Use of Antibodies against Dodecylsulfate-Denatured Heavy Meromyosins to Probe Structural Differences between Muscular Myosin Isoenzymes

Abstract

Heavy meromyosin, a tryptic myosin fragment, was purified from rabbit fast twitch muscles and rat cardiac ventricles. Both types of heavy meromyosin were denatured by sodium dodecylsulfate and used to immunize guinea‐pigs after chromatography on Sephadex G‐10 to remove excess dodecylsulfate. Micro‐complement fixation analysis showed that the antisera were specific to a denatured configuration of heavy meromyosin and myosin, and hardly recognized the native proteins. Crossreactions performed with both rabbit skeletal and rat cardiac antisera indicated that the antigenic structures of denatured myosins varied according both to species (man, rabbit, rat or mouse), and to muscle‐type (red skeletal slow twitch, white skeletal fast twitch, cardiac atria or cardiac ventricles). Denatured heavy meromyosin chromatography on Sephadex G‐200 in the presence of 0.1% sodium dodecylsulfate enabled separation of several polypeptides groups. Of these, a polypeptide of M, 29000 was the most reactive and exhibited the same immunological specificities as the whole myosin molecule. The use of antibodies against denatured heavy meromyosin in conjunction with micro‐complement fixation therefore provides a discriminant means, not only for estimating the structural relationship between several myosin isoenzymes, but also for localizing constant and variable regions in the heavy chains of these isoenzymes.

Fiber types and myosin types in human atrial and ventricular myocardium. An anatomical description.

“…This myosin, used as an immunogen, was previously denatured with 2% sodium dodecyl sulfate (Schwartz et al, 1980) and then dialyzed in 50 mM sodium pyrophosphate, pH 7.5. The mice were immunized twice subcutaneously with 100 /xg of myosin emulsified in Freund's adjuvant at intervals of a few weeks.…”

“…Both immunizations used SDS-denatured myosin (Schwartz et al, 1980) prepared from the hypertrophic left ventricle of a 5 3-year-old female who had an obstructive cardiomyopathy. The 15 cell lines were cloned and their supernatants reacted only with the myosin heavy chains and not with the myosin light chains or other trace contaminants present in the whole muscle extract or in the myosin preparation as determined by the immune replicate technique (Fig.…”

Structural and Enzymatic Comparison of Human Cardiac Muscle Myosins Isolated from Infants, Adults, and Patients with Hypertrophic Cardiomyopathy

“…Microcomplement fixation and pyrophosphate PAGE (13)(14)(15)(16) have also been used to demonstrate a change in distribution of the myosin heavy chain isoenzymes in rat hearts undergoing hypertrophy due to mechanical overload caused by aortic stenosis and regurgitation. With increasing hypertrophy there was a decrease in the proportion of VI myosin and an increase in that of V3 myosin along with an accompanying decrease in myosin Ca++-activated ATPase activity.…”

Immunogenetic basis of myocarditis: Role of fibrillary antigens

“…Monoclonal antibodies (mAbs) have been produced which can distinguish the various myosin HCs, but the variations in reactivity patterns are due to a differential binding affinity of the monoclonal with the various myosin HC isoforms [8,9]. Antigenic determinants can be associated with the native and detergent-denatured epitopes [15,58].…”