Substructure and higher structure of chicken smooth muscle alpha-actinin molecule.
Substructure of chicken gizzard smooth muscle alpha-actinin molecule was deduced by domainal mapping of the proteolytic fragments with alpha-chymotrypsin. There were three chymotryptic cleavage sites (Sites I, II, and III, from the amino terminus). Cleavage at Site I generated two fragments, i.e. an NH2-terminal 36-kDa fragment and a COOH-terminal 70-kDa fragment. The 70-kDa fragment generated either a 55-kDa fragment by cleavage at Site II or a 65-kDa fragment by cleavage at Site III. Purified NH2-terminal 36-kDa fragment bound to F-actin, whereas the 55-kDa fragment formed a dimeric molecule. Circular dichroism and electron microscopic experiments demonstrated that the alpha-helical content of the 55-kDa fragment was 14% higher than that of native gizzard alpha-actinin, coinciding with the apparently rod-shaped configuration of this fragment. A 110-kDa product was generated from two 55-kDa fragments in a cross-linking study with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Two cross-linkable sites in the 55-kDa, A- and B-site, were shown to be involved in this reaction. Further, it was demonstrated by using N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide labeling and immunoblotting analyses that the A-site on one 55-kDa fragment was cross-linked to the B-site on the other. These results suggest that smooth muscle alpha-actinin formed an antiparallel dimeric molecule in which the 55-kDa fragments connected the two actin-binding domains composed of the 36-kDa fragments.
Imamura
M
M
Institute of Basic Medical Sciences, University of Tsukuba, Japan.
Endo
T
T
Kuroda
M
M
Tanaka
T
T
Masaki
T
T
D000185:Actinin / Q000032:analysis*; D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D005450:Fluorescamine / Q000378:metabolism; D007163:Immunosorbent Techniques; D008854:Microscopy, Electron; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000032:analysis*; D010449:Peptide Mapping
D000185:Actinin; D005450:Fluorescamine