Interaction of plasma gelsolin with G-actin and F-actin in the presence and absence of calcium ions

Plasma gelsolin formed a very tight 1:2 complex with G-actin in the presence of Ca2+, but no interaction between gelsolin and G-actin was detected in the presence of excess EGTA. However, the 1:2 complex dissociated into a 1:1 gelsolin:actin complex and monomeric actin when excess EGTA was added. Plasma gelsolin bound tightly to the barbed ends of actin filaments and also severed filaments in the presence of Ca2+ and bound weakly to the filament barbed end in the presence of EGTA. The 1:2 gelsolin-actin complex bound to the barbed ends of filaments but did not sever them. By blocking the barbed end of filaments with plasma gelsolin, we determined the critical concentration at the pointed end in 1 mM MgCl2 and 0.2 mM ATP to be 4 microM. The dissociation rate constant for ADP-G-actin from the pointed end was estimated to be about 0.4 s-1 and the association rate constant to be about 5 X 10(4) M-1 s-1. Finally, we obtained evidence that plasma gelsolin accelerates but does not bypass the nucleation step and, therefore, that the concentration of gelsolin does not directly determine the concentration of filaments polymerized in its presence. Thus, gelsolin-capped filaments may not provide an absolutely reliable method for determining the rate constant for the association of ATP-G-actin at the pointed ends of filaments, but a reasonable estimate would be 1 X 10(5) M-1 s-1 in 1 mM MgCl2 and 0.2 mM ATP.     

Muscle is the major source of plasma gelsolin

Gelsolin, a Ca2+- and polyphosphoinositide-regulated actin-binding protein, is unique among vertebrate proteins in being both cytoplasmic and secreted. Plasma gelsolin, present at greater than 200 micrograms/ml in human plasma, may have a protective function by promoting the clearance of actin filaments released during tissue injury. Although there is evidence that smooth muscle tissues and HepG2 cells synthesize plasma gelsolin, the predominant secretory source is hitherto unknown. We report here that skeletal, cardiac, and smooth muscles have large amounts of plasma gelsolin mRNA and devote 0.5-3% of their biosynthetic activity to plasma gelsolin, whereas liver makes relatively little. Since skeletal muscle accounts for a large fraction of body mass and total protein synthesis, it is the major source of plasma gelsolin.     

Interaction of plasma gelsolin with ADP-actin

In the presence of Ca2+, gelsolin forms a very tight, stoichiometric complex with 2 molecules of ADP-G-actin. Removal of free Ca2+ causes the 1:2 complex to dissociate to a 1:1 complex. Gelsolin accelerates the very slow polymerization of ADP-actin, apparently by accelerating the rate of nucleation, but the number concentration of filaments formed is probably less than the gelsolin concentration, indicating that the GA2 complex is not a true nucleus. These results are similar to those obtained for the interaction of gelsolin with ATP-G-actin. Both kinetic and equilibrium measurements demonstrate that the critical concentration of gelsolin-capped ADP-actin filaments (8 microM in 1 mM MgCl2 and 0.2 mM ADP) is the same as for the uncapped filaments, proving that the critical concentration is the same at both ends of the equilibrium polymer in ADP as predicted by theory. The association and dissociation rate constants for the addition of ADP-G-actin at the pointed end of an ADP-F-actin filament are estimated to be 4.6 X 10(4) M-1 s-1 and 0.4 s-1, respectively, about 15-fold lower than the rate constants at the barbed end.     

