The quaternary structure of the plasma membrane b-type cytochrome of human granulocytes.
The quaternary structure of the plasma membrane b-type cytochrome of human granulocytes.
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DOI：
10.1016/0005-2728(88)90140-5
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发表时间：
1988
期刊：
影响因子：
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通讯作者：
Charles A. Parkos;Rodger A. Allen;Charles G. Cochrane;A. J. Jesaitis
中科院分区：
文献类型：
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作者：
Charles A. Parkos;Rodger A. Allen;Charles G. Cochrane;A. J. Jesaitis
对去污剂溶解的细胞色素 b 进行了流体动力学、交联和免疫沉淀研究，以证明分子量为 91 000（糖基化）和 22 000 [1, 2] 的两种共纯化多肽形成了分子复合物。流体动力学研究表明，细胞色素b洗涤剂复合物在Triton X-100中的沉降系数、部分比容和斯托克斯半径分别为5.25 S、0.82 cm 3/g和6.2 nm，在辛基葡萄糖苷中分别为6.05 S、0.80 cm 3/g和5.6 nm。这些研究还表明，Triton X-100 和辛基葡萄糖苷中的去污剂-蛋白质复合物的分子量分别为 202 和 188 kDa，形状不对称，摩擦系数为 1.3-1.4，并结合大量去污剂。去污剂-细胞色素复合物的蛋白质部分的分子量估计在 100 至 127 kDa 之间。用二琥珀酰亚胺基辛二酸酯和碱性可裂解的双[2-(琥珀酰亚胺氧基-羰氧基)乙基]砜进行交联研究表明，纯化的细胞色素b的M r= 91 000和M r= 22 000组分密切相关，并且可以共价结合形成多肽，通过SDS聚丙烯酰胺凝胶电泳，该多肽的M r 值为8% 和 11%(w/v) SDS 聚丙烯酰胺凝胶上分别为 110,000–120,000 和 120,000–135,000。交联物质的裂解导致 M r= 91 000 和 M r= 22 000 物质的重新出现。 H 2 O 中组成的线性蔗糖密度梯度中交联细胞色素 b 的沉降曲线与非交联对照的沉降曲线相同。通过对较小亚基特异的抗体也免疫沉淀较大亚基的能力，进一步证实了两种蛋白质种类的密切关联。旨在鉴定血红素携带亚基的实验尚无定论，因为复合物的解离导致细胞色素 b 光谱的损失。这些结果与我们的 SDS 聚丙烯酰胺凝胶电泳分子量估计相结合，为细胞色素 b 是由糖基化 M r= 91 000 和非糖基化 M r= 22 000 多肽组成的 α-β 型异二聚体提供了强有力的证据。
Hydrodynamic, crosslinking and immunoprecipitation studies were performed on detergent solubilized cytochrome b to demonstrate that the two copurifying polypeptides of molecular weight 91 000 (glycosylated) and 22 000 [1, 2] formed a molecular complex. The hydrodynamic studies indicated that the cytochrome b detergent complex had a sedimentation coefficient, partial specific volume and Stokes radius of 5.25 S, 0.82 cm 3/g and 6.2 nm in Triton X-100 and 6.05 S, 0.80 cm 3/g and 5.6 nm in octylglucoside, respectively. These studies also indicated that the detergent-protein complex has a molecular mass of 202 and 188 kDa in Triton X-100 and octylglucoside, respectively, is asymmetric in shape with a frictional coefficient of 1.3–1.4 and binds significant amounts of detergent. The molecular mass of the protein portion of the detergent-cytochrome complex was estimated to be between 100 and 127 kDa. Crosslinking studies with disuccinimidyl suberate and alkaline cleavable bis [2-(succinimidooxy-carbonyloxy) ethyl] sulfone revealed that the M r= 91 000 and M r= 22 000 components of purified cytochrome b are closely associated and can be covalently bound to form a polypeptide which, by SDS-polyacrylamide gel electrophoresis, has M r values of 110,000–120,000 and 120,000–135,000 on 8% and 11%(w/v) SDS-polyacrylamide gels, respectively. Cleavage of the crosslinked species resulted in the reappearance of the M r= 91 000 and M r= 22 000 species. Sedimentation profiles of crosslinked cytochrome b in linear sucrose density gradients made up in H 2 O were identical to those of non-crosslinked controls. A close association of the two protein species was further confirmed by the ability of antibody specific for the smaller subunit to immunoprecipitate the larger one also. Experiments aimed at identifying the heme-carring subunit (s) were inconclusive, since dissociation of the complex resulted in loss of cytochrome b spectrum. These results, in combination with our SDS-polyacrylamide gel electrophoresis molecular-weight estimates, provide strong evidence for the cytochrome b being an α-β-type heterodimer composed of a glycosylated M r= 91 000 and non-glycosylated M r= 22 000 polypeptide.