Losa R, Thoma F, Koller T
J Mol Biol. 1984 Jun 5;175(4):529-51. doi: 10.1016/0022-2836(84)90183-9.
We have attacked H1-containing soluble chromatin by alpha-chymotrypsin under conditions where chromatin adopts different structures. Soluble rat liver chromatin fragments depleted of non-histone components were digested with alpha-chymotrypsin in NaCl concentrations between 0 mM and 500 mM, at pH 7, or at pH 10, or at pH 7 in the presence of 4 M-urea. alpha-Chymotrypsin cleaves purified rat liver histone H1 at a specific initial site (CT) located in the globular domain and produces an N-terminal half (CT-N) which contains most of the globular domain and the N-terminal tail, and a C-terminal half (CT-C) which contains the C-terminal tail and a small part of the globular domain. Since in sodium dodecyl sulfate/polyacrylamide-gel electrophoresis CT-C migrates between the core histones and H1, cleavage of chromatin-bound H1 by alpha-chymotrypsin can be easily monitored. The CT-C fragment was detected under conditions where chromatin fibers were unfolded or distorted: under conditions of H1 dissociation at 400 mM and 500 mM-NaCl (pH 7 and 10); at very low ionic strength where chromatin is unfolded into a filament with well-separated nucleosomes; at pH 10 independent of the ionic strength where chromatin never assumes higher order structures; in the presence of 4 M-urea (pH 7), again independent of the ionic strength. However, hardly any CT-C fragment was detected under conditions where fibers are observed in the electron microscope at pH 7 between 20 mM and 300 mM-NaCl. Under these conditions H1 is degraded by alpha-chymotrypsin into unstable fragments with a molecular weight higher than that of CT-C. Thus, the data show that there are at least two different modes of interaction of H1 in chromatin which correlate with the physical state of the chromatin. Since the condensation of chromatin into structurally organized fibers upon raising the ionic strength starts by internucleosomal contacts in the fiber axis (zig-zag-shaped fiber), where H1 appears to be localized, it is likely that in chromatin fibers the preferential cleavage site for alpha-chymotrypsin is protected because of H1-H1 contacts. The data suggest that the globular part of H1 is involved in these contacts close to the fiber axis. They appear to be hydrophobic and to be essential for the structural organization of the chromatin fibers.(ABSTRACT TRUNCATED AT 400 WORDS)
我们在染色质呈现不同结构的条件下，用α-糜蛋白酶处理含H1的可溶性染色质。将去除了非组蛋白成分的大鼠肝脏可溶性染色质片段，在0 mM至500 mM的NaCl浓度下、pH 7或pH 10时，或在4 M尿素存在下pH 7时，用α-糜蛋白酶进行消化。α-糜蛋白酶在位于球状结构域的一个特定起始位点（CT）切割纯化的大鼠肝脏组蛋白H1，产生一个N端半段（CT-N），其包含大部分球状结构域和N端尾巴，以及一个C端半段（CT-C），其包含C端尾巴和一小部分球状结构域。由于在十二烷基硫酸钠/聚丙烯酰胺凝胶电泳中CT-C在核心组蛋白和H1之间迁移，因此可以很容易地监测α-糜蛋白酶对染色质结合的H1的切割。在染色质纤维展开或扭曲的条件下检测到了CT-C片段：在400 mM和500 mM NaCl（pH 7和10）下H1解离的条件下；在非常低的离子强度下，染色质展开成具有间隔良好的核小体的细丝；在pH 10时，与离子强度无关，此时染色质从不呈现高级结构；在4 M尿素存在下（pH 7），同样与离子强度无关。然而，在pH 7、20 mM至300 mM NaCl条件下在电子显微镜中观察到纤维的情况下，几乎未检测到任何CT-C片段。在这些条件下，H1被α-糜蛋白酶降解为分子量高于CT-C的不稳定片段。因此，数据表明染色质中H1至少有两种不同的相互作用模式，这与染色质的物理状态相关。由于随着离子强度升高，染色质凝聚成结构有序的纤维是从纤维轴上的核小体间接触（锯齿状纤维）开始的，H1似乎定位于此处，所以在染色质纤维中，α-糜蛋白酶的优先切割位点可能由于H1-H1接触而受到保护。数据表明H1的球状部分参与了靠近纤维轴的这些接触。它们似乎是疏水的，并且对于染色质纤维的结构组织至关重要。（摘要截短至400字）
