An immunological approach to the role of the low molecular weight subunits in myosin. II. Interaction of myosin and its subfragments with antibodies to the light chains.
Immunological methods, in parellel with measurement of ATPase activity, have been used to characterize the reactions of antibodies specific for light chains with myosin and its water-soluble proteolytic subfragments, heavy meromyosin (HMM) and subfragment 1 (HMM S-1). Antiserum to the 5,5'-dithiobis(2-nitro-enzoic acid) (DTNB) light chain undergoes a precipitation reaction with all of the enzyme species, in which half of the homologous light chain is selectively dissociated. The results suggest that the incomplete dissociation reflects the way in which the light chain is bound, rather than the existence of two distinct species of DTNB 1.c. Little reaction was observed with antisera to alkali-released light chains, indicating that these components in myosin and the subfragments are either largely buried or else conformationally different from the isolated light chains used as immunogens. None of the antisera produced significant changes in Ca2+- or EDTA-ATPase activities. Moreover, calcium regulation through the troponin-tropomyosin system was unaffected by removal of DTNB 1.c. from myosin, as well as from the subfragments. The absolute level of actin-activated ATPase activity was, however, consistently lower in the presence of light chain antisera (or purified IgG and antibody) than in aqueous buffer or nonimmune serum. For both alkali and DTNB 1.c. antisera, this loss in activity seemed to result from steric hindrance of actin binding by antibody bound to undissociated light chain. Experimental conditions which would be expected to weaken such an antigen-antibody interaction, as well as the use of monovalent Fab in place of IgG, decreased the inhibition of activity. Altogether the activity measurements suggest that the light chains, particularly DTNB 1.c., are probably not integral parts of either the hydrolytic or actin-binding sites.
D000199:Actins / Q000378:metabolism; D000818:Animals; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D002645:Chickens; D004228:Dithionitrobenzoic Acid; D046911:Macromolecular Substances; D009132:Muscles; D015879:Myosin Subfragments; D009218:Myosins / Q000276:immunology* / Q000378:metabolism; D010446:Peptide Fragments; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation
D000199:Actins; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010446:Peptide Fragments; D004228:Dithionitrobenzoic Acid; D009218:Myosins