THIOL-DISULFIDE EXCHANGE OF RIBONUCLEASE INHIBITOR BOUND TO RIBONUCLEASE-A - EVIDENCE OF ACTIVE INHIBITOR-BOUND RIBONUCLEASE
THIOL-DISULFIDE EXCHANGE OF RIBONUCLEASE INHIBITOR BOUND TO RIBONUCLEASE-A - EVIDENCE OF ACTIVE INHIBITOR-BOUND RIBONUCLEASE
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DOI：
10.1074/jbc.270.48.28570
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发表时间：
1995-12-01
影响因子：
4.8
通讯作者：
GARCIASEGURA, JM
中科院分区：
文献类型：
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作者：
FERRERAS, M;GAVILANES, JG;GARCIASEGURA, JM
核糖核酸酶抑制剂 (RI) 已从猪睾丸中纯化。它含有 30 个半胱氨酸，其氧化影响其结合和抑制核糖核酸酶 (RNase) 的能力，通过 N 端序列分析，睾丸 RI 显示与猪肝脏的相同，据报道，5,5'-二硫代双（2-硝基苯甲酸）（DTNB）对猪肝脏进行特征性全或无类型的 SH 氧化（Fominaya，J. M. 和 Hofsteenge，J. (1992) J. Biol. 257, 24655-24660)，在类似的反应条件下，与 RNase A 结合的睾丸 RI 没有表现出这种特殊类型的氧化；相反，结合的 RI 具有中等氧化程度（每个 RI 部分最多可氧化 14 个硫醇），而不会与 RNase 解离。此外，与部分氧化的 RI 结合的 RNase 能够表达其部分（15%）潜在活性（活性复合物）。仅当 DTNB 处理导致复杂解离（每个 RI 部分氧化 > 14 个硫醇）时，释放的 RI 分子才会表现出全氧化或全无氧化行为。通过动力学和圆二色性分析，RI-RNase 复合物从非活性形式转变为活性形式的构象变化已得到证实。 RI-RNase 结合的松弛而不引起 RI 结构的重大改变被认为是复合物激活的原因。我们根据 RI 氧化还原状态介导的 RNase 活性可逆调节模型对结果进行了讨论。
Ribonuclease Inhibitor (RI) has been purified from pig testis. It contains 30 half-cystines whose oxidation affects its ability to bind and inhibit ribonuclease (RNase), By N-terminal sequence analyses testis RI showed to be identical to that from porcine liver, for which a characteristic all-or-none type of SH-oxidation by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) has been reported (Fominaya, J. M., and Hofsteenge, J. (1992) J. Biol. Chem. 257, 24655-24660), Under comparable reaction conditions, testis RI bound to RNase A did not exhibit this particular type of oxidation; instead, bound RI got intermediate oxidation degrees (up to 14 thiols oxidized per RI moiety) without dissociating from RNase. Moreover, RNase bound to partially oxidized RI was able to express some (15%) of its potential activity (active complex). Only when DTNB treatments accounted for complex dissociation (>14 thiols oxidized per RI moiety) the released RI molecules exhibited the all-or-none oxidation behavior. By both kinetic and circular dichroism analyses, conformational changes have been evidenced for the transition from the inactive to the active form of RI-RNase complex. Relaxation of RI-RNase binding without major alterations in RI structure is proposed as responsible for complex activation. The results are discussed in terms of a model for the reversible regulation of RNase activity mediated by the redox status of RI.