Okamoto T, Akaike T, Nagano T, Miyajima S, Suga M, Ando M, Ichimori K, Maeda H
Department of Microbiology, Kumamoto University School of Medicine, Japan.
Arch Biochem Biophys. 1997 Jun 15;342(2):261-74. doi: 10.1006/abbi.1997.0127.
The involvement of nitric oxide (NO) and its reactive intermediates such as nitrogen dioxide (NO2) and peroxynitrite (ONOO-) in the activation of matrix metallo-proteinase was investigated. The human neutrophil procollagenase (matrix metalloproteinase-8) (M(r), 85 kDa) was purified to homogeneity from human neutrophils by using column chromatography. After incubation of human neutrophil procollagenase with various nitrogen oxide-generating systems, collagenolytic activity in each reaction system was measured. In addition, neutrophil collagenase activity was determined by assessment of proteolysis of human alpha 1-protease inhibitor. NO was formed by the propylamine NONOate, and NO2 was generated by oxidation of NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). NO2, formed by NONOate and carboxy-PTIO, and the synthetic ONOO- exhibited strong activation of the procollagenase at 1-20 microM. Significant activation of the procollagenase was observed with use of authentic NO2 gas as well. Constant flux infusion of ONOO- into the procollagenase solution resulted in stronger procollagenase activation than did a bolus addition of ONOO- to the reaction mixture. However, NO showed only weak activating potential under the aerobic (ambient) condition; an NO concentration of more than 10 mM was needed for appreciable activation of the procollagenase. Of considerable importance was the fact that NO participates in activation of the neutrophil collagenase through its conversion to NO2 or ONOO- in human neutrophils. These results suggest that NO2 and ONOO- may be potent activators of human neutrophil procollagenase.
研究了一氧化氮（NO）及其反应性中间体如二氧化氮（NO₂）和过氧亚硝酸盐（ONOO⁻）在基质金属蛋白酶激活中的作用。通过柱色谱法从人中性粒细胞中纯化人中性粒细胞原胶原酶（基质金属蛋白酶-8）（M(r)，85 kDa）至同质。将人中性粒细胞原胶原酶与各种氮氧化物生成系统孵育后，测量每个反应系统中的胶原分解活性。此外，通过评估人α1-蛋白酶抑制剂的蛋白水解来测定中性粒细胞胶原酶活性。丙胺亚硝基酯生成NO，NO用2-(4-羧基苯基)-4,4,5,5-四甲基咪唑啉-1-氧基3-氧化物（羧基-PTIO）氧化生成NO₂。由亚硝基酯和羧基-PTIO生成的NO₂以及合成的ONOO⁻在1-20μM时对原胶原酶表现出强烈的激活作用。使用纯NO₂气体也观察到原胶原酶的显著激活。将ONOO⁻持续通量注入原胶原酶溶液比向反应混合物中一次性加入ONOO⁻导致更强的原胶原酶激活。然而，在有氧（环境）条件下，NO仅显示出较弱的激活潜力；需要超过10 mM的NO浓度才能明显激活原胶原酶。相当重要的是，在人中性粒细胞中，NO通过转化为NO₂或ONOO⁻参与中性粒细胞胶原酶的激活。这些结果表明，NO₂和ONOO⁻可能是人中性粒细胞原胶原酶的有效激活剂。
