Proton movements across the membranes of sarcoplasmic reticulum during the uptake of calcium ions

A burst of proton ejection was observed during the initial steps of Ca²⁺ uptake by sarcoplasmic reticulum vesicles. The initial rate of this proton ejection is considerably higher than the initial rate of Ca²⁺ uptake, and is independent of the amount of accumulated Ca²⁺. The ejection of protons is a transmembrane event, since it is dissipated by the ionophore X-537A, and does not occur when the ionophore is added before the initiation of the transport of Ca²⁺. The low proton permeability of the membranes is largely increased by X-537A. The studies of facilitated diffusion of protons in the presence of the ionophore permitted the estimation of the pH within the vesicles. A fast alkalinization occurs within the vesicles during the initial steps of Ca²⁺ uptake, as revealed by sequestered bromothymol blue. The change in absorbance of this dye corresponds to a change of 0.15 pH unit within the vesicles, and a maximal transmembrane ΔpH of about 0.5 may build up. Since such a gradient may not account energetically for the transmembrane gradients of Ca²⁺, I suggest that a transmembrane electrical potential may develop as a consequence of proton ejection.     

Attractant-induced changes and oscillations of the extracellular Ca++ concentration in suspensions of differentiating Dictyostelium cells

We used a Ca++-sensitive electrode to measure changes in extracellular Ca++ concentration in cell suspensions of Dictyostelium discoideum during differentiation and attractant stimulation. The cells maintained an external level of 3-8 microM Ca++ until the beginning of aggregation and then started to take up Ca++. The attractants, folic acid, cyclic AMP, and cyclic GMP, induced a transient uptake of Ca++ by the cells. The response was detectable within 6 s and peaked at 30 s. Half-maximal uptake occurred at 5 nM cyclic AMP or 0.2 microM folic acid, respectively. The apparent rate of uptake amounted to 2 X 10(7) Ca++ per cell per min. Following uptake, Ca++ was released by the cells with a rate of 5 X 10(6) ions per cell per min. Specificity studies indicated that the induced uptake of Ca++ was mediated by cell surface receptors. The amount of accumulated Ca++ remained constant as long as a constant stimulus was provided. No apparent adaptation occurred. The cyclic AMP-induced uptake of Ca++ increased during differentiation and was dependent on the external Ca++ concentration. Saturation was found above 10 microM external Ca++. The time course and magnitude of the attractant-induced uptake of external Ca++ agree with a role of Ca++ during contraction. During development the extracellular Ca++ level oscillated with a period of 6-11 min. The change of the extracellular Ca++ concentration during one cycle would correspond to a 30-fold change of the cellular free Ca++ concentration.     

Phosphorylation of the red blood cell membrane during the active transport of C++

The phosphorylation of red blood cell membrane fragments (RBCMF) during Ca++ transport was investigated. When red cell membrane fragments are incubated with [gamma-32P]ATP under the experimental condition which minimizes the phosphorylation of Na+-K+-ATPase, RBCMF are labeled in the presence of Mg++ without Ca++. When Ca++ is added, the labeling decreases due to dephosphorylation of RBCMF. The initial reaction of phosphorylation is reversed in the presence of excess ADP. The treatment of RBCMF with n-ethylmaleimide (NEM) does not interfere with the initial phosphorylation reaction, but blocks the dephosphorylation in the presence of Ca++. These data suggest that the enzymatic sequence of the Ca++ transport mechanism may be very similar to that of the Na+ transport mechanism.     

