Muhlrad A, Peyser Y M, Ringel I
Department of Oral Biology, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.
Biochemistry. 1991 Jan 29;30(4):958-65. doi: 10.1021/bi00218a011.
Near-UV irradiation in the presence of vanadate cleaves the heavy chain of myosin subfragment 1 at three specific sites located at 23, 31, and 74 kDa from the N-terminus. Increasing the pH from 6.0 to 8.5, gradually, reduces the efficiency of the cleavage and completely eliminates the 31-kDa cut. Actin specifically inhibits the photocleavage at the sites located 31 and 74 kDa from the N-terminus. ATP strongly protects from cleavage at the 23- and 31-kDa sites and less strongly from the cut at the 74-kDa site. ADP and pyrophosphate have similar, but less pronounced, effects as ATP. Orthophosphate inhibits the photocleavage at the 23- and 74-kDa sites with a similar efficiency. In the ternary actin-S-1-ATP complex, the photocleavage is inhibited at all sites, and the effects of actin and ATP are additive. Photocleavages affect the K+(EDTA)-, Ca2(+)-, and actin-activated ATPase activity of subfragment 1. Loss of all three ATPases is caused by cleavage at the 23-kDa site, while the cut at the 74-kDa site only leads to the loss of actin-activated ATPase activity. It is concluded that subfragment 1 contains at least two distinct phosphate binding sites, the first being part of the "consensus" ATP binding site wherein the 23-kDa photocleavage site is located. This site is responsible for the binding and hydrolysis of ATP. It is possible that the 31-kDa cleavage site is also associated with the "consensus" site through a loop. The 74-kDa cleavage site is a part of another phosphate binding site which may play a role in the regulation of the myosin-actin interaction.
在钒酸盐存在的情况下，近紫外辐射会在肌球蛋白亚片段1重链上距N端23、31和74 kDa处的三个特定位点进行切割。将pH从6.0逐渐提高到8.5会降低切割效率，并完全消除31 kDa处的切割。肌动蛋白特异性抑制距N端31和74 kDa处位点的光切割。ATP强烈保护23 kDa和31 kDa处的位点不被切割，对74 kDa处的切割保护作用稍弱。ADP和焦磷酸具有与ATP相似但不太明显的作用。正磷酸盐以相似的效率抑制23 kDa和74 kDa处的光切割。在肌动蛋白-S-1-ATP三元复合物中，所有位点的光切割均受到抑制，肌动蛋白和ATP的作用是相加的。光切割会影响亚片段1的K+(EDTA)-、Ca2(+)-和肌动蛋白激活的ATP酶活性。所有三种ATP酶活性的丧失是由23 kDa处的切割引起的，而74 kDa处的切割仅导致肌动蛋白激活的ATP酶活性丧失。得出的结论是，亚片段1至少包含两个不同的磷酸盐结合位点，第一个是“共识”ATP结合位点的一部分，23 kDa光切割位点位于其中。该位点负责ATP的结合和水解。31 kDa切割位点可能也通过一个环与“共识”位点相关联。74 kDa切割位点是另一个磷酸盐结合位点的一部分，可能在调节肌球蛋白-肌动蛋白相互作用中发挥作用。
