Muno D, Sutoh N, Sekine T
J Biochem. 1987 Mar;101(3):661-9. doi: 10.1093/jb/101.3.661.
When DNP-SH1-myosin, selectively dinitrophenylated at SH1 by 1,2,4-trinitrobenzene, was irradiated with a high-pressure mercury lamp equipped with a UV cut filter, a new 220-kDa band called the X-band appeared right above the heavy chain band (200 kDa) on SDS-PAGE (Laemmli). The time course of the X-band formation was composed of two phases, the initial one being rapid, and the second slow. Immune reaction experiments using antibodies specific for heavy or light chains indicated that the X-band in the initial phase contained heavy chain alone, but no light chains. Such an extra band (106 kDa) was also observed in the initial phase of photolysis of DNP-SH1-Subfragment-1 (heavy chain: 96 kDa) obtained from DNP-SH1-myosin. Trypsinolysis of the 106-kDa product generated a 83-kDa band. N-Terminal sequence analysis and the amino acid composition of the band revealed that the X-band is an intraheavy chain cross-linking product between the 20- and the 50-kDa fragments. This presents a striking contrast to the other cross-linking from SH1 using benzophenone-4-iodoacetamide which reacted with the 25-kDa fragment alone (Lu, R.C. et al. (1986) Proc. Natl. Acad. Sci. U.S. 83, 6392-6396). Based upon the result obtained, the spatial arrangement of the three tryptic domains around SH1 is discussed.
当用1,2,4 - 三硝基苯对SH1进行选择性二硝基苯基化的DNP - SH1 - 肌球蛋白，用配备有紫外线截止滤光片的高压汞灯照射时，在SDS - PAGE（Laemmli）上，一条名为X带的新的220 kDa条带出现在重链带（200 kDa）正上方。X带形成的时间进程由两个阶段组成，初始阶段迅速，第二阶段缓慢。使用针对重链或轻链的特异性抗体进行的免疫反应实验表明，初始阶段的X带仅包含重链，不包含轻链。在从DNP - SH1 - 肌球蛋白获得的DNP - SH1 - 亚片段 - 1（重链：96 kDa）的光解初始阶段也观察到了这样一条额外的带（106 kDa）。对106 kDa产物进行胰蛋白酶消化产生了一条83 kDa的带。该带的N端序列分析和氨基酸组成表明，X带是20 kDa和50 kDa片段之间的重链内交联产物。这与使用二苯甲酮 - 4 - 碘乙酰胺从SH1进行的其他交联形成了鲜明对比，后者仅与25 kDa片段反应（Lu, R.C.等人（1986年），美国国家科学院院刊83, 6392 - 6396）。基于所得结果，讨论了SH1周围三个胰蛋白酶结构域的空间排列。
