Observation of calcium uptake by isolated sarcoplasmic reticulum employing a fluorescent chelate probe

The fluorescent chelate probe technique is employed to observe the accumulation and binding of Ca⁺⁺ to isolated sarcoplasmic reticulum from skeletal and cardiac muscle. Chlorotetracycline serves as a fluorescent chelate probe which chelates to membrane bound Ca⁺⁺ giving rise to an intensely fluorescence adduct. An increase in fluorescence of chlorotetracycline is caused by ATP induced Ca⁺⁺ transport in both skeletal and cardiac muscle microsomes. The fluorescence spectra indicate that Ca⁺⁺ lies on the membrane surface in a relatively polar environment.     

Monitoring of membrane-bound divalent cations in plant mitochondria using chlorotetracycline fluorescence

Chlorotetracycline (CTC) shows a strongly enhanced fluorescence upon addition of mitochondria isolated from Jerusalem artichoke ( Helianthus tuberosus L.) tubers in a low-cation medium. This indicates the presence of membrane-bound divalent cations. The chelation by CTC of the membrane-bound divalent cations does not affect the oxidation of exogenous NADH significantly. The removal of the bound divalent cations using ethyleneglycol-bis-(β-aminoethylether)-N,N'-tetraacetic acid (EGTA) and EDTA causes an 80% decrease in CTC fluorescence. Titration of CTC fluorescence (a direct measure of bound divalent cations) and 9-aminoacridine fluorescence (a measure of surface potential) with EGTA and EDTA gives similar curves, although CTC fluorescence responds more slowly to the addition of chelators. The same bound divalent cations appear to be monitored by CTC fluorescence or by 9-aminoacridine fluorescence.     

Induction of passive monovalent cation-exchange activity in heart mitochondria by depletion of endogenous divalent cations

Depletion of mitochondrial divalent cations by addition of the ionophore A23187 results in a marked increase in passive ${}^{42}\text{K}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ exchange activity. The exchange is activated by increasing pH and temperature and inhibited by added divalent cations. The reaction is independent of the amount of A23187 present, but depends on the concentration of external K⁺ (K_m = 25 mm). Intramitochondrial ⁴²K⁺ in cation-depleted mitochondria exchanges passively with external Na⁺ and Li⁺, but not with choline⁺. The evidence suggests that removal of mitochondrial divalent cations by A23187 activates the endogenous $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange component of the mitochondrion and that the activated exchanger promotes cation/cation exchange in the absence of a metabolic pH gradient.     

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.     

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.     

NOTPME: a 31P NMR probe for measurement of divalent cations in biological systems

1,4,7-Triazacyclononane-N,N',N'-tris(methylenephosphonate monoethylester) (NOTPME) has been synthesized, characterized and analyzed for use as a 31P NMR indicator of intracellular Mg2+ and Zn2+ ions. The 31P NMR spectrum of this chelate in the presence of metal ions shows characteristic resonances for the free chelate, Mg(NOTPME)-, Zn(NOTPME)-, and Ca(NOTPME)-. The Kd values indicate that this chelate has a 10-fold higher affinity for Mg2+ than for Ca2+ at physiological pH values. In the presence of Mg2+, NOTPME is readily loaded into red blood cells. A 31P NMR spectrum of red cells taken after several washings shows resonances characteristic of entrapped NOTPME and the Mg(NOTPME)- complex, the relative areas of which report an intracellular free Mg2+ concentration of 0.32 mM. The 31P chemical shifts of the free chelate and its metal complexes are far downfield from the typical phosphorus-containing metabolites observed in biological systems, thus making it possible to monitor intracellular cation concentrations and cell energetics simultaneously.     

Estimation of matrix pH in isolated heart mitochondria using a fluorescent probe

Isolated heart mitochondria hydrolyze the acetoxymethyl esters of the Ca2+-sensitive fluorescent probe fura-2 and the pH-sensitive 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). The resulting charged forms of the probes are retained in the mitochondrial matrix and appear well-suited for the estimation of pCa and pH in this compartment. The mitochondria esterase activity is stimulated by Ca2+, inhibited by butacaine and quinine, and shows an alkaline pH optimum. The esterase has a similar affinity for the two probes (Km about 1.5 microM) and a somewhat higher Vmax for BCECF. Intramitochondrial pH can be determined by recording the ratio of the fluorescence of matrix BCECF at its excitation maximum of 509 nm to that at 450 nm, an excitation wavelength that is unresponsive to pH. A calibration plot relating the fluorescence ratio to pH is constructed using detergent-lysed mitochondria and the excitation maximum of 500 nm for BCECF in aqueous solution. Estimates of matrix pH by BCECF fluorescence in its useful range (pH 6 to 8) agree well with values obtained using the distribution of 5,5-dimethyl-2,4-oxazolidenedione. In protocols in which the fluorescence with excitation at 450 nm does not vary, a direct recording of BCECF fluorescence with excitation at 509 nm can be used to follow the kinetics of matrix pH changes.     

