An epitope proximal to the carboxyl terminus of the alpha-subunit is located near the lobe tips of the phosphorylase kinase hexadecamer.
An epitope proximal to the carboxyl terminus of the alpha-subunit is located near the lobe tips of the phosphorylase kinase hexadecamer.
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靠近α亚基羧基末端的表位位于磷酸化酶激酶十六聚体的叶尖附近。
DOI：
10.1006/jmbi.1994.1051
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发表时间：
1994
影响因子：
5.6
通讯作者：
Carlson,GM
中科院分区：
文献类型：
--
作者：
Wilkinson,DA;Marion,TN;Tillman,DM;Norcum,MT;Hainfeld,JF;Seyer,JM;Carlson,GM
通过两种类型的免疫电子显微镜，将快肌骨骼肌磷酸化酶激酶 α 亚基的表位定位于双叶激酶分子的尖端。这是第一个确定该酶 16 个亚基在磷酸化酶激酶分子内位置的直接证据。通过传统透射电子显微镜观察磷酸化酶激酶与特异于 α 亚基的免疫球蛋白 G 单克隆抗体 (mAb 157) 的负染色复合物，并通过扫描透射电子显微镜观察未染色的酶与源自 mAb 157 的十一金标记 Fab' 片段的复合物。两种技术的图像均表明表位的对称排列，与四个 α 亚基的“头对头”堆积排列一致。在蛋白质印迹中，mAb 157 与通过用多种蛋白酶消化非变性磷酸化酶激酶获得的共迁移片段发生交叉反应，表明抗 α mAb 的表位包含在蛋白酶抗性结构域内。对 24·1 kDa 免疫反应性胰凝乳蛋白酶片段进行部分测序，将表位缩小到 α 亚基羧基末端六分之一内的某个位置。在钙调蛋白存在下、磷酸化后或与不同亚型（均具有已知的 α 亚基序列目标或差异）的情况下，对 mAb 157 与全酶的交叉反应性的研究表明，表位甚至更接近羧基末端。该表位与该酶的任何已知功能或活性无关，表明靠近α-亚基羧基末端的区域，以及因此靠近十六聚体的叶尖的区域，可能具有催化或调节以外的作用。
An epitope of the α-subunit of phosphorylase kinase from fast-twitch skeletal muscle was localized to the tips of the bilobal kinase molecule by two types of immunoelectron microscopy. This is the first direct evidence identifying the location of any of the enzyme's 16 subunits within the phosphorylase kinase molecule. Negatively stained complexes of phosphorylase kinase with an immunoglobulin G monoclonal antibody specific for the α-subunit (mAb 157) were observed by conventional transmission electron microscopy, and complexes of the unstained enzyme with undecagold-labeled Fab′ fragments derived from mAb 157 were visualized by scanning transmission electron microscopy. Images from both techniques indicate a symmetrical arrangement of the epitope, consistent with a "head-to-head" packing arrangement of the four α-subunits. In Western blots, mAb 157 crossreacted with comigrating fragments obtained by digesting non-denatured phosphorylase kinase with a variety of proteases, suggesting that the epitope for the anti-α mAb is contained within a protease-resistant domain. Partial sequencing of a 24·1 kDa immunoreactive chymotryptic fragment narrowed the epitope to somewhere within the carboxyl-terminal one-sixth of the α-subunit. Studies of the crossreactivity of mAb 157 with the holoenzyme in the presence of calmodulin, after phosphorylation or with different isoforms (all with known α-subunit sequence targets or differences), suggest that the epitope is even more proximal to the carboxyl terminus. This epitope was not implicated in any known function or activity of the enzyme, suggesting that the region proximal to the carboxyl terminus of the α-subunit, and thus to the lobe tips of the hexadecamer, may have a role other than catalytic or regulatory.