Purification and further characterization of macrophage 70-kDa protein, a calcium-regulated, actin-binding protein identical to L-plastin

Abstract

We have previously identified a macrophage 70-kDa, actin-bundling protein as a constituent of actin-based cytoplasmic gel and showed that its association with or dissociation from cytoplasmic gels was remarkably affected by submicromolar calcium. In this study, we purified the 70-kDa protein from soluble cytosolic extracts and carried out a more detailed characterization. The amino acid sequences of four peptidic fragments, obtained from the purified protein by enzymatic or chemical cleavage, were completely or nearly identical to those of L-plastin, a protein initially identified in transformed cells from solid tumors (Goldstein & Leavitt, 1985). By Western blot analysis of normal cells and tissues using specific anti-70-kDa protein antibodies, the 70-kDa molecule was detected only in hematopoietic cells. The 70-kDa protein bound to actin with apparent Kd values of 1.8 and 5.5 microM in the absence and presence of 20 microM free calcium, respectively. Cross-linking activity measured by falling-ball viscosimetry was optimal at free calcium lower than 0.15 microM but was progressively inhibited at higher calcium concentrations, within the physiological range. Half-maximal inhibition occurred at 1.6 microM free calcium. No severing of actin filaments by the 70-kDa protein was observed in any of these assays or previously (Pacaud & Harricane, 1987). Major conformational changes of the protein, as measured by the fluorescence emission intensity of tyrosine residues, occurred at free calcium concentration ranging between 0.15 and 1.5 microM. Magnesium did not mimic the calcium effect. The results suggest that the 70-kDa protein possesses both high-affinity sites and selectivity for calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin-filament cross-linking protein T-plastin increases Arp2/3-mediated actin-based movement

“…5B). Conversely, F-actin binding of the closely related L-plastin isoform was Ca 2+ sensitive when analysed under identical experimental conditions (data not shown), as observed previously (Namba et al, 1992;Pacaud and Derancourt, 1993). This result confirmed the observation that plastins differ in their Ca 2+ regulation, as previously proposed (de Arruda et al, 1990;Lin et al, 1994).…”

“…These results confirmed the F-actinstabilizing capacity of T-plastin and ABD1. It is documented that physiological Ca 2+ concentrations (1-2 µM) inhibit the F-actin binding of L-plastin (Namba et al, 1992;Pacaud and Derancourt, 1993), raising the possibility that the stabilizing effects we observed with T-plastin were also Ca 2+ -dependent. Because cell extracts contain other Ca 2+ -regulated proteins like gelsolin that might also contribute to actin turnover in comets (Samarin et al, 2003), we decided to examine the effect of Ca 2+ in the F-actin depolymerization assay (Fig.…”

Enoxacin Directly Inhibits Osteoclastogenesis without Inducing Apoptosis

“…Our data suggest that L-plastin is likely cleaved proteolytically so that the N-terminal EF-hand domain is removed in response to enoxacin. The EF-hand binds calcium and mediates inhibition of binding of L-plastin to microfilaments in the presence of calcium (51)(52)(53).…”

Identification of I-plastin, a human fimbrin isoform expressed in intestine and kidney.

“…Human tissues were (lanes) colon adenocarcinoma cell line Caco-2 (1), colonic tumor (2) and normal colon (3) from one patient, and small intestine (4) and colon (5) from another patient. Rat tissues were (lanes) jejunal epithelium (6), colon (7), ileum (8), jejunum (9), duodenum (10), uterus (11), kidney (12), spleen (13), leg muscle (14), liver (15), stomach (16), lung (17), heart (18), esophagus (19), and brain (20 demonstrated that calcium inhibits actin bundling (23)(24)(25). In the homologous region of yeast Sac6p, critical positions are occupied by residues that would prevent calcium binding, suggesting that this protein is not calcium regulated in Saccharomyces cerevisiae (1).…”