The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA

The apparent calcium association constants (K'Ca) of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and 1,2-bis 2-bis(o-amino-5-bromophenoxy)ethane-N,N,N',N'-tetraacetic acid (dibromo-BAPTA) were measured using the method described by Bers (Am. J. Physiol. 242 (1982) C404-408). The purity of the three ligands determined from the chi-intercept of Scatchard plots were 96.3%, 79.0% and 97.3% for EGTA, BAPTA and dibromo-BAPTA, respectively. The impurity of BAPTA was found to be water by drying several samples to constant weight. Increasing temperature from 1 to 36 degrees C led to an increase in K'Ca which was of similar magnitude for the three ligands. Increasing ionic strength from 0.104 to 0.304 M led to a reduction of K'Ca in all cases, though EGTA was affected much less than BAPTA or dibromo-BAPTA. Experimental results were compared with values of K'Ca calculated from the individual association constants of the ligands for calcium and protons which were modified for the experimental conditions using the Debye-Hückel limiting law and the Van't Hoff Isochore to correct for ionic strength and temperature, respectively. The experimental values of K'Ca of EGTA agree well with those in the literature and with the calculated values. Good agreement was also found between the experimental and calculated values of K'Ca for the temperature and ionic strength dependence of BAPTA and dibromo-BAPTA.     

Critical review of the methods used to measure the apparent dissociation constant and ligand purity in Ca2+ and Mg2+ buffer solutions

Using simulated Ca²⁺ and Mg²⁺ buffers, methods proposed to measure both ligand purity and the apparent dissociation constant (K_app) were investigated regarding (1) predicted accuracy of both parameters and (2) generality of the solution.

The Bers’ Ca²⁺ macroelectrode method [Bers, D. M., 1982 A simple method for the determination of free [Ca] in Ca-EGTA solutions Am. J. Physiol. 242, C404–C408] cannot be used with Mg²⁺-macroelectrodes and is partly arbitrary since the linear part of the Scatchard plot is judged subjectively. Iterative methods have therefore been introduced. Iteration based on the Bers’ method or the lumped interference in the Nicolsky–Eisenman equation also failed with Mg²⁺ macroelectrodes. The Oiki et al., method [Oiki, S., Yomamoto, T., Okada, Y., 1994. Apparent stability constants and purity of Ca-chelating agents evaluated using Ca-sensitive electrodes by the double-log optimization method Cell Calcium 15, 209–46.] cannot be applied to Mg²⁺ macroelectrodes. The pH titration method of Moisescu and Pusch (Pflügers, Arch., 355, R122, 1975) predicted EGTA purity and Ca²⁺ contamination, but K_app values for EGTA were approximate. It cannot be applied to Mg²⁺ binding. The partition method [Godt, R.E., 1974. Calcium-activated tension of skinned muscle fibres of the frog. Dependence on magnesium adenosine triphosphate concentration J. Gen. Physiol. 63, 722–739.] only approximately estimated the K_app. Calibration, maintaining contaminating [Ca²⁺]/[Mg²⁺] at <1 μmol l⁻¹, and setting standards by dilution, is the ultimate check of calculated ionised concentrations, although technically difficult. The macroelectrode method of Lüthi et al. [1997. Calibration of Mg²⁺-selective macromolecules down to 1 μmol l⁻¹ in intracellular and Ca⁺- containing extracellular solutions. Exp. Physiol. 82, 453–467] accurately predicted purity and K_app at pK_app values >4 and was independent of electrode characteristics. It is considered the method of choice.

Macroelectrode primary calibration should be carried out in solutions varying from 0.5 to 10 mmol l⁻¹ combined with either Ca–EGTA or Mg–EDTA buffers; the [Ca²⁺] and [Mg²⁺] in other buffer ligands can be measured in a secondary calibration.     

