Binding of Myosin Cross-Bridges to Thin Filaments of Rabbit Skeletal Muscle O. A. Andreev and J. Borejdo Department of Molecular Biology and Immunology, University of North Texas, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107 Received January 6, 1999 Cross-linking of myosin subfragment 1 (S1) with a molar excess of actin in vitro reveals the presence of an actin-S1-actin complex. It is absolutely essential that actin be present in molar excess over S1 so that the decoration of F-actin with S1 be incomplete. How- ever, the excess of actin may not be available in the overlap zone of sarcomeres of skeletal muscle. We therefore found it necessary to test for the presence of the actin-S1-actin complex in vivo. Myofibrils from rabbit skeletal muscle were reacted with zero-length cross-linker, the products were resolved by polyacryl- amide gel electrophoresis and analyzed by Western blots using antibodies against actin and against heavy and light chains of myosin. The cross-linking pro- duced the evidence of formation of actin-S1-actin complex. © 1999 Academic Press Key Words: skeletal muscle; contractility; actin and myosin; cross-linking. Cross-linking of myosin S1 with a molar excess of actin in vitro yields two specific adducts indicative of the formation of actin-S1-actin triplet. The first adduct, with the molecular mass of 210 KDa, is a complex of a heavy chain (HC) of S1 and two actins (1, 2). The second adduct, with molecular mass of 66 KDa, is a complex of an essential light chain 1 (A1) and actin (3). For the triplet to be produced, it is essential that actin be in molar excess over S1, i.e. that decoration of F-actin with S1 be incomplete and include regions con- taining at least two consecutive unoccupied actin monomers (3). However, there exist four reasons why actin-S1-actin triplet may not be produced in vivo: (i) in the overlap zone of skeletal sarcomere, actin may not be present in sufficient excess to offer to S1 two free adjoining sites; (ii) muscle cross-bridge consists of two S1s. Each head may compete for the same actin bind- ing site; (iii) in vertebrate skeletal muscle, actin and myosin are arranged in regular arrays, which may alter the electrostatic interactions that are important in binding (4, 5); and (iv) the binding sites on thin filaments could be occupied by troponin-tropomyosin and/or nebulin. In fact, nebulin fragments do prevent binding of S1 to actin in vitro (6). We therefore found it necessary to ask whether S1 binds to two actins in skeletal muscle. Myofibrils from rabbit skeletal muscle were reacted with zero-length cross-linker, the prod- ucts were resolved by polyacrylamide gel electrophore- sis and analyzed by Western blots using antibodies against actin and against heavy & light chains of my- osin. The data showed that S1 which was derived from myofibrillar cross-bridges by mild chymotryptic diges- tion, yielded the 210 KDa adduct. Myofibrillar A1 formed cross-links with actin in thin filaments to yield the 66 KDa adduct. These results indicate that in skel- etal muscle myosin heads form actin-S1-actin triplet. Results obtained with the two-headed heavy mero- myosin (HMM) further support the view that the bind- ing of myosin heads with thin filaments in skeletal muscle is the same as binding of S1 with excess of actin in vitro: cross-linking of HMM in vitro yielded two specific adducts, with molecular masses of 42-39 and 63 KDa, indicating binding of the regulatory light chain (RLC) with the each of the essential light chains (ELCs) and with the neighboring RLC. Identical 42-39 and 63 KDa adducts were formed in skeletal myofi- brils. No cross-linking occurred in relaxed myofibrils. A short report of these findings appeared earlier (7, 8). MATERIALS AND METHODS Chemicals. 1-Ethyl-3-(3-dimethyl-aminopropyl) carbodiimide, anti- actin and anti-HC were from Sigma (St Louis, MO). Anti-ELC and anti-RLC antibodies were gifts from Dr. S. Lowey (Brandeis University). Abbreviations used: S1, myosin subfragment-1; HC, heavy chain of myosin subfragment-1; RLC, regulatory light chain; A1, essen- tial light chain 1; A3, essential light chain 2; ELC, essential light chains; EDC, 1-ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide; Act, actin; PAGE, polyacrylamide gel electrophoresis; HMM, heavy meromyosin. Biochemical and Biophysical Research Communications 258, 628 – 631 (1999) Article ID bbrc.1999.0319, available online at http://www.idealibrary.com on 628 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.