J. Biol. Chem., Vol. 261, Issue 16, 7294-7299, Jun, 1986
Involvement of the 50-kDa peptide of myosin heads in the ATPase activity revealed by fluorescent modification with 4-fluoro-7-nitrobenz- 2-oxa-1,3-diazole
T Hiratsuka
The fluorescent reagent 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD-F)
reacted specifically with 1.9 lysyl residues/mol of the myosin
subfragment-1 (S-1) ATPase. When 1.9 lysyl residues were modified, the K+-
and Ca2+-ATPase activities were almost completely inhibited, whereas the
Mg2+-ATPase activity was increased to 180% of original activity. The
actin-activated Mg2+-ATPase activity was decreased to 30% of original
activity by this modification. However, affinity of S-1 for actin in the
presence of ATP was unchanged. The NBD fluorescence of the modified S-1 was
quenched on addition of ATP, suggesting that ATP induced conformational
changes around the NBD groups attached to S-1. Tryptic digestion of the
modified S-1 revealed that the NBD groups are attached mainly to the 50-kDa
peptide of S-1, more precisely the 45-kDa peptide. These results confirm
the recent reports that the 50-kDa peptide of S-1 is involved in the myosin
ATPase reaction (Korner, M., Thiem, N. V., Cardinaud, R., and Lacombe, G.
(1983) Biochemistry 22, 5843-5847; Hiratsuka, T. (1986) Biochemistry 25, in
press).
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