Cation chelators and their utilization in the preparation of low concentrations of calcium. Caution of use in biological systems with high affinity to calcium

Ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA)-calcium buffer is widely used in various calcium-dependent reactions where free calcium concentrations of 1 microM or less are desirable. The free calcium concentration is calculated from the association constant of EGTA . Ca2- and serves as the true available calcium in systems devoid of a constituent with high affinity to Ca2+ other than EGTA. But, it is conceivable that in systems with high affinity to Ca2+ (comparable to that of EGTA) this is not the case, because such systems will compete with EGTA for the total calcium content in the medium, so that the true available calcium for these systems is greater than that calculated from the EGTA buffer. This hypothesis was tested in three different Ca+-modulated systems: Quin 2 fluorescence, Ca2+-ATPase, and adenylate cyclase, in which the response of the system to calcium was compared between EGTA-free media, containing known amounts of added calcium, and the EGTA-Ca2+ buffer media. In all three systems, the amount of available calcium in the EGTA-Ca2+ buffer medium was much greater than the calculated free Ca2+ concentration. This indicates that in systems with high affinity to Ca2+, preparation of available Ca2+ in concentrations of 1 microM or less must account for both the EGTA and the system capacities for calcium.     

Preparation of solutions with free calcium concentration in the nanomolar range using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid

There are many uses for solutions with a known free calcium concentration ([Ca2+]free) in the nanomolar range. Most frequently ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) has been used as a buffer for the control of [Ca2+]free; however, under a variety of conditions the use of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) for this purpose would be advantageous. The theory and calculations necessary to make solutions with known [Ca2+]free appropriate for given conditions of pH, ionic strength, and temperature for use with EGTA or BAPTA are reviewed. Practical considerations and methods for making such solutions are detailed. The advantages and disadvantages associated with the use of each of the two chelators are discussed. As one example of the application of solutions with free calcium in the nanomolar range, the dissociation constant of the fluorescent indicator fura-2 for calcium has been determined in a physiologic buffer at 22 and 37 degrees C. For practical reasons, the use of BAPTA is advantageous when solutions with different known [Ca2+]free must be used on a daily basis.     

Effects of intracellular injection of calcium buffers on light adaptation in Limulus ventral photoreceptors

The calcium sequestering agent, EGTA, was injected into Limulus ventral photoreceptors. Before injection, the inward membrane current induced by a long stimulus had a large initial transient which declined to a smaller plateau. Iontophoretic injection of EGTA tended to prevent the decline from transient to plateau. Before injection the plateau response was a nonlinear function of light intensity. After EGTA injection the response-intensity curves tended to become linear. Before injection, bright lights lowered the sensitivity as determined with subsequent test flashes. EGTA injection decreased the light-induced changes in sensitivity. Ca-EGTA buffers having different levels of free calcium were pressure-injected into ventral photoreceptors; the higher the level of free calcium, the lower the sensitivity measured after injection. The effects of inotophoretic injection of EGTA were not mimicked by injection or similar amounts of sulfate and the effects of pressure injection of EGTA buffer solutions were not mimicked by injection of similar volumes of pH buffer or mannitol. The data are consistent with the hypothesis that light adaptation is mediated by a rise of the intracellular free calcium concentration.     

Buffering of calcium in the vicinity of a channel pore.

The function of calcium entry or release channels is often modulated by the cytosolic free calcium concentration. When such channels are studied in isolation, calcium buffer solutions are usually used to control the free calcium at the cytosolic face of the channel. Such solutions are generally formulated on the basis of equilibrium considerations. We calculate the gradient of [Ca2+] in the vicinity of a channel pore, in the presence of such buffers. We find that the effective degree of buffering near the pore is markedly affected by kinetic considerations. Commonly used EGTA solutions are completely ineffective in buffering [Ca2+] within macromolecular distances of the pore. In order to achieve useful buffering, the fastest buffers (e.g. BAPTA derivatives) must be used, in concentrations very much higher than those conventionally employed. Because of the diffusion limit on the maximum rate of binding of calcium to the buffer ligand, it is physically impossible to achieve good control of [Ca2+] at cytosolic levels at distances of less than a few nm from a pore conducting pico-ampere calcium current.     

Real-time measurement of the rate of sarcoplasmic reticulum calcium pumping in skinned muscle fibers

