We report measurements of the reactivity (degree of labeling, as mole of ligand per mole of protein, at constant exsure time) of the reactive thiol, "SHI", of a subfragment of myosin (S-i), and of Cys-10 of F-actin under various conditions, using N-iodo{3Hjacetyl-N(1-sulfo-5-naphthyl)ethylenediamine, a fluorescent radioactive iodoacetamide analog. When either ADP or adenyloyl imidodiphosphate (simulating unhydrolyzed ATP) is bound to the enzymatic site of S-i, the reactivity of "SHI" is slightly enhanced, but when active ATPase is going on, reactivity is reduced by about a third, presumably due to the species, (S.1)**ADPPi. The reactivity of Cys-10 alone is very low. When the complex, (S-I)F-actin, is formed, the reactivity of SHI is strongly decreased, and the reactivity of Cys-10 is strongly increased. The foregoing results explain our further observation (on glycerol-treated rabbit psoas fibers) that when fibers labeled in relaxation solution are compared with fibers labeled in rigor solution, myosin is more reactive and actin is less reactive, in the former case; a-actinin and C-protein are also less reactive in the former case.Our laboratory has used the compound N-iodo-[3H]-acetyl-N'-(l-sulfo-5-naphthyl)ethylenediamine ([3H]1,5-IAEDANS) extensively to fluorescence-label myosin and muscle fibers because this compound has a strong specificity for the reactive thiol (SHI) in the S-1 moiety of myosin.While searching for conditions in which the proteins of glycerinated psoas fibers are selectively labeled, we noted that the "relaxation" state tended to favor myosin labeling and to suppress actin labeling, while the "tension-generating" and "rigor" states tended to favor the converse. In order to clarify this reciprocity between myosin and actin, and to investigate the chemical changes that occur during myosin-actin binding, we forsook fibers and experimented with purified proteins and various nucleotides. We have thus been led into work along the lines pioneered by Barany et al. (1). We have learned that the reactivity of "SH1" with [3H]1,5-IA-EDANS depends upon the occupancy of the ATPase site; it is slightly enhanced by either ADP or adenyloyl imidodiphosphate (AMPPNP), and is significantly depressed during hydrolysis. It also depends on the occupancy of the actin-5-naphthyl)ethylenediamine; SH1, the "fast reacting" and calcium ATPase activating thiol located on the heavy chain of S-1; 5-1, subfragment resulting from papain hydrolysis of myosin; AMPPNP, adenyloyl imidodiphosphate; Cys-10 andCys-373, cysteinyl residue in position 10 (NH2-terminal peptide) and 373 (COOH-terminal peptide), respectively, of the amino-acid sequence of actin; TPCK, L-1-tosylamide-2-phenylethylchloromethyl ketone (chymotrypsin inhibitor); TLCK, N-a-p-tosyl-l-lysine chloromethyl ketone.HCI (papain inhibitor); EGTA, ethylene-glycol-bis(,3-aminoethyl ether) N,N'-tetraacetic acid; NaDodSO4, sodium dodecyl sulfate; Tes, Ntris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. binding site and is very strongly depressed when actin i...
