Isolation of brain alpha-actinin. Its characterization and a comparison of its properties with those of muscle alpha-actinins.
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)
D000185:Actinin / Q000302:isolation & purification*; D000199:Actins / Q000302:isolation & purification; D000818:Animals; D001923:Brain Chemistry; D002642:Chick Embryo; D002645:Chickens; D002942:Circular Dichroism; D009124:Muscle Proteins / Q000302:isolation & purification*; D009132:Muscles / Q000032:analysis*; D009928:Organ Specificity; D011487:Protein Conformation; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D014783:Viscosity
D000199:Actins; D009124:Muscle Proteins; D000185:Actinin