A method and a device had been developed to directly measure the accumulation of calcium in the sarcoplasmic reticulum and its release from the sarcoplasmic reticulum, depending on the free Ca²⁺ concentration in the solution. The sarcoplasmic reticulum occupies to 30% of the volume of the swim bladder muscles of the oyster toadfish Opsanus tau. To isolate and skin muscle fibers and to remove the accumulated calcium from the sarcoplasmic reticulum, a set of solutions containing EGTA as a pCa buffer was used. To measure the calcium exchange between a fiber ～10 nl in volume and the solution in a 5-μl cuvette, instead of EGTA, 50–100 μM FURA2 or bisFURA2 was used both as pCa buffer and as a fluorescent indicator of the calcium concentration in the cuvette. An increase in fluorescence intensity meant an increase in the free FURA concentration in the solution surrounding the fiber since the calcium entering the sarcoplasmic reticulum was taken from this solution. The slope of the fluorescence curve corresponded to a rate of calcium accumulation in the sarcoplasmic reticulum of 1.6 μmol per second per liter of the solution in the cuvette or 2.6 mmol per second per liter of the sarcoplasmic reticulum. A solution without oxalate and ruthenium red may exhibit oscillations of the free FURA concentration, which can be explained by calcium-activated calcium release from the sarcoplasmic reticulum.     

Twitches in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid

Single muscle fibers continue to twitch for up to 20 min when immersed in ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) solutions containing less than 10⁻⁸ M free calcium. Failure of the twitch results from reversible depolarization, which occurs after 15–20 min in EGTA. The results make it clear that external calcium or calcium in the transverse tubules play no essential part in action potential propagation or excitation-contraction coupling.     

Expanding the range of free calcium regulation in biological solutions

Many biological systems use ethylene glycol bis (beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) to regulate the free calcium concentration ([Ca(2+)](free)) in the presence of physiological levels of free Mg(2+) ([Mg(2+)](free)). Frequently, it is necessary to work at [Ca(2+)](free) beyond EGTA's buffering capabilities. Therefore, we have developed methods to extend the buffering range by adding nitrilotriacetic acid (NTA) to solutions containing EGTA. This extension results from NTA having a lower K'(dCa) than EGTA. Such equilibria are solved by pCa Calculator, a computer program designed to aid in the study of Ca(2+)-dependent physiological processes while accounting for the effects of pH, temperature, and ionic strength. With multiple chelators and pH buffers from which to choose, pCa Calculator calculates the total concentration of each species required to achieve specified free concentrations of Ca(2+), ATP, and Mg(2+). The program is intuitive, user-friendly, and flexible enough to fix or vary the [Mg-ATP(2-)] and ionic strength. Moreover, it can account for increases in experimental volume from calcium addition. A comparative analysis is reported for testing solutions in the presence and absence of NTA by measuring the calcium binding affinity of fluorescent cardiac troponin C. These findings demonstrate that EGTA, when used in conjunction with NTA, improves and expands the regulation of free calcium in solution.     

Prevention of the cortical reaction in fertilized sea urchin eggs by injection of calcium-chelating ligands.

Eggs from the sea urchin, Lytechinus pictus, were injected with either EGTA or EDTA, and were subsequently fertilized. EGTA prevented cortical vesicle discharge and formation of the fertilization membrane. EDTA had either no effect, or sometimes retarded the elevation of the fertilization membrane, or reduced the percentage of eggs with elevated membranes. Theoretical considerations lead to estimates of the probable effects of EGTA and EDTA on the internally released calcium which triggers the cortical reaction. Whether or not cytoplasmic calcium buffers are considered, it is concluded: (1) that normally several times the threshold calcium concentration for the cortical reaction is released into a subsurface space; (2) that if a rapidly-equilibrating high-affinity buffer is present, it is locally saturated by the calcium released internally; (3) the injected EDTA reduces the subsurface free calcium concentration normally reached to approximately threshold for the cortical reaction, while injected EGTA reduces the calcium concentration to below this threshold; and (4) a rise in the internal ionic calcium concentration is a necessary step in the activation of the cortical reaction at fertilization.     

Intracellular calibration of the fluorescent calcium indicator Fura-2

We present the techniques we have used and the problems we have encountered in our laboratories in the in vivo calibration of the fluorescent Ca2(+)-indicator Fura-2. These techniques include the use of potentiometric methods for the precise control and determination of Ca2+ levels in bathing solutions, in association with methods for the equilibration of internal and external solutions with ionophores (Br-A23187, ionomycin, monensin and nigericin). A by-product of these techniques has been the development of a simple procedure that utilizes Fura-2 as a general indicator of ionized Ca2+ concentrations within the physiological range (pCa 7.5 to 5.5), in other experimental solutions. The major advantages of this relatively simple procedure are that it is (i) rapidly performed, (ii) independent of the total EGTA concentration within each experimental solution, (iii) independent of the absolute EGTA purity, and (iv) unaffected by a large number of potentially interfering cations (i.e. Mg2+, H+, K+, Na+) within the test solutions.     

