Divalent Cation Exchange Research Articles

A23187—Channel behaviour: Fluorescence study

Pyranine entrapped soylipid liposomes have been used as a model system to study the proton transport across membrane in the presence of A23187, a carboxylic ionophore specific for electroneutral exchange of divalent cations. An apparent rate constant (kapp) for transport of protons has been determined from the rate of change of fluorescence intensity of pyranine by stopped flow rapid kinetics in the presence of proton gradient The variation of kapp has been studied as a function of ionophore concentration and the results have been compared with gramicidin—a well known channel former under the similar experimental conditions. The rates thus obtained showed that A23187 is not only a simple carrier but also shows channel behaviour at high concentration of ionophore.

Influence of the high affinity divalent cation on actin tryptophan fluorescence.

This study demonstrates that the intrinsic tryptophan fluorescence of actin provides an effective and convenient way of measuring divalent cation exchange and polymerization of native actin. In the measurement of divalent cation exchange, this method is as sensitive as those previously used (8) and provides further evidence of the importance of the tightly bound divalent cation to the properties of actin. In monitoring polymerization, this method cannot compete with the sensitivity of the commonly used pyrene-actin fluorescence. However, some proteins (e.g., profilin) and other agents (e.g., cytochalasin D) that bind to actin are affected by the presence of fluorescent labels, making actin intrinsic fluorescence potentially useful in investigating the interaction of these agents with actin, and in validating data obtained using labeled actin. Our laboratory routinely checks the quality of our native actin preparations using this technique and, more recently, we have used actin tryptophan fluorescence to monitor the nucleating and severing effects of gelsolin on actin. The simplicity of the technique is most appealing, and we expect that a variety of innovative and routine uses will be developed.

Actin-bound nucleotide/divalent cation interactions.

At this point, it may be worthwhile to list, in summary form, the important aspects of divalent cation and nucleotide binding to actin that have been reviewed here: 1) High affinity divalent cation binding to actin is very tight, with equilibrium dissociation constant KCa approximately 1 nM and KMg approximately 5 nM at pH 7.0. 2) The binding kinetics of Ca++ are diffusion limited. Dissociation is slow, with k-Ca approximately 0.015 sec at pH 7.0 (and low ionic strength). 3) The binding kinetics of Mg++ are limited by the characteristics of the Mg++ aquo-ion and are much slower than for Ca++; k-Mg approximately 0.0012 at pH 7.0. 4) Increase in pH or ionic strength weakens divalent cation binding at the high affinity site, primarily by increasing k-Ca and k-Mg. 5) Exchange of Mg++ for Ca++ (or vice versa) at the high affinity site is by a competitive pseudo-first order process with an apparent rate constant (kapp) intermediate between k-Ca and k-Mg and dependent upon the cation concentration ratio [Ca]/[Mg] present. 6) High affinity ATP binding is modulated by the high affinity divalent cation. The cation concentration range over which this modulation occurs is about 100-fold higher for Mg++ than for Ca++, again because of the different characteristics of the Mg++ and Ca++ aquo-ions. 7) At low divalent cation concentrations, ATP dissociation from actin is limited by dissociation of the tightly-bound divalent cation. 8) At high divalent cation concentrations, ATP dissociation probably occurs via dissociation of the divalent cation-nucleotide complex and is quite slow, with dissociation rate constant approximately 0.0005 sec-1. 9) Competitive nucleotide exchange on actin may be described by a pseudo-first order model analogous to that for divalent cation exchange. The pseudo-first order rate constants depend upon the divalent cation concentration. The overall nucleotide exchange rate constant kex depends upon these constants and the solution nucleotide concentration ratio, e.g. [ATP]/[ADP]. The following circumstances develop from the characteristics of the high affinity binding of divalent cation and nucleotide to actin: 1) The standard methods for actin preparation convert in vivo Mg-actin into Ca-actin. 2) Converting Ca-actin back to Mg-actin is not easy. A very low ratio of [Ca]/[Mg] is necessary, which usually requires the use of Ca-cheltors, and a long time (5-10 min) must be allowed for complete exchange.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of Cations on the Volume and Elemental Composition of Nematocysts Isolated from Acontia of the Sea Anemone Calliactis polypus.