Irreversible effects of calcium ions on the plasma membrane calcium pump

The calcium pump of human red cells can be irreversibly activated by preincubation of the membranes in the presence of calcium ions, with a pattern reminiscent of that produced by controlled trypsin attack. With 1 mm Ca²⁺, the activity of the basal enzyme increases three to fourfold over 30 to 60 min, to levels about half those obtained in the presence of calmodulin. On the whole, the effect occurs slowly, with a very low Ca²⁺ affinity at 37°C and is unaffected by serine-protease inhibitors. The activation caused by 1 mm Ca²⁺ is little affected by leupeptin (a thiol-protease inhibitor) and that obtained at 10 m Ca²⁺ is not inhibited. Preincubations at 0°C also lead to activation, to a level up to half that seen at 37°C, and the effect is not affected by leupeptin or antipain. No activation is observed by preincubating soluble purified Ca,Mg-ATPase in Ca²⁺-containing solutions at 37°C. Instead, calcium ions protect the detergent-solubilized enzyme from thermal inactivation, the effect being half-maximal between 10 and 20 m Ca²⁺. We conclude that the activation of the membrane-bound Ca,Mg-ATPase by Ca²⁺ should result from an irreversible conformational change in the enzyme and not from attack by a membrane-bound protease, and that this change presumably arises from the release of inhibitory particles existing in the original membrane preparations.We thank The Wellcome Trust for a research grant, the Medical Research Council for an equipment grant and the Regional Transfusion Service (Sheffield) for bank blood supplies.     

Interaction of plasma gelsolin with tropomyosin.

Horse plasma gelsolin labelled with benzophenone-4-isothiocyanate can be photochemically cross-linked to rabbit cardiac tropomyosin. The cross-linking proceeds with greater efficiency in calcium-containing buffers. Further evidence for interaction between these proteins is provided by retention of fluorescently labelled gelsolin on tropomyosin-agarose affinity columns and by the ability of tropomyosin to cause an increase in the fluorescence intensity of gelsolin labelled with fluorescein-5-isothiocyanate. Both of these effects require the presence of calcium ions.     

Global structure changes associated with Ca2+ activation of full-length human plasma gelsolin

Gelsolin regulates the dynamic assembly and disassembly of the actin-based cytoskeleton in non-muscle cells and clears the circulation of filaments released following cell death. Gelsolin is a six-domain (G1-G6) protein activated by calcium via a multi-step process that involves unfolding from a compact form to a more open form in which the three actin-binding sites (on the G1, G2, and G4 subdomains) become exposed. To follow the global structural changes that accompany calcium activation of gelsolin, small-angle x-ray scattering (SAXS) data were collected for full-length human plasma gelsolin at nanomolar to millimolar concentrations of free Ca2+. Analysis of these data showed that, upon increasing free Ca2+ levels, the radius of gyration (Rg) increased nearly 12 A, from 31.1+/-0.3 to 43+/-2 A, and the maximum linear dimension (Dmax) of the gelsolin molecule increased 55 A, from 100 to 155A. Structural reconstruction of gelsolin from these data provided a striking visual tracking of the gradual Ca2+-induced opening of the gelsolin molecule and highlighted the critical role played by the flexible linkers between homologous domains. The tightly packed architecture of calcium-free gelsolin, seen from both SAXS and x-ray crystallographic models, is already partially opened up in as low as 0.5 nM Ca2+. Our data confirm that, although the molecule springs open from 0 to 1 microM free Ca2+, even higher calcium concentrations help to stabilize a more open structure, with increases in Rg and Dmax of approximately 2 and approximately 15 A, respectively. At these higher calcium levels, the SAXS-based models provide a molecular shape that is compatible with that of the crystal structures solved for Ca2+/gelsolin C-terminal and N-terminal halves+/-monomeric G-actin. Placement of these crystal structures within the boundaries of the SAXS-based model suggests a movement of the G1/G2 subunits that would be required upon binding to actin.     

Glu 69 is essential for the high sensitivity of muscle fructose-1,6-bisphosphatase inhibition by calcium ions

Muscle fructose-1,6-bisphosphatase (FBPase) is highly sensitive toward inhibition by AMP and calcium ions. In allosteric inhibition by AMP, a loop 52-72 plays a decisive role. This loop is a highly conservative region in muscle and liver FBPases. It is feasible that the same region is involved in the inhibition by calcium ions. To test this hypothesis, chemical modification, limited proteolysis and site directed mutagenesis Glu(69)/Gln were employed. The chemical modification of Lys(71-72) and the proteolytic cleavage of the loop resulted in the significant decrease of the muscle FBPase sensitivity toward inhibition by calcium ions. The mutation of Glu(69)-->Gln resulted in a 500-fold increase of muscle isozyme I(0.5) vs. calcium ions. These results demonstrate the key role that the 52-72 amino acid loop plays in determining the sensitivity of FBPase to inhibition by AMP and calcium ions.     

