Katayama T, Ohtsuka T, Wakamura K, Yoshida K, Okamura N, Ishibashi S
Department of Physiological Chemistry, Hiroshima University School of Medicine, Japan.
Arch Biochem Biophys. 1990 May 1;278(2):431-6. doi: 10.1016/0003-9861(90)90281-3.
In an attempt to elucidate properties and activation mechanisms of the NADPH oxidase system, which is known to be responsible for the production of superoxide anion (O2-) in cell membranes of polymorphonuclear leukocytes (PMNL), intact guinea pig PMNL were treated with glutaraldehyde, a protein crosslinking reagent, before or after stimulation with phorbol 12-myristate 13-acetate (PMA). Then, PMNL were disrupted and NADPH oxidase activity was measured. After the treatment of resting PMNL with glutaraldehyde, NADPH oxidase was no longer activated by PMA. On the other hand, the NADPH oxidase activity enhanced by PMA in advance was markedly retained by the glutaraldehyde treatment of such PMA-stimulated PMNL as compared to that in untreated cells. Similar retention by glutaraldehyde of the stimulated NADPH oxidase activity was observed in PMNL stimulated by formyl-methionyl-leucyl-phenylalanine (FMLP) and cytochalasin D. Furthermore, the oxidase activity of glutaraldehyde-treated PMNL was stable during incubation at 37 degrees C, the half life of the oxidase activity of the treated PMNL being more than 90 min whereas that of the untreated PMNL is about 15 min. This ability of the glutaraldehyde treatment to retain the activity was also observed against inactivation by high concentrations of NaCl and by positively charged alkylamine.
为了阐明已知在多形核白细胞（PMNL）细胞膜中负责产生超氧阴离子（O2-）的NADPH氧化酶系统的特性和激活机制，在用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）刺激之前或之后，完整的豚鼠PMNL用蛋白质交联剂戊二醛处理。然后，破坏PMNL并测量NADPH氧化酶活性。用戊二醛处理静息的PMNL后，NADPH氧化酶不再被PMA激活。另一方面，与未处理的细胞相比，通过戊二醛处理预先被PMA增强的NADPH氧化酶活性在这种PMA刺激的PMNL中明显保留。在用甲酰甲硫氨酰亮氨酰苯丙氨酸（FMLP）和细胞松弛素D刺激的PMNL中观察到戊二醛对受刺激的NADPH氧化酶活性有类似的保留作用。此外，戊二醛处理的PMNL的氧化酶活性在37℃孵育期间是稳定的，处理过的PMNL的氧化酶活性的半衰期超过90分钟，而未处理的PMNL的半衰期约为15分钟。戊二醛处理保留活性的这种能力在抵抗高浓度NaCl和带正电荷的烷基胺的失活方面也观察到。