Interaction of seminalplasmin with chlortetracycline, a fluorescent chelate probe of Ca2+

The interaction of seminalplasmin with chlortetracycline, a fluorescent chelate probe of Ca2+, was studied. The results indicate that seminalplasmin binds to chlortetracycline. The binding is not influenced by salt. Both Ca2+ and seminalplasmin probably bind to the same site on chlortetracycline. Seminalplasmin also reduced the Tb3+-associated fluorescence of bovine spermatozoal plasma membrane. These results are discussed in relation to the inhibitory effect of seminalplasmin on the uptake of Ca2+ in bovine spermatozoa.     

Proton transport is necessary for divalent metal cations release from deenergized mitochondria

The release of divalent cations (Ca2+ and Sr2+) from rat liver mitochondria after membrane depolarization with protonophore (carbonyl cyanide m-chlorophenyl hydrazone, CCCP), sodium azide and K(+)-ionophore (valinomycin) was studied. It is stated that membrane depolarization itself is not sufficient for cations release from mitochondrial matrix (provided that mitochondrial permeability transition pore is blocked by cyclosporin A). Complete delivering of divalent cations is observed only after protonophore (CCCP) addition to suspension of deenergized mitochondria. The data show that membrane permeabilisation to hydrogen ions (H+) is necessary for complete cation release from the mitochondrial matrix. The enhancement in K(+)-conductivity of mitochondrial membrane (by valinomycin), on the contrary, is not able to provide complete delivering of cations from mitochondria. It is shown that quantity of divalent metal cation released from mitochondria (depolarized and permeabilized for K+ as well) is proportional to the concentration of protonophore (but not K(+)-ionophore) introduced in the incubation medium. The data obtained lead to the conclusion that H(+)-permeabilization of the mitochondrial membrane is necessary for the complete release of Ca2+ and Sr2+ from mitochondria after membrane depolarization. The possible mechanism of divalent metal cations release from deenergized mitochondria is discussed.     

Regulation of reverse uniport activity in mitochondria by extramitochondrial divalent cations. Dependence on a soluble intermembrane space component.

We previously reported that uncoupling Ca2(+)-loaded mitochondria in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) produces a partial expression of the permeability transition. From this and related observations, it was proposed that the absence of external free Ca2+ is inhibitory to reverse activity of the Ca2+ uniporter (Igbavboa, U., and Pfeiffer, D.R. (1988) J. Biol. Chem. 263, 1405-1412). By using Sr2(+)-instead of Ca2(+)-loaded mitochondria, the transition is avoided upon treatment with EGTA plus uncoupler, and inhibition of reverse uniport activity can be observed directly. In the presence of physiological Mg2+ concentrations, reverse uniport of Sr2+ is eliminated by external EGTA following a brief period of rapid activity. It is proposed that binding of Mg2+ rather than Sr2+ (Ca2+) at an external site is responsible for the inhibition. Regulation at the external site is modified by the size of the Sr2+ load. EGTA, in the presence of Mg2+, does not inhibit the reverse uniport-dependent release of Sr2+ from mitoplasts. The inhibitory effect can be recovered by adding back the soluble components obtained as the intermembrane space fraction following removal of the outer membrane. The soluble factor could be a regulatory subunit which contains the external cation binding site. Adjustments to uniporter activity due to regulation by the binding site and/or the soluble factor may be slow and may be significant in determining how mitochondria respond to rapid Ca2+ transients in vivo.     

Aggregation of nucleosomes by divalent cations.

Conditions of precipitation of nucleosome core particles (NCP) by divalent cations (Ca(2+) and Mg(2+)) have been explored over a large range of nucleosome and cation concentrations. Precipitation of NCP occurs for a threshold of divalent cation concentration, and redissolution is observed for further addition of salt. The phase diagram looks similar to those obtained with DNA and synthetic polyelectrolytes in the presence of multivalent cations, which supports the idea that NCP/NCP interactions are driven by cation condensation. In the phase separation domain the effective charge of the aggregates was determined by measurements of their electrophoretic mobility. Aggregates formed in the presence of divalent cations (Mg(2+)) remain negatively charged over the whole concentration range. They turn positively charged when aggregation is induced by trivalent (spermidine) or tetravalent (spermine) cations. The higher the valency of the counterions, the more significant is the reversal of the effective charge of the aggregates. The sign of the effective charge has no influence on the aspect of the phase diagram. We discuss the possible reasons for this charge reversal in the light of actual theoretical approaches.     