Protons resolve dual effects of calcium on miniature end-plate potential frequency at frog neuromuscular junctions

Inhibition of transmitter release by protons (H+) was studied at the frog neuromuscular junction at various extracellular concentrations of calcium ([Ca++]o) and potassium ([K+]o) by recording miniature end-plate potential (MEPP) frequency with the intracellular microelectrode. H+ decreased K+ -stimulated MEPP frequency. A double logarithmic graph of MEPP frequency at 7.5 mM K+ vs. [H+]o yielded a straight line with negative slope. At 10 mM K+, there was a parallel shift to the right of the graph. According to the surface charge model, K+ acts solely to depolarize the prejunctional membrane in accordance with the Nernst equation. By decreasing the prejunctional negative surface charge, H+ decreases K+ -stimulated MEPP frequency by decreasing [Ca++]o at the Ca++ channel. An estimated pKa of 4.20 may represent an acidic site at the Ca++ channel associated with Ca++ influx. As [Ca++]o increased above 1 mM for pH 7.40 and 10 mM K+, MEPP frequency decreased, i.e., the inhibitory component of dual effects of Ca++ occurred. At pH 6.40, the inhibitory component was abolished, unmasking the stimulatory effect of Ca++ on MEPP frequency. Reversal of Ca++ action by H+ could not be explained by surface charge theory alone. A double logarithmic graph of MEPP frequency vs. [K+]o at 8.5-10.5 mM was linear with a slope of 4. There were parallel shifts to the right of this graph for changes in pH from 7.40 to 6.90 and in [Ca++]o from 1 to 2.5 mM. These results are explained on the hypothesis that K+ also acts at an acidic prejunctional site to increase Ca++ -dependent quantal transmitter release. This action of K+ was inhibited by H+ and raised Ca++. Based on kinetic theory, the estimated pKa of the acidic prejunctional K+ site was 6.31. Based on free energy calculations, its cation preference was H+ greater than K+ greater than Ca++.     

The high affinity binding site for calcium on the oxidizing side of photosystem II

Preparations of photosystem II complex from spinach chloroplasts with Triton X-100 were treated with 1 M KCl to release 17 KDa and 23 KDa polypeptides. The inhibited oxygen evolution activity could be reactivated by adding high concentration (mM) of Ca++ or by reconstituting 17 KDa and 23 KDa polypeptides which were found to promote high affinity binding of Ca++ to the reconstituted membranes (Ghanotakis et al. FEBS (1984) 170, 169-173). Inclusion of 50 mM Ca++ during KCl treatment did not prevent the release of 17 KDa and 23 KDa polypeptides but protected oxygen evolution from being inactivated. It is explained by preservation of the high affinity binding site for Ca++ if, Ca++ is present during the salt treatment even though depletion of 17 KDa and 23 KDa polypeptides usually results in replacement by a low affinity (mM) binding site for Ca++. It also implies that the high affinity binding site is not located on 17 KDa and 23 KDa polypeptides.     

Sucrose uptake by pinocytosis in Amoeba proteus and the influence of external calcium

The relationship between Ca++ and pinocytosis was investigated in Amoeba proteus. Pinocytosis was induced with 0.01% alcian blue, a large molecular weight dye which binds irreversibly to the cell surface. The time-course and intensity of pinocytosis was monitored by following the uptake of [3H]SUCROSE. When the cells are exposed to 0.01% alcian blue, there is an immediate uptake of sucrose. The cells take up integral of 10% of their initial volume during the time-course of pinocytosis. The duration of pinocytosis in the amoeba is integral of 50 min, with maximum sucrose uptake occurring 15 min after the induction of pinocytosis. The pinocytotic uptake of sucrose is reversibly blocked at 3 degrees C and a decrease in pH increases the uptake of sucrose by pinocytosis. The process of pinocytosis is also dependent upon the concentration of the inducer in the external medium. The association between Ca++ and pinocytosis in A. proteus was investigated initially by determining the effect of the external Ca++ concentration on sucrose uptake induced by alcian blue. In Ca++-free medium, no sucrose uptake is observed in the presence of 0.01% alcian blue. As the Ca++ concentration is increased, up to a maximum of 0.1 mM, pinocytotic sucrose uptake is also increased. Increases in the external Ca++ concentration above 0.1 mM brings about a decrease in sucrose uptake. Further investigations into the association between Ca++ and pinocytosis demonstrated that the inducer of pinocytosis displaces surface calcium in the amoeba. It is suggested that Ca++ is involved in two separate stages in the process of pinocytosis; an initial displacement of surface calcium by the inducer which may increase the permeability of the membrane to solutes and a subsequent Ca++ influx bringing about localized increases in cytoplasmic Ca++ ion activity.     

