Isolation of brain .alpha.-actinin. Its characterization and a comparison of its properties with those of muscle .alpha.-actinins

Abstract

A rapid purification procedure has been developed for the isolation of .alpha.-actinin from chicken brain. Brains were homogenized in cold water containing 0.5 mM phenylmethanesulfonyl fluoride (PMSF), the homogenate was centrifuged, and the .alpha.-actinin was extracted from the pelleted material in a low ionic strength buffer for 30 min at 22.degree. C. Purification of the protein to homogeneity on sodium dodecyl sulfate containing polyacrylamide gels required an ammonium sulfate precipitation step followed by chromatography on columns of DEAE-cellulose, hydroxylapatite and Sepharose CL-6B. The .alpha.-actinins from chicken pectoral muscle (skeletal) and gizzard (smooth muscle) were purified in a similar fashion but without the DEAE-cellulose chromatography step. All 3 .alpha.-actinins have an identical Stroke radius of 7.1 nm determined by gel filtration chromatography. The individual proteins are homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but do not comigrate; however, all 3 .alpha.-actinins have identical retardation coefficients, obtained from electrophoretic mobilities at different acrylamide concentrations; this indicates that they all have similar subunit MW (.apprx. 105,000). All 3 proteins behave similary on isoelectric focusing gels (pI [isoelectric point] of native proteins .simeq. 4.7-4.9) and have similar UV and CD [circular dichromism] spectroscopic properties. Significant differences exist both in their amino acid composition and in their peptide maps, obtained from limited proteolysis, which indicates that the proteins are all unique gene products. In the absence of Ca2+, all 3 .alpha.-actinins increase the viscosity of an F-actin solution; however, Ca2+ in micromolar concentration inhibits the increased viscosity of F-actin solutions induced by .alpha.-actinin from brain but not from muscle. Purified .alpha.-actinins were labeled by reductive methylation to specific activities of > 105 dpm/.mu.g. .alpha.-Actinins so modified bind to F-actin at 4.degree. C in a highly cooperative fashion in the absence of Ca2+ and saturate microfilaments at a molar ratio of 1 .alpha.-actinin to 9-11 actin monomers. Binding of the muscle .alpha.-actinins to F-actin is not affected by Ca2+ concentrations up to 2 mM. The cooperative nature of the binding of brain .alpha.-actinin to F-actin is diminished by 2 mM Ca2+, although binding still occurs.