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Cell Motil Cytoskeleton. 1986;6(6):649-61.

Characterization of alpha-actinin from Acanthamoeba.

Abstract

Characterization of a protein from Acanthamoeba that was originally called gelation protein [T.D. Pollard, J. Biol. Chem. 256:7666-7670, 1981] has shown that it resembles the actin filament cross-linking protein, alpha-actinin, found in other cells. It comprises about 1.5% of the total amoeba protein and can be purified by chromatography with a yield of 13%. The native protein has a molecular weight of 180,000 and consists of two polypeptides of 90,000 Da. The Stokes' radius is 8.5 nm, the intrinsic viscosity is 0.35 dl/dm, and the extinction coefficient at 280 mm is 1.8 X 10(5)M-1 X cm-1. Electron micrographs of shadowed specimens show that the molecule is a rod 48 nm long and 7 nm wide with globular domains at both ends and in the middle of the shaft. On gel electrophoresis in sodium dodecylsulfate the pure protein can run as bands with apparent molecular weights of 60,000, 90,000, 95,000, or 134,000 depending on the method of sample preparation. Rabbit antibodies to electrophoretically purified Acanthamoeba alpha-actinin polypeptides react with all of these electrophoretic variants in samples of purified protein and cell extracts. By indirect fluorescent antibody staining of fixed amoebas, alpha-actinin is distributed throughout the cytoplasmic matrix and concentrated in the hyaline cytoplasm of the cortex. The protein cross-links actin filaments in the presence and absence of Ca++. It inhibits slightly the time course of the spontaneous polymerization of actin monomers but has no effect on the critical concentration for actin polymerization even though it increases the apparent rate of elongation to a small extent. Like some other cross-linking proteins, amoeba alpha-actinin inhibits the actin-activated ATPase of muscle myosin subfragment-1. Although Acanthamoeba alpha-actinin resembles the alpha-actinin from other cells in shape and ability to cross-link actin filaments, antibodies to amoeba and smooth muscle alpha-actinins do not cross react and there are substantial differences in the amino acid compositions and molecular dimensions.

PMID:
2948678
[PubMed - indexed for MEDLINE]
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