Oxalate, calcium uptake and ATPase activity of sarcoplasmic reticulum vesicles.

Ca++-uptake and Mg++-Ca++-dependent ATPase activity of skeletal muscle sarcoplasmic reticulum vesicles were reciprocally affected by increasing the oxalate concentration from 0 to 4 mM. At 0-0.1 mM oxalate approximately 17% of the calcium was removed by the vesicles from the medium while the ATPase activity was maximal (approximately 0.66 mumoles Pi mg-1 protein min-1). Between 0.1 to 0.2 mM oxalate the ATPase activity was reduced to one-fifth but the uptake rose sharply and 100% of the 45Ca++ was removed from the medium. The uptake was maintained at this level at oxalate concentrations greater than 0.4 mM but the ATPase activity remained inhibited. The kinetics of Ca++-uptake and ATPase activity were also differentially affected by oxalate. In the presence of oxalate, ruthenium red had only a very slight inhibitory effect on the calcium uptake. Addition of 0.1 mM EGTA removed 80% of the Ca++ from preloaded vesicles within 10 min. The formation of insoluble Ca-oxalate salt on the surface of the vesicle is suggested by these results. Calculations based on the Ksp of the calcium oxalate salt are presented to show its formation and the possible speciation of a Ca-oxalate complex which may affect the Ca++-uptake and ATPase activity.     

Utilization of X-537A to distinguish between intravesicular and membrane-bound calcium ions in sarcoplasmic reticulum.

The Ca2+ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca2+ and surface membrane-bound Ca2+ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca2+ in the absence of ATP, 10-12 h are necessary for measurable amount of Ca2+ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca2+ from 'loaded' vesicles only after this period of incubation. A fraction of Ca2+ of 50-60 nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg2+ and K+ in the medium and is readily released by ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca2+ (30-40 nmol/mg protein) is rapidly released X-537A. The results indicate that most of the Ca2+ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca2+ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.     

Immune opsonin-independent phagocytosis by pulmonary macrophages

The uptake of albumin-coated latex particles by hamster pulmonary macrophages (PM) in vitro was investigated by using a new technique that combined flow cytometry and fluorescence microscopy to differentiate and quantitate bound vs ingested particles. In the absence of serum, PM avidly bound and ingested particles, whereas phagocytosis by hamster polymorphonuclear leukocytes (PMN) was less marked. In the presence of serum, phagocytosis by PM was slightly depressed, whereas phagocytosis by PMN was stimulated more than 10-fold. The binding of particles to PM in the absence of serum was pH, temperature, and trypsin sensitive and was dependent on the presence of extracellular Ca++ but not Mg++. The ingestion of particles by this immune opsonin-independent pathway was also temperature sensitive but was not affected by either pH or extracellular Ca++. Particle ingestion, but not binding, was inhibited by cytochalasin D and the divalent cation ionophore A23187.     

Metabolic characteristics of pancreatic beta-cells exposed to calcium-transporting ionophores.

