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Flexibility and Fine Structure of Smooth‐Muscle α‐Actinin
Author(s) -
Winkler Jörg,
Lünsdorf Heinrich,
Jockusch Brigitte M.
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.00193.x
Subject(s) - spectrin , antiparallel (mathematics) , crystallography , thermolysin , dimer , molecule , actin , microfilament , negative stain , biophysics , chemistry , ionic strength , electron microscope , cytoskeleton , biology , biochemistry , physics , trypsin , organic chemistry , quantum mechanics , magnetic field , optics , cell , enzyme , aqueous solution
The microfilament protein α‐actinin exists as a dimer. The N‐terminal regions of both polypeptides, arranged in antiparallel orientation, comprise the actin‐binding regions, while the C‐terminal, larger parts consist of four spectrin‐like repeats that interact to form a rod‐like structure. To elucidate the fine structure of smooth‐muscle α‐actinin, we used energy‐filtered transmission electron microscopy in conjunction with negative staining. Survey pictures of the protein purified from chicken gizzard revealed discrete, elongated particles whose length and width varied with the ionic strength of the buffer. It was determined to to 29.3 nm × 4.8 nm in 0.05 M KC1 and 32.6 nm × 4.4 nm in 0.15 M KC1. Both ends of the molecule displayed hook‐like structures consisting of globular domains, which were highly variable in their orientation with respect to the long axis of the molecule. Their location at the ends of the molecule, and the finding that these hooks were missing from particles obtained by thermolysin digestion indicated that they probably correspond to the N‐terminal actin‐binding regions. The rod‐like center of the molecule revealed discrete globular masses which probably comprise the spectrin‐like repeats. Their arrangement was compatible with the interpretation that three spectrin repeats of each polypeptide chain can form pairs with the respective sequences of the other chain. The rod‐like 53‐kDa fragment obtained after thermolysin digestion largely retained this structural organization but appeared wider (22.5 nm × 5.9 nm). Our results help to clarify previous discrepancies on the quatenary organization of α‐actinin and suggest that effective actin‐binding and cross‐linking of α‐actinin is based on the high flexibility of the terminal hooks.