Macrophage alpha-actinin is not a calcium-modulated actin-binding protein.
alpha-Actinin was purified from rabbit macrophages to apparent homogeneity by a procedure designed to remove other actin-binding proteins. Large bundles of filaments were formed when 1 molecule of alpha-actinin interacted with 10-12 actin monomers. This process involved the successive occupancy of two classes of actin-binding sites with different affinities. The apparent Kd of alpha-actinin for F-actin was unaffected by the addition of 25 microM free Ca2+. Analysis of the influence of increasing Ca2+ concentrations on alpha-actinin-F-actin interactions by low-speed sedimentation assays, low-shear viscosity, and electron microscopy indicated that Ca2+ had a small inhibitory effect in the approximate range of 50-1000 microM. Furthermore, the ability of alpha-actinin to assemble actin filaments into bundles was apparently inhibited only at Ca2+ concentrations which also affected the physical properties of F-actin alone. alpha-Actinin immobilized on a nitrocellulose membrane did not bind detectable amounts of Ca2+. Nevertheless, Ca2+ or Mg2+ binding to alpha-actinin induced small decreases in the fluorescence emission intensity of tryptophan and tyrosine residues. The maximal change induced by Mg2+ was smaller than that observed with Ca2+, but Ca2+ and Mg2+ effects were abolished by the addition of 140 mM KCl. Under near-physicological ionic conditions, Ca(2+)-binding sites with an apparent Kd higher than 80-100 microM could not be detected. The results on the functional and physical properties of alpha-actinin are consistent with the hypothesis that Ca2+ decreases alpha-actinin--F-actin interactions by acting both on actin filaments and on cross-linking molecules. Although this conclusion is in contradiction with the generally accepted idea that alpha A is a Ca(2+)-regulated actin-binding protein, it could be predicted from the primary sequence of the two EF-hand-like motifs in the alpha-actinin monomer [Arimura et al. (1988) Eur. J. Biochem. 177, 649-655] based on the crucial role of some Ca(2+)-binding residues as recently demonstrated by point mutations in Ca(2+)-binding sites of calmodulin [Haiech et al. (1991) J. Biol. Chem. 266, 3427-3431]. It is also in agreement with our previous finding that Ca2+ does not affect the behavior of alpha-actinin in actin gel networks from macrophage cytosolic extracts [Pacaud & Harricane (1987) J. Cell Sci. 88, 81-94].
Pacaud
M
M
Centre de Recherches de Biochimie Macromoléculaire, CNRS UPR 9008, INSERM U 249, Université de Montpellier I, France.
Harricane
M C
MC
D000185:Actinin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006863:Hydrogen-Ion Concentration; D016676:Macrophages, Alveolar / Q000737:chemistry*; D008274:Magnesium / Q000378:metabolism; D008854:Microscopy, Electron; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014783:Viscosity
D000199:Actins; D000185:Actinin; D008274:Magnesium; D002118:Calcium