The effects of the ionophores A-23187 and X-537 A on glucose metabolism, ATP content and sucrose permeability in pancreatic islets microdissected from obese-hyperglycemic mice were studied. The formation of 14CO2 from 10 mM D-[U-14C] GLUCOSE WAS INHIBITED BY OMISSION OF Ca2+ from the medium. A-23187 (10 muM) induced a further decrease of 14CO2 formation whereas X-537 A (10 muM) had no effect. At 20 mM glucose both A-23187 (48 muM) and X-537 A (43 muM) decreased the 14CO2 formation in the absence of Ca2+ whereas only X-537 A inhibited in the presence of Ca2+. X-537 A (43 muM) also decreased the formation of 3H2O from 20 mM D-[5-3H] glucose. The islet content of ATP was not changed after incubation in media deficient in either Mg2+ or Ca2+. However, omission of both Mg2+ and Ca2+ resulted in about 50% decrease of the ATP content. A-23187 and X-537 A induced dose-dependent decreases of the islet ATP content. X-537 A was much more potent than A-23187. Both ionophores induced stronger depression of the ATP content when Ca2+ was omitted. X-537 A (43 muM) but not A-23187 (48 muM) increased the beta-cell membrane permeability as indicated by an increased sucrose space in relation to the urea space of islets. Such an effect was not obtained with X-537 A at 1 muM or by omission of Ca2+. It is suggested that the marked metabolic effects of the ionophores reflect an impaired mitochondrial metabolism. These metabolic changes should be considered in interpretations of ionophore action on insulin secretion.     

Some effects of the ionophore X-537A on the isolated rat tail artery

The effects of micromolar concentrations of the ionophore X-537A (RO 2-2985) were studied using isolated preparations of the rat tail artery. The ionophore causes complete release of catecholamines from adrenergic nerves, which is the sole cause of the transient contractile response. The amines are released by a nonexocytotic process which seems to be related to the ability of X-537A to act as an efficient transmembrane carrier of Na+, k+, and H+. The ionophore also causes an almost complete and irreversible loss of the cocaine-sensitive component of metaraminol uptake by the tissue. X-537A dissipates the transmembrane concentration gradients of Na and K in the smooth muscle component of the preparation. This effect is unrelated to the release of endogenous catecholamines, and it can also be observed after the Na pump has been inhibited with ouabain. It is fully reversible, though not readily, and it can be induced repeatedly. In catecholamine-depleted strips, X-537A dissipates the transmembrane Na+ and K+ gradients without causing any change in tension. Stimulation of the rate of O2 consumption by X-537A in catecholamine-depleted tissue is reversible, and it is unaffected by ouabain and (or) removal of external Ca2+.     

State of translocated Ca2+ by sarcoplasmic reticulum inferred from kinetic analysis of calcium oxalate precipitation

Uptake of Ca²⁺ by sarcoplasmic reticulum in the presence of oxalate displays biphasic kinetics. An initial phase of normal uptake is followed by a second phase coincident with precipitation of calcium oxalate inside the vesicles. The precipitation rate induced by actively transported Ca²⁺ is depressed by increasing the added Ca²⁺ concentration. This correlates linearly with the reciprocal of precipitation rate. Therefore, a maximal limit rate could be extrapolated at zero Ca²⁺ ($\text{V}{}_0$). The rate of precipitation, also a function of added amount protein, gives a linear correlation in a double reciprocal plot. Thus, it was possible to estimate the maximal precipitation rate occurring at infinite protein concentration ($\text{V}{}^{\mathrm{\infty }}$). With the combined extrapolated values a maximal expected precipitation rate could be calculated ($\text{V}{}_0{}^{\mathrm{\infty }}$). Kinetics of calcium oxalate precipitation was studied in the absence of calcium uptake and empirical equations relating the rate of precipitation with the added Ca²⁺ were established. Entering $\text{V}{}_0{}^{\mathrm{\infty }}$ in the equations, an internal free Ca²⁺ concentration of approx. 2.5 mM was estimated. Additionally, it is shown that the ionophore X-537A does not supress the Ca²⁺ uptake, if added during the oxalate-dependent phase, albeit the uptake proceeds at a slower rate after the release of approx. 70 nmol Ca²⁺/mg protein. This amount presumably equals the internal free Ca²⁺ not sequestered by oxalate, producing a maximal concentration approx. 14 mM. Taking into account low affinity binding of internal binding sites and the transmembrane Ca²⁺ gradients built up during the uptake of Ca²⁺, values of free Ca²⁺ ranging from 3 to 6 mM, approaching those estimated by the precipitation analysis, could be estimated.     