The hypothesis that exchange of intracapsular divalent cations with Na+ in seawater increases the internal osmotic pressure during discharge of nematocysts of marine cnidarians was tested by examining effects of externally applied cations on the volume and elemental composition of nematocysts isolated from acontia of the sea anemone Calliactis polypus. The volume of isolated nematocysts increased with increasing concentrations of cations if the cation was monovalent but appeared to decrease if the cation was divalent. Ca2+ reduced the internal osmotic pressure of the nematocysts more efficiently than Mg2+. X-ray microanalysis of nematocysts incubated in 1 M solutions of various salts showed that Ca2+ in isolated nematocysts was only partially replaced, if at all, by externally applied Na+ and Mg2+ while most Mg2+ was replaced by Na+ and Ca+. The present results suggest that exchange of intracapsular divalent cations with external monovalent cations increases the internal osmotic pressure, and that selective binding of Ca2+ to polyanions in the capsule decreases it. Whether the increase in the internal osmotic pressure caused by the cation exchange is large enough to trigger discharge remains to be investigated.

High affinity divalent cation exchange on actin. Association rate measurements support the simple competitive model.

Each actin molecule has one high affinity site which binds a divalent cation. It has been proposed that an isomerization of the actin molecule is involved in divalent cation exchange at this site ("isomerization model," Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886); we have maintained that exchange is by a simple competitive mechanism (Estes, J. E., Selden, L. A., and Gershman, L. C. (1987) J. Biol. Chem. 262, 4952-4957). Here, using fluorescent-labeled actin, we measure the apparent rate constant for exchange (kapp) as a function of the ratio of free Ca2+ and Mg2+ concentrations, ([Ca]/[Mg]), and show that both models are consistent with the data. The major parameter controlling this relationship in the simple competitive exchange model, the ratio of the association rate constants for Ca2+ and Mg2+ to actin (kCa/kMg), is found to have a value of about 90. We have verified this parameter by direct measurements of kCa and kMg, finding that kCa = 1.9 x 10(7) M-1 s-1 and kMg = 2.3 x 10(5) M-1 s-1, consistent with the characteristics of the Ca2+ and Mg2+ aquo ions. The corresponding parameter derived from the isomerization model is not verifiable. We conclude that high affinity divalent cation exchange on actin proceeds by a simple competitive mechanism.

Effects of obstruction on renal functions.

Following ureteral obstruction there is a progressive fall in glomerular filtration rate (GFR) due to a reduction in single nephron glomerular filtration rate (SNGFR) and a reduced number of filtering nephrons. Renal plasma flow also declines after a transient, prostaglandin-dependent increase, due to afferent and efferent arteriolar vasoconstriction. The vasoactive hormones thromboxane A2 and angiotensin II are implicated in the pathogenesis of the vasoconstriction following ureteral obstruction and they also reduce the glomerular ultrafiltration coefficient by causing mesangial contraction. Ureteral obstruction also leads to profound changes in renal tubular cell function. These include altered sodium and water handling resulting in a post-obstructive diuresis and natriuresis and a failure to dilute or concentrate the urine. Potassium and divalent cation exchange is also affected, as is urinary acidification. Furthermore, the response of the tubule to hormones such as antidiuretic hormone and parathyroid hormone is impaired. The pathophysiology of these alterations in renal function is discussed.

Tight binding of divalent cations to monomeric actin. Binding kinetics support a simplified model.

Using the fluorescent Ca2+ selective chelator Quin2 to induce and measure the dissociation of Ca2+ from actin, we have recently found that actin binds Ca2+ and Mg2+ much more tightly than previously thought (Gershman, L.C., Selden, L.A., and Estes, J.E. (1986) Biochem. Biophys. Res. Commun. 135, 607-614). In this report, we show that the kinetics of dissociation of Ca2+ from Ca-actin and Mg2+ from Mg-actin closely parallel the fluorescence changes in 1,5-I-N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS)-actin, suggesting that the 1,5-I-AEDANS-actin fluorescence directly reflects slow first-order cation exchange rather than a slow Mg2+-induced isomerization as originally proposed by Frieden (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886). Measuring divalent cation exchange directly, we have determined the dissociation rate constants for Ca2+ (k-Ca) and Mg2+ (k-Mg), the equilibrium dissociation constants for Ca2+ (KCa), and the ratio of cation binding affinities, KMg/Kca, to actin over the pH range 7-8. We have found that k-Ca is 5-10 times greater than k-Mg and KMg is about 4 times greater than KCa. From the data we calculate the association rate constants for Ca2+ (kCa) and Mg2+ (kMg) to be about 7 X 10(6) M-1 s-1 and 2 X 10(5) M-1 s-1, respectively. kCa appears to be diffusion-limited, but kMg is significantly smaller due to the characteristics of the Mg2+ aquo ion. These findings are consistent with a simple first-order binding model for the tight binding of divalent cations to actin.

