Calponin binds G-actin and F-actin with similar affinity

Calponins are actin-binding proteins that are implicated in the regulation of actomyosin. Calponin binds filamentous actin (F-actin) through two distinct sites ABS1 and ABS2, with an affinity in the low micromolar range. We report that smooth muscle calponin binds monomeric actin with a similar affinity (K(d) of 0.15 microM). We show that the arrangement of binding is similar to that of F-actin by a number of criteria, most notably that the distance between Cys273 on calponin and Cys374 of actin is 29A when measured by fluorescent resonance energy transfer, the same distance as previously reported for F-actin.     

Conformational changes induced by Mg2+ on actin monomers. An immunologic attempt to localize the affected region

The effect of Mg2+ ions on the conformation of G-actin and in particular on the accessibility of its antigenic regions has been tested. Experiments were performed with G-actin coupled to Sepharose 4B which was, therefore, maintained in the monomeric state. The results presented her show that the 2mM MgCl2-perturbed antigenic site is located in a central region of the actin sequence.     

Arsenic is decreased in target organs during viral infection in mice

Arsenic (As), a potentially toxic trace element, has been shown to influence viral replication and resistance to microbial infection. However, the impact of infection on the normal As status in target organs involved in the disease process has not been studied to date. In the present study, As was measured through inductively coupled plasma mass spectrometry in the plasma, liver, spleen, kidney, heart, pancreas and brain at days 1 and 3 of coxsackievirus B3 infection in female Balb/c mice. The severity of the infection was assessed from clinical signs of disease. The infection changed plasma As in a biphasic pattern with a small increase (n.s.) at day 1 that turned into a decreasing trend (13%, p<0.05) by day 3. In the liver, spleen, heart, pancreas and kidney As was unchanged at day 1 but, at day 3, it had decreased by 71% (p<0.01), 64% (p<0.01), 55% (p<0.01), 63% (p<0.01) and 73% (p<0.01), respectively. In the brain, As went unchanged. The pathophysiological interpretation of these findings requires further research.     

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.     

Structural variations in actins. A study of the immunological reactivity of the N-terminal region.

The antigenicity of the N-terminal region of skeletal-muscle actin was analysed. Two epitopes, corresponding to the 1-7 and 18-28 sequences, were determined. The antibodies specific for the first epitope discriminate skeletal-muscle actin from cardiac-muscle and smooth-muscle actins. The antibodies specific for the second epitope interact with all the actins tested, ranging from invertebrate to higher-vertebrate actins.     

Sarcomeric disorganization in post-mortem fish muscles.

1. The post-mortem evolution of protein pattern in fish striated muscle was followed by SDS-PAGE, after different conditions of storage time and temperature. 2. Sarcoplasmic and sarcomeric fractions were analyzed respectively by low and high ionic strength extractions of fish muscle samples. 3. No evident modification of electrophoretic patterns was observed during the pre-rigor mortis period. 4. The high mol. wt proteins titin and nebulin were highly sensitive to proteolysis during the rigor mortis period. 5. Myosin extraction was predominantly influenced by the storage temperature. The myosin content of the extracts decreased during the rigor mortis period at storage temperatures greater than 8 degrees C. 6. alpha-Actinin was very resistant to proteolysis, but could be released from Z-disc structure during post-mortem aging.     

An approach to the antigenic structure of arginine kinase and creatine kinase. Physical, chemical and immunological study of some modified derivatives.

The antigenic structure of arginine kinase and creatine kinase has been approached using chemical modifications and enzymatic cleavage. Mild performic oxidation that, with a restricted number of oxidized amino acid residues, results for both enzymes in a severe decrease of the helical structure and in a large increase of the protein-solvent interactions, affects differently their antigenic reactivity: when compared with antisera to the homologous native enzymes, arginine kinase and its oxidized derivative cross-react fully, while creatine kinase and its oxidized derivative cross-react only about 30%. The persistence of the antigenic reactivity of arginine kinase through drastic structural alterations is confirmed by the high inhibitory capacity (about 80%) of crude tryptic hydrolyzates towards the combination of argining kinase with its specific antibodies. Tryptic peptides of creatine kinase, obtained in the same conditions, inhibit weakly (about 12%) the homologous antigen-antibody interaction. The participation of the lysines in the antigenicity of arginine kinase and creatine kinase is suggested by the enhanced inhibitory capacity of the tryptic hydrolyzates when the cleavage is restricted to the arginyl peptide bounds, and was verified for arginine kinase through assays with lysine-modified derivatives.     

Co-operation of domain-binding and calcium-binding sites in the activation of gelsolin.

Gelsolin is an abundant calcium dependent actin filament severing and capping protein. In the absence of calcium the molecule is compact but in the presence of calcium, as its six similar domains alter their relative position, a generally more open configuration is adopted to reveal the three actin binding sites. It is generally held that a 'helical-latch' at the C-terminus of gelsolin's domain 6 (G6), binds domain 2 (G2) to keep gelsolin in the calcium-free compact state, and that the crutial calcium binding site(s) reside in the C-terminal half of gelsolin perhaps involving the C-terminal helix itself has to be bound to release this latch. Here we provide evidence for a calcium dependent conformational change within G2 (Kd = approximately 15 micro m). We also report a calcium dependent binding site for the C-terminus (G4-6) within G2 and delimit this further to a specific region formed by residues 203-225 and 159-193. It is known that the activation of gelsolin involves multiple calcium binding events (around 6) the first of which (in G6) may release the latch. We propose that the calcium-dependent conformational change in G2 may be a subsequent step that is necessary for the dissociation of G2 from G4-6, and that this movement occurs in sympathy with calcium induced conformational changes within G6 by the physical coupling of the two calcium binding sites within G2 and G6. Additional calcium binding in other domains then result in the complete opening and activation of the gelsolin molecule.