The calcium dependence of pigment translocation in freshwater shrimp red ovarian chromatophores

The roles of calcium in cell signaling consequent to chromatophorotropin action and as an activator of mechanochemical transport proteins responsible for pigment granule translocation were investigated in the red ovarian chromatosomes of the freshwater shrimp Macrobrachium olfersii. Chromatosomes were perfused with known concentrations of free Ca++ (10(-3) to 10(-9) M) prepared in Mg(++)-EGTA-buffered physiological saline after selectively permeabilizing with 25 microM calcium ionophore A23187 or with 10(-8) M red pigment concentrating hormone (RPCH). The degree of pigment aggregation and the translocation velocity of the leading edges of the pigment mass were recorded in individual chromatosomes during aggregation induced by RPCH or A23187 and dispersion induced by low Ca++. Aggregation is Ca++ dependent, showing a dual extracellular and intracellular requirement. After perfusion with reduced Ca++ (10(-4) to 10(-9) M), RPCH triggers partial aggregation (approximately 65%), although the maximum translocation velocities (approximately 16.5 microns/min) and velocity profiles are unaffected. After aggregation induced at or below 10(-5) M Ca++, spontaneous pigment dispersion ensues, suggesting a Ca++ requirement for RPCH coupling to its receptor, or a concentration-dependent, Ca(++)-induced Ca(++)-release mechanism. The Ca(++)-channel blockers Mn++ (5 mM) and verapamil (50 microM) have no effect on RPCH-triggered aggregation. An intracellular Ca++ requirement for aggregation was demonstrated in chromatosomes in which the Ca++ gradient across the cell membrane was dissipated with A23187. At free [Ca++] above 10(-3) M, aggregation is complete; at 10(-4) M, aggregation is partial, followed by spontaneous dispersion; below 10(-5) M Ca++, pigments do not aggregate but disperse slightly. Aggregation velocities diminish from 11.6 +/- 1.2 microns/min at 5.5 mM Ca++ to 7.4 +/- 1.3 microns/min at 10(-4) M Ca++. Half-maximum aggregation occurs at 3.2 x 10(-5) M Ca++ and half-maximum translocation velocity at 4.8 x 10(-5) M Ca++. Pigment redispersion after 5.5 mM Ca(++)-A23187-induced aggregation is initiated by reducing extracellular Ca++: slight dispersion begins at 10(-7) M, complete dispersion being attained at 10(-9) M Ca++. Dispersion velocities increase from 0.6 +/- 0.2 to 3.1 +/- 0.5 microns/min. Half-maximum dispersion occurs at 7.6 x 10(-9) M Ca++ and half-maximum translocation velocity at 2.9 x 10(-9) M Ca++. These data reveal an extracellular and an intracellular Ca++ requirement for RPCH action, and demonstrate that the centripetal or centrifugal direction of pigment movement, the translocation velocity, and the degree of pigment aggregation or dispersion attained are calcium-dependent properties of the granule translocation apparatus.     

Properties of vagal excitation transfer to the heart atrium]

A mathematical model of pre- and postganglionic parasympathetic nerve fiber excitation transfer is developed. This model gives a measure Q of acetylcholine (ACh) release from presynaptic preganglionic boutons and postganglionic varicosities. When increasing Ca++ the measure Q increases too. Na-ions exert a competitive inhibition. The relationship between Q and the Ca/Na2-quotient is a hyperbolic one. Mn++ inhibits the release of ACh non-competitively. Q increases both by excess potassium and Cs+ depolarization. The ACh release is diminished by Mg++. Ba++ cannot replace the effect of Ca++ on ACh release in Ca++ depleting conditions. Q increases with decreasing pH-level. The ACh release is not significantly influenced by increasing pH, Verapamil (4 mg/l), prostaglandins E2 and F2alpha (20 ng/ml) and substitution of nonpermeable anions for Cl-.     

