IRREVERSIBLE INHIBITION OF CREATINE KINASE BY PEROXYNITRITE

肌酸激酶（CK）是参与心肌细胞凋亡的一种必需酶。 调节肌肉、大脑和其他组织的能量代谢。 由于在其活性中心存在反应性硫醇基团， CK易被氧化剂灭活。 过氧亚硝酸盐是一种 超氧化物和一氧化氮反应的产物， 在许多细胞类型中形成。 我们用的是兔子的CK 肌肉，和3-吗啉基丙酮亚胺（SIN-1），其分解为 释放超氧化物和一氧化氮形成过氧亚硝酸盐。 SIN-1（100 20 min内，巯基对CK的抑制率为对照的1.9%， 还原剂谷胱甘肽（1 mM）和二硫苏糖醇（1 mM）修复 抑制酶的活性分别为对照的22+/-4和17+/-2%， 分别 而CK活性则受 S-亚硝基硫醇和过氧化氢被完全回收， 硫醇还原剂。 CK与SIN-1和超氧化物的孵育 歧化酶（500 U/ml）阻止SIN-1诱导的抑制，表明 一氧化氮本身并不能抑制酶。 SIN-1诱导 一氧化氮供体也可阻止CK抑制， 精胺NONO（3-25微摩尔），但盐酸精胺不是 有效 超氧自由基生成系统（黄嘌呤+黄嘌呤 氧化酶）中度抑制CK，并加入一氧化氮， 孵育混合物形成过氧亚硝酸盐加重CK 抑制作用 我们得出结论，过氧亚硝酸盐是主要不可逆的 CK的抑制剂，并且比超氧化物或氢更有效 过氧化物

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.