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 degrees 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 three alpha-actinins have an identical Stokes 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 three alpha-actinins have identical retardation coefficients, obtained from electrophoretic mobilities at different acrylamide concentrations, which indicates that they all have similar subunit molecular weights (about 105 000). All three proteins behave similarly on isoelectric focusing gels (pI of native proteins congruent to 4.7-4.9) and have similar UV and CD 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of two isoforms of human blood platelet α‐actinin

“…The binding ratio is approximately 1 molecule of x-actinin (aa, bb or cc) per 9 -11 actin units in the actin filament. This binding ratio seems to be quite general since several authors reported similar ratios despite the Pact that they used different methods, different temperatures and they studied the interaction between muscle actin and various x-actinins from skeletal muscle [3, 281, Ehrlich tumor cells [29], brain [30] or macrophages [31].…”

A chick skeletal‐muscle α‐actinin gene gives rise to two alternatively spliced isoforms which differ in the EF‐hand Ca2+‐binding domain

“…However, the significance of this spacer region is unclear, and it is notably absent from D. discoideum a-actinin which is considered to be the archetypal non-muscle isoform (Noegel et al, 1987). Finally, non-muscle a-actinins are calcium sensitive with respect to binding F-actin, whereas muscle isoforms are calcium insensitive (Burrdige and Feramisco, 1981;Bennett et al, 1984;Duhaiman and Bamburg, 1984;Landon et al, 1985). EF-hand calcium-binding domains contain a helix-loop-helix motif, where coordination of the calcium ion is dependent on the residues at the X, Y, Z, -X, -Y and -Z vertices in the loop.…”

“…in the presence of Ca2+, whereas the smooth-muscle and skeletal-muscle isoforms are CaZ + insensitive (Burrdige and Feramisco, 1981;Bennett et al, 1984;Duhaiman and Bamburg, 1984;Landon et al, 1985). The smooth-muscle and non-muscle isoforms of a-actinin arise by alternative splicing of the transcript from a single gene (Arimura et al, 1988;Waites et al, 1992).…”

Unanticipated temporal and spatial effects of sarcomeric α-actinin peptides expressed in PtK2 cells

“…The only functional difference detected thus far between s-␣-actinin and non-s-␣-actinin is that binding of the former to actin is Ca 2ϩ insensitive, whereas binding of the latter is Ca 2ϩ sensitive [Burridge and Feramisco, 1980;Duhaiman and Bamburg, 1984;Bennett et al, 1984;Landon et al, 1985]. Thus, fluorescently labeled non-s-␣-actinin injected into fibroblasts is selectively and rapidly incorporated into preexisting dense bodies along stress fibers and into focal adhesion plaques [Feramisco, 1979;McKenna et al, 1985;Pavalko and Burridge, 1991].…”