Effects of the thyroid status on the sarcoplasmic reticulum in slow skeletal muscle of the rat

The effects of the thyroid status on the Ca++-transporting capabilities of rat slow skeletal muscle (m.soleus) were studied. The oxalate supported Ca++-uptake activity and Ca++-loading capacity of muscle homogenates from hyperthyroid rats showed an approximate 4.2 and 2.5 fold increase, respectively, as compared to values found in the hypothyroid group. Muscle homogenates of euthyroid rats gave intermediate values. The specific activity of oxalate supported Ca++ uptake, but not the Ca++-loading capacity, of membrane preparations enriched with respect to sarcoplasmic reticulum (SR) increased in proportion to the thyroid status. This was paralleled by a 3.5 fold increase in the amount of active Ca++ pumps in the SR preparations in the transition from hypothyroidism to hyperthyroidism as determined by measurement of Ca++-dependent 32P incorporation. These observations are not explained by differences in degree of purification of the examined SR preparations. Protein profiles of the membrane preparations obtained by gel electrophoresis indicated a thyroid-hormone dependent increase in Ca++-pump content relative to other SR proteins. The results suggest that thyroid hormone stimulates the proliferation of the SR and possibly also increases the Ca++-pump density in the SR membrane.     

Evidence for a calcium-sensitive factor which alters the alkaline pH sensitivity of sarcoplasmic reticulum calcium transport

Oxalase-supported, ATP-dependent Ca2+ uptake by cardiac and skeletal muscle sarcoplasmic reticulum (SR) exhibits a pH profile with the maximal rate of Ca2+ uptake at pH 6.6-6.8 and marked inhibition (90-95%) at pH 7.4-7.6, a point at which Ca2+-dependent ATPase activity is optimal. These observations are noted when the SR is first preincubated in media containing no added Ca2+. This alkaline pH inhibition is not caused by an irreversible perturbation since the Ca2+ uptake rate is fully restored by changing the alkaline pH preincubation medium to pH 6.8. When SR is preincubated with added Ca2+, Ca2+ uptake at alkaline pH (7.4-7.6) is only inhibited by 10-30%. Ca2+ uptake at pH 6.8 is the same regardless of preincubation conditions. A depressed oxalate permeability is not a factor in the observed alkaline pH inhibition of Ca2+ uptake. At alkaline pH, the relationship between the preincubation Ca2+ concentration and the rate of Ca2+ uptake is hyperbolic; the half-maximal free Ca2+ concentration for stabilization of Ca2+ uptake is 8-15 microM with a Vmax equal to the velocity at the optimal pH. The Hill coefficient is 1.0, implying a single class of Ca2+-requiring sites for stabilization at alkaline pH. In contrast to its effect on Ca2+ uptake, the presence of Ca2+ during preincubation does not alter the pH sensitivity of Ca2+-dependent ATPase activity. Thus, the presence of Ca2+ during preincubation may stabilize a state of the CaATPase, conducive to the coupling of net Ca2+ translocation to Ca2+-dependent ATPase activity, which is ordinarily opposed by alkaline pH. The data suggest a single class of Ca2+-requiring sites which favors this coupled state.     

Calcium transport and exchange in mouse 3T3 and SV40-3T3 cells

The kinetics of Ca++ uptake have been evaluated in 3T3 and SV40-3T3 mouse cells. The data reveal at least two exchangeable cellular compartments in the 3T3 and SV40-3T3 cell over a 50-min exposure to 45Ca++. A rapidly exchanging compartment may represent surface-membrane-localized Ca++ whereas a more slowly exchanging compartment is presumably intracellular. The transition of the 3T3 cell from exponential growth (at 3 day's incubation) to quiescence (at 7 days) is characterized by a 7.5-fold increase in the size of the fast component. Quiescence of the 3T3 cell is also characterized by a 3.2-fold increase in the unidirectional Ca++ influx into the slowly exchanging compartment and a 3.6-fold increase in its size. The increase in size of the slow compartment at quiescence may result from a redistribution of intracellular Ca++ to a more readily exchangeable compartment, possibly reflecting a release of previously bound Ca++. In contrast, no significant change in any of these parameters is observed in the proliferatively active SV40-3T3 cells after corresponding period of incubation, even though these cells attained higher growth densities and underwent postconfluence.     

