pH 7.4), regardless of the cation concentration or whether there is aggregation of the vesicles. A mass-action kinetic analysis of the data provides estimates for the rate of aggregation, C11, and for the rate of fusion per se, f11. Values of f11 increase dramatically with reduction in monovalent cation concentration and are primarily determined by binding ratios of Ca2+ or Mg2+ per PS. With 300 mM of monovalent cations, the fusion per se is essentially rate-limiting to the overall fusion process and values of f11 are significantly larger with the monovalent cations which bind the least, i.e., according to the sequence tetramethylammonium greater than K+ greater than Na+ greater than Li+. With monovalent cations in concentrations of 100 mM or less, the aggregation is rate-limiting to the fusion and the overall initial fusion rates are determined by an interplay between aggregation and fusion rates. Under conditions of fast aggregation, the Ca2+-induced fusion of small PS vesicles can occur within milliseconds or less.     

Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate

Inositol trisphosphate (IP3) was previously shown to release Ca2+ from a nonmitochondrial store in sea urchin eggs. In this study, egg homogenates and purified microsomes were monitored with either fura 2 or Ca2+-sensitive minielectrodes to determine whether other stimuli would induce Ca2+ release. Pyridine nucleotides (whose concentrations are known to change at fertilization) were found to release nearly as much Ca2+ as did IP3. Average releases/ml of homogenate were: 0.6 microM IP3, 10.9 nmol of Ca2+; 50 microM NADP, 7.3 nmol of Ca2+; and 100 microM NAD, 6.5 nmol of Ca2+ (n = 6). Specificity was demonstrated by screening a series of other phosphorylated metabolites, and none was found to reproducibly release Ca2+. Calcium release induced by IP3 or NADP was immediate, whereas a lag of 1-4 min occurred with NAD. This lag before NAD-induced Ca2+ release led to the discovery that a soluble egg factor (Mr greater than 100,000) converts NAD into a highly active metabolite that releases Ca2+ without a lag. The NAD metabolite (E-NAD) was purified to homogeneity by high pressure liquid chromatography and produced half-maximal Ca2+ release at about 40 nM. Injection of E-NAD into intact eggs produced both an increase in intracellular Ca2+ (as assayed with indo-1) and a cortical reaction. Following Ca2+ release by each of the active agents (IP3, NAD, and NADP), the homogenates resequestered the released Ca2+ but were desensitized to further addition of the same agent. A series of desensitization experiments showed that homogenates desensitized to any two of these agents still responded to the third, indicating the presence of three independent Ca2+ release mechanisms. This is further supported by experiments using Percoll density gradient centrifugation in which NADP-sensitive microsomes were partially separated from those sensitive to IP3 and NAD.     

Calcium-induced aggregation of archaeal bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

Previously, we showed that the proton permeability of small unilamellar vesicles (SUVs) composed of polar lipid fraction E (PLFE) from the thermoacidophilic archaeon Sulfolobus acidocaldarius was remarkably low and insensitive to temperature (Komatsu and Chong 1998). In this study, we used photon correlation spectroscopy to investigate the time dependence of PLFE SUV size as a function of Ca2+ concentration. In the absence of Ca2+, vesicle diameter changed little over 6 months. Addition of Ca2+, however, immediately induced formation of vesicle aggregates with an irregular shape, as revealed by confocal fluorescence microscopy. Aggregation was reversible upon addition of EDTA; however, the reversibility varied with temperature as well as incubation time with Ca2+. Freeze-fracture electron microscopy showed that, after a long period of incubation (2 weeks) with Ca2+, the PLFE vesicles had not just aggregated, but had fused or coalesced. The initial rate of vesicle aggregation varied sigmoidally with Ca2+ concentration. At pH 6.6, the threshold calcium concentration (Cr) for vesicle aggregation at 25 and 40 degrees C was 11 and 17 mM, respectively. At pH 3.0, the Cr at 25 degrees C increased to 25 mM. The temperature dependence of Cr may be attributable to changes in membrane surface potential, which was -22.0 and -13.2 mV at 25 and 40 degrees C, respectively, at pH 6.6, as determined by 2-(p-toluidinyl)naphthalene-6-sulfonic acid fluorescence. The variation in surface potential with temperature is discussed in terms of changes in lipid conformation and membrane organization.     