Calcium diffusion modeling in a spherical neuron. Relevance of buffering properties.

We have developed a calcium diffusion model for a spherical neuron which incorporates calcium influx and extrusion through the plasma membrane as well as three calcium buffer systems with different capacities, mobilities, and kinetics. The model allows us to calculate the concentration of any of the species involved at all locations in the cell and can be used to account for experimental data obtained with high-speed Ca imaging techniques. The influence of several factors on the Ca2+ transients is studied. The relationship between peak [Ca2+]i and calcium load is shown to be nonlinear and to depend on buffer characteristics. The time course of the Ca2+ signals is also shown to be dependent on buffer properties. In particular, buffer mobility strongly determines the size and time course of Ca2+ signals in the cell interior. The model predicts that the presence of exogenous buffer, such as fura-2, modifies the Ca2+ transients to a variable extent depending on its proportion relative to the natural, intrinsic buffers. The conclusions about natural calcium buffer properties that can be derived from Ca imaging experiments are discussed.     

Presynaptic calcium signals during neurotransmitter release: detection with fluorescent indicators and other calcium chelators.

Synthetic calcium buffers, including fluorescent calcium indicators, were microinjected into squid 'giant' presynaptic nerve terminals to investigate the calcium signal that triggers neurotransmitter secretion. Digital imaging methods, applied in conjunction with the fluorescent calcium indicator dye fura-2, reveal that transient rises in presynaptic calcium concentration are associated with action potentials. Transmitter release terminates within 1-2 ms after a train of action potentials, even though presynaptic calcium concentration remains at micromolar levels for many seconds longer. Microinjection of the calcium buffer, EGTA, into the presynaptic terminal has no effect on transmitter release evoked by single presynaptic action potentials. EGTA injection does, however, block the change in calcium concentration measured by fura-2. Therefore, the calcium signal measured by fura-2 is not responsible for triggering release. These results suggest that the rise in presynaptic calcium concentration that triggers release must be highly localized to escape detection with fura-2 imaging. Unlike EGTA, microinjection of BAPTA--a calcium buffer with an equilibrium affinity for calcium similar to that of EGTA--produces a potent, dose-dependent, and reversible block of action-potential evoked transmitter release. The superior ability of BAPTA to block transmitter release apparently is due to the more rapid calcium-binding kinetics of BAPTA compared to EGTA. Because EGTA should bind calcium within a few tens of microseconds under the conditions of our experiments, the inability of EGTA to block release indicates that transmitter release is triggered within a few tens of microseconds after the entry of calcium into the presynaptic terminal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diffusion barriers limit the effect of mobile calcium buffers on exocytosis of large dense cored vesicles

Fast exocytosis in melanotropic cells, activated by calcium entry through voltage-gated calcium channels, is very sensitive to mobile calcium buffers (complete block at 800 microM ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA)). This indicates that calcium diffuses a substantial distance from the channel to the vesicle. Surprisingly, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), having a similar KD for calcium as EGTA but a approximately 100 times faster binding rate, blocked exocytosis only twice as effectively as EGTA. Using computer simulations, we demonstrate that this result cannot be explained by free diffusion and buffer binding rates. We hypothesized that local saturation of calcium buffers is involved. A diffusion barrier for both calcium and buffer molecules, located 50-300 nm from the membrane and reducing diffusion 1000 to 10,000 times, generated similar calcium concentrations for specific concentrations of EGTA and BAPTA. With such barriers, calcium rise phase kinetics upon short step depolarizations (2-20 ms) were faster for EGTA than for BAPTA, implying that short depolarizations should allow exocytosis with 50 microM EGTA but not with 25 microM BAPTA. This prediction was confirmed experimentally with capacitance measurements. Coupling exocytosis to calcium dynamics in the model, we found that a barrier with a approximately 3000 times reduced diffusion at approximately 130 nm beneath the membrane best explains the experimentally observed effects of EGTA and BAPTA on block and kinetics of release.     