Free energy of the accumulation of a fluorescent cation probe within lymphocyte mitochondria

Fluorescent probe-cation 4-(p-dimethylaminostyryl)-1-methylpyridinium (DSM) can be accumulated in mitochondria of rat thymus lymphocytes. A method is proposed for microfluorometric measuring of DSM concentrations ratio inside the thymocyte mitochondria and in the external medium. This method requires no knowledge about the cell and mitochondria volume, the number of accumulated probe and formation of ionic gradients in the cell by carriers. DSM concentration in energisized mitochondria of the thymocyte exceeds its concentration in the external medium by 10(4) times. This corresponds to the free energy accumulated by DSM equaling 5.2 +/- 0.2 kcal/mole. If such work on the probe accumulation is performed by the electrostatic fields on the plasma and mitochondrial cell membranes, the sum of the potentials of these two fields is 230 +/- 10 mV.     

Metabolism of calcium and effect of divalent cations on respiratory activity of yeast mitochondria

The adsorption of Ca²⁺ to the mitochondria ofSaccharomyces cerevisiae was investigated and it was found that, in contrast with animal mitochondria, Ca²⁺ is not accumulated through an energydependent process but is more probably adsorbed to mitochondrial membranes. The adsorption magnitude depends both on the amount of added calcium and on the ionic composition of the medium. It was found by study of the effect of divalent cations on the respiratory activity of yeast mitochondria that (a) Ca²⁺ and Mg²⁺ inhibit their oxidation competitively with succinate or citrate, the oxidation of NADH not being affected; (b) stimulation of oxidation of NADH and inhibition of oxidation of citrate and succinate may be observed with Ca²⁺ in the mitochondria ofTorulopsis utilis and with Co²⁺ in the mitochondria ofSaccharomyces cerevisiae; (c) Zn²⁺ inhibits the oxidation of NADH and of citrate; (d) the rate of oxidation of NADH in the presence of Cd²⁺ is several-fold greater than State 3 activity—on the other hand, oxidation of suceinate and citrate is inhibited by cadmium. In comparison with animal mitochondria, the fate of Ca²⁺ as well as the effects of other divalent cations on the respiratory activity of yeast mitochondria are different.     

Regulation of smooth muscle phosphatase-II by divalent cations

Summary Smooth Muscle Phosphatases II (SMP-I1) which has been purified from turkey gizzards and previously classified as protein phosphatase 2C, is inactive in the absence of divalent cations. Study of the activation of SMP-II by Mg²⁺ and Mn²⁺ revealed differences in the modes of activation by these cations. The maximal activation elicited by Mg²⁺ is 1.5–2.5-fold higher than the maximal Mn²⁺ activation. However, the latter is achieved at a lower concentration than the maximal Mg²⁺-activation. Furthermore, at low cation concentrations ( 2 mM), the Mn²⁺-activated activity is higher than the Mg²⁺-activated activity. In the presence of both cations, the effect of Mn²⁺ predominates suggesting that the affinity of the enzyme for Mn²⁺ is greater than for Mg²⁺. In contrast to Mg²⁺ and Mn²⁺, Ca²⁺ does not activate SMP-II but it was observed to antagonize the effects of Mg²⁺ and Mn²⁺. Ca²⁺ acts as a competitive inhibitor of Mg²⁺. However, the inhibitory effect at high Ca²⁺ concentrations is not completely reversed by increasing the Mg²⁺ concentration. Mn²⁺ activation is also inhibited by Ca²⁺ but to a lesser extent. Ca²⁺ cannot completely inhibit Mn²⁺-activation suggesting that SMP-I1 has greater affinity for Mn²⁺ than for Ca²⁺. The finding that Ca²⁺ inhibits the activation of SMP-II raises the possibility that Ca²⁺ may be a regulator of SMP-II in vivo.Abbreviations SMP-II Smooth Muscle Phosphatase-II - MOPS 3-[N-Morpholine]propane Sulfonic Acid - PLC Phosphorylated Myosin Light Chains