Aggregation of smooth muscle membranes and its use in the preparation of plasma membrane enriched fraction from gastric fundus smooth muscle

Microsomal membranes isolated from rat gastric fundus smooth muscle by differential centrifugation aggregate substantially in the presence of the divalent metal ion Mg2+ or Ca2+. The magnitude of cation-induced membrane aggregation is higher for Ca2+ than for Mg2+, but the ion concentration required for half-maximum membrane aggregation (K0.5 value) is similar for Mg2+ and Ca2+. Cation-induced membrane aggregation is suppressed by high ionic strength and low pH of the medium. Cation-induced membrane aggregation of mitochondrial membrane and plasma membrane enriched fractions differ in the rate of aggregate formation, metal ion concentration dependence, and pH dependence. Such different properties of membrane aggregation were used to prepare a plasma membrane enriched fraction by conventional differential centrifugation. Subfractionation of the heterogeneous microsomal membranes by free-flow electrophoresis indicated that smooth muscle plasma membranes showed a higher electrophoretic mobility than the intracellular membranes. These results suggest that ionic interactions on the cell membrane surfaces differ from those on the intracellular membrane surfaces and that induction of membrane aggregation by Ca2+ or Mg2+ is a useful procedure for an effective and rapid preparation of plasma membrane enriched fraction from smooth muscle.     

The role of autophosphorylation in activation of the type II calmodulin-dependent protein kinase

Autophosphorylation of the type II calmodulin-dependent protein kinase is known to remove the dependence of this enzyme on Ca2+ and calmodulin. The enzymatic activity in the presence of Ca2+, on the other hand, was reported to be unaffected or decreased by this interconversion. The role of autophosphorylation in the kinase reaction was reinvestigated using short assay times and low ATP concentrations to decrease the extent and rate of this process. Under these conditions, the ATP dependence of the kinase reaction with syntide-2 as the substrate (but not the autophosphorylation reaction) exhibited kinetic cooperativity due to a lag in the progress curve of syntide-2 conversion. Partial autophosphorylation of the protein kinase prior to phosphorylation of the peptide substrate completely abolished this hysteretic response without affecting the final rate of substrate conversion. These observations suggest that autophosphorylation is an obligatory step in the response of this kinase to activation by calmodulin.     

Clotting of bovine fibrinogen. Calcium binding to fibrin during clotting and its dependence on release of fibrinopeptide B

Polymerization of bovine fibrinogen acted upon by thrombin is accompanied by binding of Ca2+ and a concomitant decrease of the free Ca2+ concentration. The latter can be recorded by a Ca2+-selective electrode as a shift in the electrode potential. The shift shows marked dependence on the initial free Ca2+ concentration, being maximal at about 10(-4.1) M and decreasing sharply on either side of this. Thus, the effect is limited to the 10(-3)-10(-5) M free Ca2+ concentration range. From the initial and the final value of the electrode potential during a clotting experiment, the amount of Ca2+ bound to fibrinogen and fibrin, respectively, can be calculated. The difference between the two, plotted against free Ca2+ concentration, gives a bell-shaped curve. This indicates that the reason for the Ca2+ binding is a shift of the pK of some groups from a lower to higher value. The recordings can be used for evaluation of the kinetics of the Ca2+ uptake. However, they have to be corrected for the effect of the continuous shift in the free Ca2+ concentration during the experiment. The reaction does not follow simple kinetics, showing a lag period. Therefore, rates were estimated from inverse half-reaction times. Half-times of the corrected curves show that the reaction is first order with respect to thrombin. Moreover, the rate of Ca2+ uptake is identical with that of the conformational change seen in differential scanning calorimetry [Donovan, J.W., & Mihalyi, E. '1985) Biochemistry 24, 3434]. The inverse rate and the final corrected Ca2+ uptake increase linearly with the initial fibrinogen concentration. Concomitant estimates of fibrinopeptide A and B release showed that the Ca2+ uptake runs parallel to the release of fibrinopeptide B. Fibrinopeptide A was released largely during the lag period of the Ca2+ uptake. In agreement with this, clotting with Ancrod, an enzyme that liberates only fibrinopeptide A, was not accompanied by binding of Ca2+. Thus, polymerization is not sufficient for the Ca2+ uptake to occur; liberation of fibrinopeptide B seems to be obligatory. Further support for this was obtained with experiments with the polymerization inhibitor Gly-Pro-Arg-Pro. The tetrapeptide inhibits polymerization and also, proportional to this, release of fibrinopeptide B [Hurlet-Jensen, A., Cummins, H.Z., Nossel, H.L., & Liu, C.Y. (1982) Thromb. Res. 27, 419; Lewis, S.D., Shields, P.P., & Shafer, J.A. (1985) J. Biol. Chem. 260, 10192]. Calcium uptake was also depressed by the tetrapeptide in a way similar to its effect upon fibrinopeptide B release.     

