The enzymic reduction and kinetics of oxidation of cytochrome b-245 of neutrophils.
The enzymic reduction and kinetics of oxidation of cytochrome b-245 of neutrophils.
复制标题
中性粒细胞细胞色素 b-245 的酶还原和氧化动力学。
作者：
A. Cross;F. K. Higson;O. Jones;A. Harper;A. Segal
1.测定人中性粒细胞质膜还原-氧化细胞色素b-245的吸收系数[δε（mM；559-540 nm）= 21.6 cm-1]。 2. 中性粒细胞多形核白细胞（中性粒细胞）由人血、牛血、马血和猪血制备。在每种情况下，发现质膜部分都含有低电位细胞色素 b。当马中性粒细胞膜与 NADH 或 NADPH 厌氧孵育时，细胞色素 b 减少。先前用佛波醇肉豆蔻酸酯乙酸酯刺激细胞并没有增加分离膜中细胞色素b的减少速率或程度，但确实增加了Triton处理的细胞中NADPH的减少速度和程度。 3.细胞色素b也存在于人中性粒细胞的特定颗粒部分中。发现其 Em (pH 7.0) 为 -248 mV，与质膜细胞色素 b 非常相似。 4.使用停流技术测定空气饱和缓冲液对还原细胞色素b-245的氧化速率。在完整的膜中，氧化的 t 1/2 为 4.7 ms。该速率足够快，足以支持细胞色素 b-245 是中性粒细胞呼吸爆发中的氧化酶的观点。 5. 人中性粒细胞的质膜细胞色素 b 与 CO 形成复合物。在室温和 1 atm 的 CO 条件下，大约40%的细胞色素形成复合物；约。 60% 结合是在 5 摄氏度下溶解的 CO 浓度增加时测量的。产生 50% 结合的 CO 浓度为 1.18 mM。
1. The absorption coefficient of human neutrophil plasma-membrane reduced-minus-oxidized cytochrome b-245 was determined [delta epsilon (mM; 559-540 nm) = 21.6 cm-1]. 2. Neutrophil polymorphonuclear leucocytes (neutrophils) were prepared from human, ox, horse and pig blood. In each case plasma-membrane fractions were found to contain low-potential cytochrome b. When membranes from horse neutrophils were incubated anaerobically with either NADH or NADPH the cytochrome b became reduced. Prior stimulation of the cells with phorbol myristate acetate did not increase the rate or extent of cytochrome b reduction in isolated membranes, but did increase both the rate and extent of reduction by NADPH in Triton-treated cells. 3. A cytochrome b was present also in the specific granule fraction of human neutrophils. Its Em (pH 7.0) was found to be -248 mV, very similar to that of the plasma-membrane cytochrome b. 4. The rate of oxidation of reduce cytochrome b-245 by air-saturated buffer, was determined by using stopped-flow techniques. In intact membranes t 1/2 for oxidation was 4.7 ms. This rate is sufficiently rapid to support the view that cytochrome b-245 is the oxidase in the respiratory burst of neutrophils. 5. Plasma-membrane cytochrome b of human neutrophils formed a complex with CO. At room temperature and 1 atm of CO approx. 40% of the cytochrome formed a complex; approx. 60% binding was measured at the increased concentration of dissolved CO achieved at 5 degrees C. The concentration of CO giving 50% binding was 1.18 mM.