Thiolysis of dinitrophenylated sulfhydryl groups in gizzard myosin. Restoration of regulatory properties in a reconstituted actomyosin.

Dinitrophenylated chicken gizzard myosin which had incorporated 5.2 mol of l-fluoro-2,4-dinitrobenzene/4.7 x 10' g of protein failed to form a calcium-sensitive actomyosin. Thiolysis of 2 mol of the dinitrophenyl group from modified gizzard myosin with 2-mercaptoethanol restored its ability to form a calcium-sensitive actomyosin. It was determined that the dinitrophenyl group was removed from the 17,000-dalton light chain and some heavy chains (Bailin, G., and Lopez, F. (1981) Biochim Biophys. Acta 668,46-56). Sulfopropyl (SP) Sephadex chromatography of trypsin-pepsin digests of dinitrophenylated gizzard myosin in the presence and absence of 2-mercaptoethanol yielded two major peptide fractions (I and III) and at least two minor ones (IX and X). Peptide I was also found in similar digests of the 17,000-dalton dinitrophenylated light chain. These peptides contained only S-dinitrophenylcysteine. Peptide I11 (and I to some extent) contained several amino acids that were stoichiometrically equivalent to its S-dinitrophenylcysteine content. Some of the reactive sulfhydryl groups of gizzard myosin were different than those of rabbit skeletal myosin. They were located predominantly in the 17,000dalton light chain of gizzard myosin. Treatment of dinitrophenylated gizzard myosin with N-ethylmaleimide followed by thiolysis with 2-mercaptoethanol mainly resulted in the labeling of the heavy chains. Both heavy and light chains were modified in gizzard myosin treated with N-ethylmaleimide alone. Conformational changes, induced by l-fluoro-2,4-dinitrobenzene, resulted in major changes in the reactivity of "SH groups and enzymic activity of the myosin. Gizzard myosin contains sites on the light and heavy chains that are involved in the regulation of its actinactivated ATPase activity.