The with Presence of Two Skeletal Muscle a-Actinins Correlates Troponin-Tropomyosin Expression and Z-line Width

Two species of ~-actinin from rabbit fast skeletal muscles were identified with a monospecific antisera. Designated a-actininlf and c~-actinin~f, their distribution in muscles does not correlate with histochemically defined fast fiber type. Rather, the presence of each correlates with Z-line width and with the expression of different thin filament Ca2+-regulatory complexes, a-Actininlf is expressed with troponin Tlf-a/~ tropomyosin, and a-actinin2f with troponin T2f-a2 tropomyosin. CNBr peptide maps show that the fast a-actinin species differ in primary structure. In contrast, the slow c~-actinin is indistinguishable from a-actininlf. Further evidence for the similarity of ~-actininlf and slow a-actinin comes from electron microscopic studies which show that fibers that express these species exhibit thick Z-lines. So, unlike other contractile proteins, the multiple forms of c~-actinin do not reflect the distinction between fastand slow-twitch muscles. Multiple forms of most of the major skeletal muscle contractile proteins have been identified in mammalian muscles. In the case of myosin (l l), troponin T (7), a-tropomyosin subunits (4), and C-protein (6), these homologous species are thought to be part of the molecular basis responsible for the differences between fastand slow-twitch muscle. Similarly, Suzuki et al. (33, 34) described two porcine skeletal muscle forms of a-actinin: one found in fast muscle and the other in slow muscle. In the studies described here, two forms of aactinin in fast skeletal muscles of rabbit are characterized. Identified with a monospecific antisera on immunoblots, they differ extensively in primary structure and have been designated a-actininlf and a-actinin2f. a-Actinin is a major component of the Z-line in skeletal muscle (5). Because differences in Z-line structure have been associated with the two histologically and ultrastructurally defined types of fast fibers (9, 10, 12), the relationship between Z-line width, histochemical fiber type, and the expression of these two fast a-actinin species was investigated. No simple correlation between histochemically defined fast fiber type and the expression of either fast a-actinin species was found. Instead, Z-line width appears to be at least partly determined by the a-actinin species expressed. In addition to this morphological correlate, comparison of the pattern of fast aactinin expression with that of the two major forms of the fast skeletal thin filament protein troponin T (described by Briggs et al. [3]) shows that the presence of each a-actinin is THE JOURNAL OF CELL BIOLOGY . VOLUME 101 SEPTEMBER 1985 1001-1008 © The Rockefeller University Press • 0021-9525185/09/1001/08 $1.00 linked to the presence of a different fast troponin T. An investigation of the relationship between the two forms of fast a-actinin and slow a-actinin by CNBr peptide mapping reveals no differences in the CNBr fragments of a-actininlf and a-actinins. Because there is commonly more homology among fast myofibrillar proteins than between fast and slow homologs, the differences between the peptide maps of the two fast a-actinin species, coupled with the similarity of one of the fast a-actinin species and slow a-actinin, was unexpected. It suggests that the selective pressures that resulted in the multiple forms of a-actinin differed from those that led to the multiplicity of other myofibrillar proteins. MATERIALS AND METHODS Reagents: The monospecific antisera to a-actinin was a gift from Dr. Keith Burridge and was characterized as previously described (18). Second antibodies for immunoblot reactions were obtained from either Tago Inc. (Burlingame, CA) or Vector Laboratories, Inc. (Burlingame, CA). Trasylol was purchased from Mobay Chemical Corp. (Pittsburg, PA) and Tris-HCl and Tris base were from Sigma Chemical Company (St. Louis, MO). Pepstatin A, leupeptin, antipain, and chymostatin were purchased from the Peptide Institute (Osaka, Japan). Glutaraldehyde was purchased from Tousimis (Rockville, MD). All other chemicals were reagent grade. Nitrocellulose paper was purchased from Schleicher & Schuell, Inc. (Keene, NH). Myofibril and Single Fiber Preparations: Dissection of muscles and single muscle fibers and preparation of myofibrils has been described previously (4, 30, 31 ). Gel Electrophoresis: Polyacrylamide gel electrophoresis in the pres1001 on Jne 1, 2017 D ow nladed fom Published September 1, 1985