Factors influencing the response of human blood platelets to analogues of ADP which may act as partial agonists at the ADP receptor.

1. The prior addition of non-aggregating concentrations of the divalent cation ionophore, A-23187, causes human platelets to aggregate in response to a subsequent addition of the 2',3'-dialdehyde and 2',3'-dialcohol derivatives of ADP (oADP and or ADP). Previous studies [Pearce et al. (1978) Eur. J. Biochem. 88, 543--555] have shown that these derivatives act as partial agonists at the platelet ADP receptor inducing only the transition from discoid to globular morphology ('shape change'). A secretion response is also observed on addition of a low concentration of ionophore A-23187 prior to orADP. These responses are not observed if ionophore A-23187 is added prior to the 2',3'-dialdehyde and 2',3'-dialcohol derivatives of ATP (oATP and or ATP) and are markedly inhibited by prior addition of the ADP antagonist, adenosine 5'-[beta, gamma-methylene]triphosphate. 2. The aggregation response to oADP in the presence of ionophore A-23187 is reduced but not eliminated by addition of 3 mM EGTA when studies are performed in heparinised platelet-rich plasma. Additions of 3 mM EGTA in citrated platelet-rich plasma, or of 4 mM EDTA in either system completely inhibits this response. Inhibitors which are reported to elevate the intracellular concentration of adenosine 3':5'-monophosphate (cyclic AMP) or to prevent Ca2+ movement also inhibit the aggregation response to oADP which is observed in the presence of ionophore A-23187. 3. Prior addition of inhibitors of adenylate cyclase fails to cause an aggregation response to subsequent addition of oADP or orADP. Certain of these inhibitors enhance and prolong the shape change response to oADP or orADP but only at concentrations an order of magnitude in excess of those required to antagonise inhibition by agents such as prostaglandin E1, which act by increasing the concentration of cyclic AMP. 4. The concentration of prostaglandin E1, adenosine or papaverine required to inhibit shape change induced by oADP is one to two orders of magnitude lower than that required to inhibit shape change induced by ADP. 5. Prior addition of oADP decreases the lag phase in the response of human platelets to arachidonate while also increasing the concentration required to observe half-maximal response, and causing a decrease in the extent of the response. Prior addition of oATP also diminishes the extent of this response and increases the concentration of arachidonate required but has no effect on the lag phase. 6. The data suggest that oADP and orADP are capable only of acting as partial agonists at the ADP receptor because of a defective ability to increase cytosolic Ca2+ concentration. The defect is rectified by the presence of low concentrations of ionophore A-23187, which promotes mobilisation of Ca2+ from an intracellular store. The results do not appear consistent with the thesis that a decrease in platelet cyclic AMP is an initiating event in aggregation induced by ADP, but do support a model which implicates cyclic AMP in depletion of cytosolic Ca2+.     

Cytoplasmic Ca2+ release induced by microinjection of Ca2+ and effects of microinjected divalent cations on Ca2+ sequestration and exocytosis of cortical alveoli in the medaka egg

Intracellular release of Ca2+ by microinjection of Ca2+ was analyzed by measuring the luminescence of aequorin loaded in eggs of the medaka (Oryzias latipes). Microinjection of Ca2+ into the cortical cytoplasm induced propagative waves of cytoplasmic Ca2+ release and exocytosis of cortical alveoli initiated at the injection point. The Ca2+ wave was initiated with a time lag after some was sequestered at the region of the microinjection. Microinjection of Mg2+ or Mn2+ failed to trigger Ca2+ release and exocytosis. When the aequorin-loaded eggs were inseminated after microinjection of Mg2+, Mn2+, or Co2+ into a restricted region of the vegetal hemisphere, the wave of Ca release was propagated through the injected region toward the vegetal pole, but neither Ca sequestration (fall in Ca-aequorin luminescence) nor exocytosis occurred at the area of cortex where the eggs were injected with these divalent cations. These results suggest that a significant period is required to induce Ca2+ release from cytoplasmic stores by the increased Ca2+ concentration and that both the phenomena of Ca2+ release and Ca sequestration are involved in the process of exocytosis.