Modification of myosin a by 1-dimethylaminonaphthalene-5-sulfonyl chloride

Abstract

Summary 1. 1-Dimethylaminonaphthalene-5-sulfonyl chloride reacted with myosin A to give a fluorescent conjugate. A slight blue shift of the absorption maximum at 279 m μ was observed at degrees of labeling greater than one mole dye per mole myosin A; and a molar extinction coefficient of 1.97·10 4 at 250 m μ was calculated for the conjugated dye. 2. The ATPase activity of myosin A (ATP phosphohydrolase, EC 3.6.1.3) was markedly inhibited by the reaction with one molecule of the fluorescent dye, and the actin-binding ability of myosin A was partially inhibited. Both the clearing response and the super-precipitation of actomyosin were inhibited by labeling of myosin A with the dye. 3. The dye gave non-competitive inhibition to both the ATPase and ITPase of myosin A, and the dissociation constants ( K i ) for these were 11.0·10 −5 M and 7.3.10 −5 M respectively. The ATPase of dye-labeled myosin A showed scarcely any depression near the neutral region in its pH-dependency curve, and was not activated by low concentrations of EDTA or p -chloromercuribenzoate. 4. About 3.5 moles SH groups were masked by the reaction with one molecule of the dye; however, the dye may possibly bind to lysine or histidine residues rather than SH groups in the active center of myosin A. The masked SH groups were recovered in part by treatment with low concentrations of urea or ethanol. 5. The chemical nature and approximate size of the biological active center of myosin A are discussed.