IRREVERSIBLE INHIBITION OF CREATINE KINASE BY PEROXYNITRITE

肌酸激酶(CK)是一种重要的酶。 调节肌肉、脑和其他组织的能量代谢。 由于其活性中心中存在活性硫基， CK很容易被氧化剂灭活。过亚硝酸根是一种 超氧化物和一氧化氮反应的产物，即 在体内形成的多种细胞类型。我们用的是兔子的CK 肌肉，和3-吗啉基四氢呋喃亚胺(SIN-1)，它分解成 释放超氧化物和一氧化氮，形成过氧亚硝酸盐。SIN-1(100 微摩尔)在20min内使CK抑制到对照的1.9%，而硫醇 还原型谷胱甘肽(1 MM)和二硫苏糖醇(1 MM)修复 抑制酶活性分别为对照的22+/-4和17+/-2%， 分别进行了分析。而CK的活性则受到抑制。 S-亚硫醇和过氧化氢的完全回收 硫醇还原剂。CK与SIN-1和超氧化物歧化的孵育 歧化酶(500U/ml)可阻断SIN-1诱导的抑制信号 一氧化氮本身并不会使该酶失活。SIN-1诱导 一氧化氮供体对CK的抑制作用也可被阻止。 精胺-NONO(3-25微摩尔)，而盐酸精胺不是 有效。超氧阴离子自由基发生系统(黄嘌呤+黄嘌呤 氧化酶)适度抑制CK，并在此基础上加入一氧化氮 形成过氧亚硝酸根的混合培养物加剧了CK 抑制力。我们得出结论，过氧亚硝酸盐是一种主要不可逆的 CK的抑制剂，比超氧化物或氢更有效 过氧化氢。

Creatine kinase (CK) is an essential enzyme involved in the regulation of energy metabolism in muscle, brain, and other tissues. Because of the presence of reactive thiol groups in its active center, CK is vulnerable to inactivation by oxidants. Peroxynitrite is a product of the reaction between superoxide and nitric oxide which is formed in vivo in many cell types. We have used CK from rabbit muscle, and 3-morpholinosydnonimine (SIN-1) which decomposes to release superoxide and nitric oxide forming peroxynitrite. SIN-1 (100 micromole) inhibited CK to 1.9% of control within 20 min, and thiol reducing agents glutathione (1 mM) and dithiothreitol (1 mM) repaired the activity of inhibited enzymes to 22+/- 4 and 17+/- 2% of control, respectively. However, the activity of CK inhibited with S-nitrosothiols and hydrogen peroxide was recovered completely by thiol reducing agents. Incubation of CK with SIN-1 and superoxide dismutase (500 U/ml) prevented the SIN-1 induced inhibition indicating that nitric oxide itself did not inactivate the enzyme. SIN-1 induced inhibition of CK was also prevented by nitric oxide donor, spermineNONO (3-25 micromole), but spermine hydrochloride was not effective. Superoxide radical generating system (xanthine+xanthine oxidase) moderately inhibited CK, and addition of nitric oxide to this incubation mixture to form peroxynitrite exacerbated the CK inhibition. We conclude that peroxynitrite is a mainly irreversible inhibitor of CK, and is more potent than either superoxide or hydrogen peroxide.