PURIFIED CYTOCHROME-B FROM HUMAN GRANULOCYTE PLASMA-MEMBRANE IS COMPRISED OF 2 POLYPEPTIDES WITH RELATIVE MOLECULAR-WEIGHTS OF 91,000 AND 22,000
PURIFIED CYTOCHROME-B FROM HUMAN GRANULOCYTE PLASMA-MEMBRANE IS COMPRISED OF 2 POLYPEPTIDES WITH RELATIVE MOLECULAR-WEIGHTS OF 91,000 AND 22,000
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DOI：
10.1172/jci113128
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发表时间：
1987-09-01
影响因子：
15.9
通讯作者：
JESAITIS, AJ
中科院分区：
文献类型：
--
作者：
PARKOS, CA;ALLEN, RA;JESAITIS, AJ
开发了一种从受刺激的人粒细胞中纯化细胞色素 b 的新方法，该方法具有从实际数量的全血中获得高产量的优点。用氟磷酸二异丙酯处理多形核白细胞，通过氮空化作用脱粒和破碎。通过差速离心制备富含细胞色素b的膜。 1-M 盐洗涤后，细胞色素从膜上完全溶解在辛基葡萄糖苷中。麦芽凝集素偶联的 Sepharose 4B 特异性结合溶解的细胞色素 b，并进行三重纯化。固定化麦芽凝集素的洗脱液通过固定化肝素色谱进一步富集。通过蔗糖密度梯度中的速度沉降，最终实现了 b 型细胞色素的 260 倍纯化，产率为 20-30%。纯化制品的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)揭示了Mr 91,000和Mr 22,000的两种多肽。用肽：N-糖苷酶F处理125I标记的纯化制剂，去除N-连接糖，将较大物种的相对分子量降低至约125I。 50,000，而β-消除（去除O-连接糖）对Mr-91,000 多肽的迁移率影响很小或没有影响。两种去糖基化条件均对 Mr-22,000 多肽的电泳迁移率没有任何影响。通过 SDS-PAGE，二琥珀酰亚胺基辛二酸酯将两种多肽交联成新的 120,000-135,000 Mr。纯化制剂产生免疫沉淀光谱活性的抗体，在蛋白质印迹中，与Mr-22,000多肽结合，但不与Mr-91,000多肽结合。对 X 连锁慢性肉芽肿病患者的粒细胞进行蛋白质印迹分析，结果显示 Mr-22,000 多肽完全缺失。这些结果 (a) 表明这两种多肽密切相关并且是细胞色素 b 的一部分，(b) 为之前报道的该蛋白质的分子量差异提供了解释，(c) 进一步支持细胞色素参与人中性粒细胞中超氧化物的产生。
A new method has been developed for purification of cytochrome b from stimulated human granulocytes offering the advantage of high yields from practical quantities of whole blood. Polymorphonuclear leukocytes were treated with diisopropylfluorophosphate, degranulated and disrupted by nitrogen cavitation. Membranes enriched in cytochrome b were prepared by differential centrifugation. Complete solubilization of the cytochrome from the membranes was achieved in octylglucoside after a 1-M salt wash. Wheat germ agglutinin-conjugated Sepharose 4B specifically bound the solubilized cytochrome b and afforded a threefold purification. Eluate from the immobilized wheat germ agglutinin was further enriched by chromatography on immobilized heparin. The final 260-fold purification of the b-type cytochrome with a 20-30% yield was achieved by velocity sedimentation in sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified preparation revealed two polypeptides of Mr 91,000 and Mr 22,000. Treatment of the 125I-labeled, purified preparation with peptide:N-glycosidase F, which removes N-linked sugars, decreased relative molecular weight of the larger species to .apprx. 50,000, whereas beta-elimination, which removes O-linked sugars, had little or no effect on the mobility of the Mr-91,000 polypeptide. Neither of the deglycosylation conditions had any effect on electrophoretic mobility of the Mr-22,000 polypeptide. Disuccinimidyl suberate cross-linked the two polypeptides to a new Mr of 120,000-135,000 by SDS-PAGE. Antibody raised to the purified preparation immunoprecipitated spectral activity and, on Western blots, bound to the Mr-22,000 polypeptide but not the Mr-91,000 polypeptide. Western blot analysis of granulocytes from patients with X-linked chronic granulomatous disease revealed a complete absence of the Mr-22,000 polypeptide. These results (a) suggest that the two polypeptides are in close association and are part of the cytochrome b, (b) provide explanation for the molecular weight discrepancies previously reported for the protein, and (c) further support the involvement of the cytochrome in superoxide production in human neutrophils.