Nieziołek Magdalena, Kot Marcin, Pyka Krzysztof, Mak Paweł, Kozik Andrzej
Faculty of Biotechnology, Jagiellonian University, Krakó, Poland.
Acta Biochim Pol. 2003;50(3):753-63.
Kininogens are multifunctional proteins involved in a variety of regulatory processes including the kinin-formation cascade, blood coagulation, fibrynolysis, inhibition of cysteine proteinases etc. A working hypothesis of this work was that the properties of kininogens may be altered by oxidation of their methionine residues by reactive oxygen species that are released at the inflammatory foci during phagocytosis of pathogen particles by recruited neutrophil cells. Two methionine-specific oxidizing reagents, N-chlorosuccinimide (NCS) and chloramine-T (CT), were used to oxidize the high molecular mass (HK) and low molecular mass (LK) forms of human kininogen. A nearly complete conversion of methionine residues to methionine sulfoxide residues in the modified proteins was determined by amino acid analysis. Production of kinins from oxidized kininogens by plasma and tissue kallikreins was significantly lower (by at least 70%) than that from native kininogens. This quenching effect on kinin release could primarily be assigned to the modification of the critical Met-361 residue adjacent to the internal kinin sequence in kininogen. However, virtually no kinin could be formed by human plasma kallikrein from NCS-modified HK. This observation suggests involvement of other structural effects detrimental for kinin production. Indeed, NCS-oxidized HK was unable to bind (pre)kallikrein, probably due to the modification of methionine and/or tryptophan residues at the region on the kininogen molecule responsible for the (pro)enzyme binding. Tests on papain inhibition by native and oxidized kininogens indicated that the inhibitory activity of kininogens against cysteine proteinases is essentially insensitive to oxidation.
激肽原是多功能蛋白质，参与多种调节过程，包括激肽形成级联反应、血液凝固、纤维蛋白溶解、抑制半胱氨酸蛋白酶等。本研究的一个工作假设是，在被招募的中性粒细胞吞噬病原体颗粒的炎症灶中释放的活性氧可通过氧化甲硫氨酸残基来改变激肽原的特性。使用两种甲硫氨酸特异性氧化试剂，N-氯代琥珀酰亚胺（NCS）和氯胺-T（CT），来氧化人激肽原的高分子量（HK）和低分子量（LK）形式。通过氨基酸分析确定修饰蛋白中甲硫氨酸残基几乎完全转化为甲硫氨酸亚砜残基。血浆和组织激肽释放酶从氧化的激肽原产生激肽的量显著低于（至少70%）从天然激肽原产生的量。这种对激肽释放的淬灭作用主要可归因于激肽原中与内部激肽序列相邻的关键Met-361残基的修饰。然而，人血浆激肽释放酶几乎不能从NCS修饰的HK形成激肽。这一观察结果表明存在其他对激肽产生有害的结构效应。事实上，NCS氧化的HK无法结合（前）激肽释放酶，可能是由于激肽原分子上负责（前）酶结合区域的甲硫氨酸和/或色氨酸残基发生了修饰。对天然和氧化激肽原抑制木瓜蛋白酶的测试表明，激肽原对半胱氨酸蛋白酶的抑制活性对氧化基本不敏感。