Excitation and adaptation of Limulus ventral photoreceptors by inositol 1,4,5 triphosphate result from a rise in intracellular calcium

Single pressure injections of 1-10 pl of inositol 1,4,5 triphosphate (IP3) or inositol 4,5 bisphosphate [I(4,5)P2] excite Limulus ventral photoreceptors by inducing rapid bursts of inward current. After excitation by IP3, responses to subsequent injections of IP3 or light flashes are often reversibly diminished (adapted). Single injections of IP3 and I(4,5)P2 are effective at concentrations in the injecting pipette of 20 microM to 1 mM. Single injections of inositol 1,4 bisphosphate are ineffective at concentrations of 100-500 microM. Excitation by IP3 or I(4,5)P2 is accompanied by a rise in intracellular free calcium, as indicated by aequorin luminescence. Prior injection of calcium buffer solutions containing 100 mM EGTA greatly diminishes the total charge transferred across the plasma membrane during excitation by IP3 or I(4,5)P2, which suggests that a rise in Cai is necessary for excitation by the inositol polyphosphates. Adaptation of the response to light by IP3 is also abolished by prior injection of EGTA. In the same cells, the response to brief light flashes is slowed and diminished in amplitude by the injection of calcium buffer, but the charge transferred during the response is not significantly diminished. This suggests that light has access to a pathway of excitation in the presence of EGTA that is not accessible to intracellularly injected IP3.     

Decay of the slow calcium current in twitch muscle fibers of the frog is influenced by intracellular EGTA

The mechanism(s) of the decay of slow calcium current (ICa) in cut twitch skeletal muscle fibers of the frog were studied in voltage-clamp experiments using the double vaseline-gap technique. ICa decay followed a single exponential in 10 mM external Ca2+ and 20 mM internal EGTA solutions in all pulse protocols tested: single depolarizing pulses (activation protocol), two pulses (inactivation protocol), and during a long pulse preceded by a short prepulse (400 ms) to 80 mV (tail protocol). In single pulses the rate constant of ICa decay was approximately 0.75 s-1 at 0 mV and became faster with larger depolarizations. ICa had different amplitudes during the second pulses of the inactivation protocol (0 mV) and of the tail protocol (-20 to 40 mV) and had similar time constants of decay. The time constant of decay did not change significantly at each potential after replacing 10 mM Ca2+ with a Ca2+-buffered solution with malate. With 70 mM intracellular EGTA and 10 mM external Ca2+ solutions, ICa also decayed with a single-exponential curve, but it was about four times faster (approximately 3.5 s-1 at 0 mV pulse). In these solutions the rate constant showed a direct relationship with ICa amplitude at different potentials. With 70 mM EGTA, replacing the external 10 mM Ca2+ solution with the Ca2+-buffered solution caused the decay of ICa to become slower and to have the same relationship with membrane potential and ICa amplitude as in fibers with 20 mM EGTA internal solution. The mechanism of ICa decay depends on the intracellular EGTA concentration: (a) internal EGTA (both 20 and 70 mM) significantly reduces the voltage dependence of the inactivation process and (b) 70 mM EGTA dramatically increases the rate of tubular calcium depletion during the flow of ICa.     

Calcium regulation by and buffer capacity of molluscan neurons during calcium transients

The properties of Ca-regulation and -buffering of physiological levels of Ca-transients were examined in the soma of Archidoris monteryensis neurons. The rate of recovery from a Ca-transient was examined with two experimental protocols; in one the pulse duration was kept constant and its amplitude was varied, and in the other the duration was varied while the amplitude was kept constant. These experiments revealed that the recovery from a Ca-transient was approximately a first order process and the apparent first order rate constant was dependent on the duration of Ca-influx. The calcium buffer capacity of the cytoplasm was determined by an indirect method which utilised measured amounts of intracellular EGTA to reduce transient changes in free calcium. An equation for the cytoplasmic buffer capacity was derived on the assumption that the capacities of exogenous and endogenous Ca buffers summate linearly. The resting cytoplasmic Ca buffer capacity was 45.2 microM/delta pCa, when it was assumed that the incoming Ca diffuses a distance of 10 microns into the cytoplasm. For a diffusion distance of 5 microns it was 34.5 microM/delta pCa. In both cases, the buffer capacity increased with an increase in the size of Ca transient.     

