Abstract
The reactive lysyl residues located in the subfragment-1 (S-1)/subfragment-2 (S-2) link region of cardiac myosin heavy chains (HCs) were preferentially labeled with a fluorescent reagent, N-methyl-2-anilino-6-naphthalenesulfonyl chloride(Mns-Cl). The effect of Mns-labeling of these residues on the ability of actomyosin to superprecipitate and on the actin-activated ATPase of myosin was investigated using two types of labeled myosins; one was myosin labeled in the absence of divalent metal ions, where one or two reactive lysyl residues were labeled (termed E-1-Mns-M or E-2-Mns-M, respectively) and the other was myosin labeled in the presence of Ca2+, where both reactive residues were intact but another lysyl residue was labeled (termed C-1-Mns-M).
Both the rate and extent of superprecipitation of actomyosin reconstituted from actin and two types of labeled myosins were reduced. Although the Mg2+-ATPase activity of E-1-Mns-M or E-2-Mns-M was practically unchanged, its actin-activated ATPase activities at both low and high ionic strengths were reduced. This reduction was saturated at an incorporation of about two moles of Mns groups per mol of myosin. Neither partial release nor partial labeling of light chain-2, which occurred during the labeling of the reactive lysyl residues, was responsible for the reduction in the actin-activated ATPase activity of myosin. The apparent dissociation constant for actin, Km, obtained from the double-reciprocal plot of actin-activated ATPase activity of E-1-Mns-M or E-2-Mns-M at low ionic strength was increased 4- or 6-fold over that of unlabeled myosin, respectively. On the other hand, changes in the actin-activated ATPase activity and in the Km of myosin were hardly observed when Ca2+ protected the reactive residues from the labeling, and another residue was labeled.
These results suggest that in the presence of ATP the actin binding site “senses” changes in the S-1/S-2 link region of the cardiac myosin HC including the reactive lysyl residue.