Effect of calcium ions on plasma membrane structure

The structure of thymocyte's plasma membranes was studied as affected by calcium ions (0-1.126 mM). The fluorescence intensity of 1-anilinonaphthalene-8-sulphonate, the epimerization degree of pyrene and fluorescence anisotropy of membrane proteins were studied. The change of electrochemical properties of membranes, conformation of membrane proteins and lipid fluidity has been shown.     

Amplified label-free electrocatalytic detection of DNA in the presence of calcium ions

A new label-free electrocatalytic method for the detection of DNA, is presented, in which DNA is the catalyst. The method takes advantage of the catalytic properties of the electrooxidised adenines within DNA toward the oxidation of NADH. This catalytic event results in an enhancement in the oxidation current of the electrooxidised adenines within DNA. Further improvement in this analytical signal is achieved in the presence of Ca(2+) ions. Parameters affecting the electrocatalytic current, such as pH or concentration of Ca(2+) ions have been investigated and optimised. Finally, the analytical features of the developed method are obtained. This method constitutes a more sensitive and reproducible alternative to other methods that use the oxidation current of the electrooxidised adenines without coupling to any catalytic event. A limit of detection of 33 fmol deoxyadenylic acid icosanucleotide (dA)(20), is obtained without labels.     

Disulfide-bonded polymerization of plasma fibronectin in the presence of metal ions

Incubation of human plasma fibronectin in the presence of low concentrations of FeCl3 or CuSO4 led to the formation of disulfide-bonded multimers as revealed by analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing or reducing conditions. The polymers induced by FeCl3 did not enter the spacer gel, and those induced by CuSO4 migrated to the top of the running gel, indicating that the former polymers were larger than the latter, which in gel filtration experiments appeared to be larger than Mr 670,000. The polymerization occurred between pH 7 and 9 and more rapidly at 22 or 37 degrees C than at 4 degrees C and was inhibited by metal-chelating reagents. NaCl, heparin, spermine, urea, or guanidine hydrochloride did not appreciably affect the reaction, whereas dithioerythritol enhanced the CuSO4-induced polymerization of fibronectin. When incubated in the presence of FeCl3, the Mr 30,000 NH2-terminal, Mr 40,000 gelatin-binding, and the Mr 120,000-140,000 COOH-terminal fragments of fibronectin formed disulfide-bonded polymers, whereas only the Mr 140,000 fragment was polymerized in the presence of CuSO4. Disulfide-bonded polymers were also formed in the presence of FeCl3 but not CuSO4 when the free sulfhydryl groups of fibronectin were blocked by N-ethylmaleimide. The results suggest that in the presence of CuSO4, disulfide-bonded polymerization of fibronectin may involve predominantly the free sulfhydryl groups, whereas in the presence of FeCl3, also the intramolecular disulfides may exchange to form disulfides between separate fibronectin molecules. Thus, under different conditions, different parts of fibronectin may be susceptible to disulfide-bonded polymerization.     

The activation of gelsolin by low pH: the calcium latch is sensitive to calcium but not pH.

Gelsolin is a multidomain and multifunction protein that nucleates the assembly of filaments and severs them. The activation of gelsolin by calcium is a multistep process involving many calcium binding sites that act to unfold the molecule from a tight structure to a more loose form in which three actin-binding sites become exposed. Low pH is also known to activate gelsolin, in the absence of calcium and this too results in an unfolding of the molecule. Less is known how pH-activation occurs but we show that there are significant differences in the mechanisms that lead to activation. Crucially, while it is known that the bonds between G2 and G6 are broken by co-operative occupancy of calcium binding sites in both domains [Lagarrique, E., Maciver, S. K., Fattoum, A., Benyamin, Y. & Roustan, C. (2003) Eur. J. Biochem. 270, 2236-2243.], pH values that activate gelsolin do not result in a weakening of the G2-G6 bonds. We report the existence of pH-dependent conformational changes within G2 and in G4-6 that differ from those induced by calcium, and that low pH overrides the requirement for calcium for actin-binding within G4-6 to a modest extent so that a Kd of 1 micro m is measured, compared to 30-40 nm in the presence of calcium. Whereas the pH-dependent conformational change in G2 is possibly different from the change induced by calcium, the changes measured in G4-6 appear to be similar in both calcium and low pH.     