Calcium and the mechanical properties of soybean hypocotyl cell walls: Possible role of calcium and protons in cell-wall loosening

The role of calcium in the mechanical strength of isolated cell walls of soybean (Glycine max (L.) Merr. cv. Wayne) hypocotyls has been investigated, using the Instron technique to measure the plastic extensibility (PEx) of methanol-boiled, bisected hypocotyl sections and epidermal strips, and atomic absorption spectroscopy to measure wall calcium. Plastic extensibility was closely correlated with the growth rate of intact soybean hypocotyls. Removal of calcium from isolated cell walls by ethylene glycol-bis(2-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) or low pH increased PEx, while addition of calcium decreased PEx; both effects were reversible. The amount of calcium removed and the increase in PEx at pH 4.5 were strongly dependent upon the chelating ability of the buffer anion. There was a direct correlation between the amount of calcium removed from the wall by EGTA or acid and the increase in PEx. Removal of up to 60% of the calcium increased PEx of half-section up to two fold, but further loss of calcium caused a much greater increase in PEx. With epidermal strips, PEx increased only when calcium was reduced below a threshold. At pH 3.5, there was an additional increase in PEx after a lag of about 2 h; this additional increase may be the result of acid-induced cleavage of a different set of load-bearing bonds. We conclude that calcium bridges are part of the load-bearing bonds in soybean hypocotyl cell walls, and that breakage of these crosslinks by apoplastic acid participates in wall loosening. Acid-induced solubilization of wall calcium may be one mechanism involved in wall loosening of dicotyledonous stems.Abbreviations EGTA ethylene glycol-bis(2-aminoethyl ether)-N,N,N,N-tetraacetic acid - PEx Instron plastic extensibility     

Calcium-labile mitotic spindles isolated from sea urchin eggs (Lytechinus variegatus)

We isolated calcium-labile mitotic spindles from eggs of the sea urchin Lytechinus variegatus, using a low ionic strength, EGTA lysis buffer that contined 5.0 mM EGTA, 0.5 mM MgCl2, 10-50 mM PIPES, pH 6.8, with 1% Nonidet P-40 (detergent) and 20-25% glycerol. Isolated spindles were stored in EGTA buffer with 50% glycerol for 5-6 wk without deterioration. The isolated spindles were composed primarily of microtubules with the chromosomes attached. No membranes were seen. Isolated spindles, perfused with EGTA buffer to remove the detergent and glycerol, had essentially the same birefringent retardation (BR) as spindles in vivo at the same mitotic stage. Even in the absence of glycerol and exogenous tubulin, the isolated spindles were relatively stable in the EGTA buffer: BR decayed slowly to about half the initial value within 30-45 min. However, both the rate and extent of BR decay increased with concentrations of Ca2+ above 0.2-0.5 muM as assayed using Ca-EGTA buffers (0.2 mM EGTA, 0.5 mM MgCl2, 50 mM PIPES, pH 6.8, plus various amounts of CaCl2). Microtubules depolymerized almost completely in < 6 min at Ca2+ concentrations of 2 muM and within several seconds at 10 muM Ca2+. Of several divalent cations tested, only Sr2+ caused comparable changes in BR. The absence of membranes in the isolated spindles appeared to be associated with a lack of calcium- sequestering ability. Our results suggest that calcium ions play an important role in the depolymerization of spindle microtubules and that membrane components may function within the mitotic apparatus of living cells to sequester and release calcium ions during mitosis.     

Force and stiffness in glycerinated rabbit psoas fibers. Effects of calcium and elevated phosphate

Force (F) and stiffness (K) were measured in glycerinated psoas fibers at various calcium levels with 0, 10, 20, and 30 mM orthophosphate (Pi) added to the bathing solutions. The concentrations of bathing solution constituents were as follows: 110 mM potassium, 40 mM sodium, 4 mM MgATP, 10 mM total EGTA, and variable amounts of MOPS (pH buffer). The pH was 7.0, the ionic strength was 200 mM, and the temperature was 10 degrees C. Calcium levels were established by adding various amounts of CaCl2. All solutions contained 4% Dextran T-500. Fiber K was measured by imposing sinusoidal length changes (0.03-0.1%) at 1 kHz and by applying rapid steps in length and measuring the resulting F changes. At all [Pi] tested, K was more sensitive to calcium than F. Elevating bathing solution [Pi] caused a decrease in the calcium sensitivity of both F and K, while the slopes of F-calcium and K-calcium relations increased. In maximally activating calcium, raising [Pi] caused a continuous decrease in F over the range tested, while from very low to 10 mM Pi K remained constant. Above 10 mM Pi K declined, but to a lesser extent than did F. The results suggest that under our experimental conditions strongly attached crossbridges can exist in both force-producing and non-force-producing states, and that the relative population of these states may be calcium dependent.