Probing myosin light chain 1 structure with monoclonal antibodies

Abstract

Five monoclonal antibodies that react with different regions of myosin light chain 1 from human ventricular myocardial muscle were used to obtain information on interactions between the light chain 1 and heavy chains and generally on the tertiary structure of the light chain 1 within the myosin head. We performed Western blot assays of the five antibodies with myosins from different cardiac and skeletal muscles, with different proteolytic fragments of bovine ventricular myosin light chain 1 (LC1) and to different recombinant fragments of human ventricular LC1 and rat fast skeletal light chain LC1/LC3. The five antibodies were mapped in three different regions of the light chain 1: two antibodies mapped within the first eight amino-terminal residues, two between residues 71 and 74, and one between residues 129 and 134. The apparent dissociation constants of the last three antibodies, determined by antibody-antigen equilibria in solution, were lower than when isolated light chains were used as antigens. It is probable that the corresponding amino acids involved in the antibody epitopes were either involved in interactions between the light and heavy myosin subunits, or somehow hindered by the myosin heavy chain bulk. In contrast, the apparent dissociation constants measured for both other antibodies were higher when myosin, rather than isolated light chains, was used as antigen. Thus LC1 fixation to heavy chains within the myosin molecule induced conformation changes at the amino-terminal end of the light chain 1. No difference in the accessibility of this mobile LC1 segment was detected in the presence of actin. Finally, observed differences in epitope accessibility on the light chain LC1 in myosin, as compared with chymotryptic subfragment 1 (SF1), indicated conformational differences between native myosin and extensively studied SF1 molecules.

Dependence of cross‐bridge kinetics on myosin light chain isoforms in rabbit and rat skeletal muscle fibres

“…The antibody for detecting the alkali myosin light chain isoforms MLC1f, MLC3f and MLC1s was purchased from Alexis (Grünberg, Germany; the monoclonal antibody to human ventricular light chain 1). The specific reactivity of this antibody with skeletal muscle alkali light chain isoforms was shown in a previous study (Cornillon et al 1992). The antibody for detecting the regulatory myosin light chain isoforms MLC2f and MLC2s was purchased by Sigma (St Louis, USA; myosin light chain 20 K).…”

Probing Conformational Changes within LC2 Domains of Cardiac Myosin

“…Monoclonal antibodies were obtained according to the method of Kohler and Milstein (1975). The procedures for mouse immunization with human ventricular LCs, fusion, screening, production, and purification of antibodies have been described previously (Dechesne et al, 1985;Cornillon et al, 1992).…”

“…Association Constant Measurements. Apparent association constants (Kd) of the antigen-antibody equilibrium in solution were measured as initially reported by Friguet et al (1985) and extensively discussed by Cornillon et al (1992). Various doses of antigen were mixed with constant quantities of Mab in 0.12 M NaCl and 20 mM Tris-HCl (pH 7.5) buffer.…”

“…The constructions were controlled by sequencing with a T7 sequencing kit (Pharmacia). Expression in an Escherichia coli JM 109 strain and fusion protein isolation were described previously (Cornillon et al, 1992).…”

Expression of muscle‐specific myosin heavy chain and myosin light chain 1 in the electric tissue of Electrophorus electricus (L.) in comparison with other vertebrate species

“…Myosin is the major constituent of the thick filaments in striated muscle, where it consists of two heavy chains (HC) associated with two alkali light chains (LC1 and LC3) and two regulatory light chains (LC2). Skeletal muscle expresses subtypes LC1, LC2, and LC3 and cardiac muscle expresses only subtypes LC1 and LC2 (Cornillon et al, 1992). Moreover, LCs can be classified as fast or slow subtypes (Pernelle et al,'90;Schiaffino and Reggiani,'94,'96), and can be expressed as different isoforms in specific cells.…”