Reactions of [C-14]-3,4-dichloroisocoumarin with subunits of pituitary and spleen multicatalytic proteinase complexes (proteasomes)
Reactions of [C-14]-3,4-dichloroisocoumarin with subunits of pituitary and spleen multicatalytic proteinase complexes (proteasomes)
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DOI：
10.1021/bi970666e
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发表时间：
1997-11-11
期刊：
影响因子：
2.9
通讯作者：
Powers, JC
中科院分区：
文献类型：
--
作者：
Orlowski, M;Cardozo, C;Powers, JC
暴露于从牛垂体和脾脏中分离的多催化蛋白酶复合物（MPC）中的[C-14]-3,4-二氯异香豆素（DCI）会导致标记并入几个β型亚基，导致凝乳胰蛋白酶样（ht -l）活性的快速失活，以及MPC其他活性的缓慢失活。垂体和脾脏MPCs的不同之处在于，前者几乎完全包含X、Y和Z亚基，而后者这些亚基主要被LMP2、LMP7和MECL1所取代。尽管LMP7亚基与X亚基的氨基酸序列同源性更高，但用两种具有ht - l活性的肽基醛抑制剂进行预孵化可以保护垂体MPC中的X亚基，以及脾脏MPC中的LMP2亚基，使其免受标签合并的影响。氨基酸测序显示，这两个亚基的氨基酸产量都有所损失。DCI-蛋白键的稳定性表明DCI与苏氨酸OH基团反应形成酰基衍生物。短暂暴露于[C-14]-DCI导致标签优先结合到LMP2和X亚基中，这与ChT-L活性的高失活速率常数一致。Z-LLF-CHO是ChT-L活性抑制剂，而Z-GPFL-CHO是支链氨基酸偏好组分抑制剂，可以阻止放射性物质掺入到X亚基中，而这两种抑制剂都可以阻止标签掺入到LMP2中，这表明两种成分对抑制的敏感性存在差异。这些数据和其他数据与X亚基和LMP2亚基分别参与垂体和脾脏MPC中ChT-L活性的表达一致，并提示另外两个β亚基的催化功能。
Exposure to [C-14]-3,4-dichloroisocoumarin (DCI) of multicatalytic proteinase complexes (MPC) isolated from bovine pituitary and spleen leads to label incorporation into several beta-type subunits, to rapid inactivation of the chymotrypsin-like (ChT-L) activity, and to a slower inactivation of other activities of the MPC. The pituitary and spleen MPCs differ in that the first contains almost exclusively the X, Y, and Z subunits, whereas in the latter these subunits are largely replaced by LMP2, LMP7, and MECL1. Preincubation with two peptidyl aledhyde inhibitors of the ChT-L activity protected the X subunit in the pituitary MPC and unexpectedly the LMP2 subunit in the spleen MPC from label incorporation, despite the greater amino acid sequence homology of the LMP7 subunit to that of the X subunit, Losses in the yield of amino acids in both subunits, shown by amino acid sequencing, and lability of the DCI-protein bond indicated formation of an acyl derivative by reaction of DCI with the threonine OH group. Brief exposure to [C-14]-DCI led to preferential incorporation of label into the LMP2 and X subunits, consistent with the high inactivation rate constants of the ChT-L activity. Z-LLF-CHO, an inhibitor of ChT-L activity, but not Z-GPFL-CHO, an inhibitor of the branched chain amino acid preferring component, prevented incorporation of radioactivity into the X subunits, whereas both inhibitors prevented label incorporation into LMP2, indicating differences in susceptibility to inhibition between the two components. These and other data are consistent with involvement of the X and LMP2 subunits in expression of the ChT-L activity in the pituitary and spleen MPC, respectively, and suggest the catalytic functions of two other beta-subunits.