The tightly bound divalent cation regulates actin polymerization

The polymerization characteristics of Ca ++-actin and Mg ++-actin were studied by measuring initial rates of polymerization upon addition of phalloidin-stabilized nuclei and neutral salt. Under conditions where the effects of divalent cation exchange were minimized, CaCl 2 and MgCl 2 were found to be equally effective in polymerizing actin. Mg ++-actin was found to nucleate and polymerize more readily than Ca ++-actin, having a forward rate constant about twice that of Ca ++-actin under a variety of polymerizing conditions. The critical concentration for Ca ++-actin is approximately 20 times that for Mg ++-actin under equivalent conditions. These data imply that the polymer of Mg ++-actin must be more stable than that of Ca ++-actin, having a depolymerization rate constant about 10 fold lower. Since Mg ++ is probably the tightly-bound cation in vivo , whereas Ca ++-actin has been more widely studied in vitro , it would appear that actin in its physiological state is probably more polymerizable and more stable in the polymer form than previously considered.

Regulation of ion permeabilities of isolated rat liver cells by external calcium concentration and temperature

Regulation of ion transport through the plasma membrane was studied on single cell suspensions of hepatocytes, obtained after perfusion of rat liver with collagenase/hyaluronidase solution. Steady-state intracellular K and Na contents were shown to be markedly dependent on external Ca concentration and temperature, the sum of both ion concentrations remaining nearly constant. In contrast, steady-state intracellular chloride content was found to be independent of external Ca concentration, but dependent on temperature. Using the constant field relations, the passive permeabilities PK and PCl for potassium and chloride, respectively, were derived from the experimental data. At temperatures at and above 37 degrees C, with increasing external Ca concentration, PK, exhibits a sharp decrease at about 10(-4)M. In contrast, PCl at 37 degrees C was found to be independent of Ca concentration within experimental error. Earth alkali ions other than Ca, show marked but different effects on PK if compared at equal concentrations. Preincubation of the cells with cholesterol leads to a broadening of the dependence of PK on external Ca concentration. The above results, as well as those on the dependence of PK on external Ca concentration obtained by other authors, could be quantitatively described by a theoretical model of the plasma membrane proposed earlier. This model postulates regulatory binding sites, which cooperatively undergo a cation exchange of divalent cations by K+ ions from the external medium if the cation composition of the latter is altered.

Heterovalent Cation Exchange Equilibria in Soils with Variable and Heterogeneous Charge

AbstractDivalent cations were incrementally displaced, from limed and unlimed samples of three soils, by suspending the soils in increasing volumes of 2mM KCl. Mass action coefficients for exchange between Ca and Mg were constant. But for the exchange of divalent cations by K, the exchange coefficients of Kerr, Vanselow, Gapon, Krishnamoorthy and Overstreet, and Gaines and Thomas increased markedly as potassium saturation or pH increased.For each cation species, separate fractions which were relatively tightly or relatively loosely adsorbed could be estimated by a simple regression analysis. The loosely adsorbed Ca, which may control Ca availability to plants, could be independently estimated by a leaching procedure. Mass action exchange coefficients for the loosely adsorbed cations were approximately constant under all conditions tested.The variable charge mobilized by liming differed from the fixed charge in having a slightly higher proportion of sites with high divalent affinity, in an Oxisol, and a much lower proportion in a Vertisol and a Mollisol.

Behavior of divalent cations and nucleotides bound to F-actin

1. 1. Nucleotides and divalent cations bound to F-actin were found to be slowly exchanged in the absence of any agitation such as sonic vibration; the half-life time, τ 1 2 , was about 5 h at 37°. 2. 2. The rate of exchange of divalent cation (Ca 2+) was always a little faster than that of nucleotide (ADP); however, the general manner of the exchange of divalent cations was similar to that of nucleotides. 3. 3. The rate of exchange was almost independent of protein concentration. High temperature or high pH increased the rate of exchange. Activation enthalpies for the exchange of ADP and Ca 2+ were both about 25 kcal/mole at neutral pH. 4. 4. The release of divalent cations or nucleotides occurred in divalent-cation- or nucleotide-free solvent. The incorporation of divalent cations and nucleotides into divalent-cation- and nucleotide-free F-actin was examined, and their binding constants were estimated. 5. 5. In the absence of ATP, the rate of exchange was decreased by myosin, H-meromyosin and tropomyosin. A large increase of Ca 2+ and ADP exchange was observed with superprecipitation. 6. 6. The mechanism of exchange or release is discussed on the basis of two models: the cycle of partial destruction of the F-actin structure, and the G-F equilibrium cycle at the peripheral region of F-actin.