Hiratsuka T
J Biol Chem. 1986 Jun 5;261(16):7294-9.
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 (Körner, M., Thiem, N. V., Cardinaud, R., and Lacombe, G. (1983) Biochemistry 22, 5843-5847; Hiratsuka, T. (1986) Biochemistry 25, in press).
荧光试剂4-氟-7-硝基苯并-2-恶唑-1,3-二氮杂茂（NBD-F）与肌球蛋白亚片段1（S-1）ATP酶的1.9个赖氨酸残基/摩尔发生特异性反应。当1.9个赖氨酸残基被修饰时，K⁺-和Ca²⁺-ATP酶活性几乎完全被抑制，而Mg²⁺-ATP酶活性增加到原来活性的180%。这种修饰使肌动蛋白激活的Mg²⁺-ATP酶活性降低到原来活性的30%。然而，在ATP存在的情况下，S-1对肌动蛋白的亲和力没有改变。加入ATP后，修饰后的S-1的NBD荧光被淬灭，这表明ATP诱导了与S-1相连的NBD基团周围的构象变化。对修饰后的S-1进行胰蛋白酶消化表明，NBD基团主要连接在S-1的50 kDa肽段上，更确切地说是45 kDa肽段上。这些结果证实了最近的报道，即S-1的50 kDa肽段参与了肌球蛋白ATP酶反应（科尔纳，M.，蒂姆，N. V.，卡尔迪瑙，R.，和拉孔布，G.（1983年）《生物化学》22，5843 - 5847；平冢，T.（1986年）《生物化学》25，即将发表）。