Calcium distribution in Amoeba proteus

A preliminary investigation of the distribution of cellular calcium in Amoeba proteus was undertaken. Total cellular calcium under control conditions was found to be 4.59 mmol/kg of cells. When the external Ca++ concentration is increased from the control level of 0.03 to 20 mM, a net Ca++ influx results with a new steady-state cellular calcium level being achieved in integral of 3 h. At steady state the amount of calcium per unit weight of cells is higher than the amount of calcium per unit weight of external solution when the external concentration of Ca++ is below 10 mM. At external Ca++ concentrations above this level, total cellular calcium approaches the medium level of Ca++. Steady-state calcium exchange in Amoeba proteus was determined with 45Ca. There is an immediate and rapid exchange of integral of 0.84 mmol/kg of cells or 18% of the total cellular calcium with the labelled Ca++. Following this initial exchange, there was very little if any further exchange observed. Most of this exchanged calcium could be eliminated from the cell with 1 mM La+++, suggesting that the exchanged calcium is associated with the surface of the cell. Increase in either the external Ca++ concentration of pH raise the amount of exchangeable calcium associated with the cell. Calcium may be associated with the cell surface as a co-ion in the diffuse double layer or bound to fixed negative sites on the surface of the cell. If Ca++-binding sites do exist on the cell surface, there may be more than one type and they may have different dissociation constants. The cytoplasmic Ca++ ion activity is probably maintained at very low levels.     

Inhibition of Ca++-stimulated respiration by uncouplers.

In a phosphate medium at pH 6.6 low concentrations of uncouplers such as p-trifluoromethoxyphenylhydrazone carbonylcyanide and 2,4-dinitrophenol inhibit the oxidation of beta-hydroxybutyrate and succinate, when added during Ca++-accumulation. The inhibition depends on the amount of accumulated Ca++, and is released by N,N,N',N'-tetramethyl-p-phenylendiamine plus ascorbate as substrate. Under identical conditions the uncouplers have no inhibitory effect when added to mitochondria during state 3 respiration or during accumulation of Sr++. Inhibition of respiration by the decrease of transmembranal succinate transport or by accumulation of oxaloacetate can be excluded. It is suggested that accumulation of Ca++ in the presence of phosphate induces structural alteration of the mitochondrial membrane, which on the one hand changes the accessibility or sensitivity of dehydrogenases to uncouplers and causes leakage of accumulated Ca++ on the other.     

A calcium- and pH-regulated protein from Dictyostelium discoideum that cross-links actin filaments

We have purified an actin binding protein from amebas of Dictyostelium discoideum which we call 95,000-dalton protein (95K). This protein is rod shaped, approximately 40 nm long in the electron microscope, contains two subunits measuring 95,000 daltons each, and cross-links actin filaments. Cross-linking activity was demonstrated by using falling-ball viscometry, Ostwald viscometry, and electron microscopy. Cross-linking activity is optimal at 0.1 microM Ca++ and pH 6.8, but is progressively inhibited at higher Ca++ and pH levels over a physiological range. Half-maximal inhibition occurs at 1.6 microM free Ca++ and pH 7.3, respectively. Sedimentation experiments demonstrate that elevated Ca++ and pH inhibit the binding of 95K to F-actin which explains the loss of cross-linking activity. Electron microscopy demonstrates that under optimal conditions for cross-linking, 95K protein bundles actin filaments and that this bundling is inhibited by microM Ca++. Severing of actin filaments by 95K was not observed in any of the various assays under any of the solution conditions used. Hence, 95K protein is a rod-shaped, dimeric, Ca++- and pH-regulated actin binding protein that cross-links but does not sever actin filaments.