Bailin G, Shah T, Huang J R
Department of Biochemistry, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Piscataway 08854-5635.
Arch Biochem Biophys. 1990 Aug 15;281(1):6-12. doi: 10.1016/0003-9861(90)90405-n.
Chicken gizzard myosin treated with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) resulted in a 65% inhibition of the K(+)-ATPase (myosin ATP phosphohydrolase (actin translocating), EC 3.6.1.32) activity and 3.5 mol of the reagent was bound per 4.7 x 10(5) g protein. The labeling was limited to the heavy chain region and none of the light chains were lost. MgATP had no effect on the inactivation or labeling pattern. Thiolysis of NBD-myosin with dithiothreitol restored the K(+)-ATPase activity and concurrently, 1 mol of the NBD group was removed from the heavy chain region. Cysteine residues were modified in NBD-myosin at sites other than the active site when the enzyme activity was inhibited. There was a difference in the extent of NBD-Cl modification of gizzard myosin at 0.6 M KCl (6 S elongated state) when compared to that at 0.15 M KCl (10 S folded state). This was also seen in the heavy meromyosin-like chymotryptic fragments and tryptic fragments of NBD-myosin. The reagent NBD-Cl can detect changes in the conformation of gizzard myosin by way of its reaction with thiol groups of the heavy chain region.
用7-氯-4-硝基苯并-2-恶唑-1,3-二氮杂茂（NBD-Cl）处理鸡胗肌球蛋白，导致K(+)-ATP酶（肌球蛋白ATP磷酸水解酶（肌动蛋白移位），EC 3.6.1.32）活性受到65%的抑制，每4.7×10(5) g蛋白质结合3.5 mol该试剂。标记仅限于重链区域，轻链均未丢失。MgATP对失活或标记模式无影响。用二硫苏糖醇对NBD-肌球蛋白进行巯基裂解可恢复K(+)-ATP酶活性，同时，1 mol NBD基团从重链区域去除。当酶活性受到抑制时，NBD-肌球蛋白中活性位点以外的半胱氨酸残基被修饰。与0.15 M KCl（10 S折叠状态）相比，0.6 M KCl（6 S伸长状态）下鸡胗肌球蛋白的NBD-Cl修饰程度存在差异。在NBD-肌球蛋白的重酶解肌球蛋白样胰凝乳蛋白酶片段和胰蛋白酶片段中也观察到了这种差异。试剂NBD-Cl可通过与重链区域的巯基反应来检测鸡胗肌球蛋白构象的变化。