The role of calcium ions during mitosis

Calcium-containing solutions were microinjected into dividing PtK1 cells to assess the effect of calcium ion concentration on the morphology and physiology of the mitotic spindle. Solutions containing 50 microM or more CaCl2 are immediately and irreversibly toxic to PtK1 cells. Those containing 5-10 microM CaCl2 cause reversible reduction in spindle birefringence followed by normal anaphase and cytokinesis. Microinjection of 5 microM or less CaCl2 into anaphase PtK1 cells has no detectable effect on the rate or extent of chromosome movement. Metaphase cells tend to enter anaphase 4-5 min after injection with 1-10 microM CaCl2, compared with an average of 16 min after injection with calcium-free buffer. Reducing the intracellular calcium concentration by injection of EGTA-CaCl2 buffers increases the lag between injection and anaphase to 20 min or more. Microinjection of calcium solutions does not promote precocious chromatid separation in nocodazole-arrested metaphase cells, indicating that the increase in calcium concentration does not induce centromere separation directly. An increase in the concentration of free calcium ions during metaphase appears to stimulate the onset of anaphase. Such an increase, regulated by the cell itself, may contribute to the initiation of chromosome separation in mammalian cells.     

High lactation index is associated with insulin sensitivity

The aim of the study was to evaluate the contribution of lactation to insulin sensitivity in women 12 to 18 month postpartum using an oral glucose tolerance test (OGTT). Mean lactation index (LI), a scoring system that considers the establishment and maintenance of the lactation was used. Lactation index was calculated according to the number of months of breast-feeding per child with a maximum of 72 points. The mean LI was calculated by dividing the total number of points by the number of children. A cutoff point of 72 was considered for the LI. We investigated the inverse of the homeostasis model assessment (HOMA_Sens) and the Cederholm index. Healthy women went through standardized interview and anthropometry. After a 10- to 12-h overnight fast, a 2-h OGTT was performed. Multiple regression analysis was performed with HOMA_Sens and Cederholm index, which were adjusted for parity, percentage body fat, LI and presence/absence of breast-feeding. Both HOMA_Sens and Cederholm index were negatively associated with percentage body fat (P<.01), and Cederholm index was positively associated with LI (P=.01). Mean 120-min insulin levels were significantly lower in women with LI=72 when compared with LI<72 women. Insulin sensitivity measured by the Cederholm index is positively associated with prolonged and sustained lactation, while percentage body fat presented a negative association. In this way, sustained lactation-associated metabolic changes are considered protective to women's health.     

Homo- and heterodimer formation with prothrombin and prothrombin fragment 1 in the presence of calcium ions

The purpose of the current study is to present further evidence for prothrombin self-association as assessed by chemical crosslinking. When the self-association (evaluated by covalent crosslinking with dithiobis(succinimidylpropionate) of prothrombin or fragment 1 was evaluated at the same molar concentration of protein, similar rates of dimer formation were observed for either protein. When prothrombin and fragment 1 were incubated together with the crosslinking reagent and calcium ions, a heterodimer consisting of prothrombin and fragment 1 was observed in addition to prothrombin dimer and fragment 1 dimer. Similar experiments with prethrombin 1 showed neither significant self-association nor effect on prothrombin self-association. Comparison of the formation of prothrombin fragment 1 heterodimer formation with the effect of fragment 1 on prothrombin activation by factor Xa suggests that the anticoagulant activity of fragment 1 is not solely a result of the formation of a heterodimer between prothrombin and fragment 1.     

Decreased levels of the gelsolin plasma isoform in patients with rheumatoid arthritis

Introduction  

Gelsolin is an intracellular actin-binding protein involved in cell shape changes, cell motility, and apoptosis. An extracellular gelsolin isoform, plasma gelsolin circulates in the blood of healthy individuals at a concentration of 200 ± 50 mg/L and has been suggested to be a key component of an extracellular actin-scavenging system during tissue damage. Levels of plasma gelsolin decrease during acute injury and inflammation, and administration of recombinant plasma gelsolin to animals improves outcomes following sepsis or burn injuries. In the present study, we investigated plasma gelsolin in patients with rheumatoid arthritis.