The microheterogeneity of rabbit testosterone-binding globulin is due to differential glycosylation of its single protomer.
The microheterogeneity of rabbit testosterone-binding globulin is due to differential glycosylation of its single protomer.
复制标题
兔睾酮结合球蛋白的微观异质性是由于其单一原体的差异糖基化造成的。
DOI：
10.1095/biolreprod41.5.957
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发表时间：
1989
影响因子：
3.6
通讯作者：
Bell,BW
中科院分区：
文献类型：
--
作者：
Danzo,BJ;Black,JH;Bell,BW
亲和纯化的兔睾酮结合球蛋白（rbTeBG）是一种同二聚体，通过化学交联蛋白的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，其分子量（Mr）约为92，000。当对非交联rbTeBG进行SDS-PAGE时，分离出单个原聚体（Mr = 44，400 ± 400和Mr = 42，000 ± 1300）。原聚体以约2（重）：1（轻）的比例存在。对天然rbTeBG或已用[1，2 - 3 H]17β-羟基-4，6-雄甾二烯-3-酮进行光亲和标记的rbTeBG进行酶促去糖基化。然后使用与rbTeBG交叉反应的单克隆抗体进行免疫印迹或通过荧光法鉴定产物。这些分析表明，rbTeBG含有唾液酸和天冬酰胺（Asn）连接的寡糖，并提供了分子上存在丝氨酸/苏氨酸（O）连接聚糖的证据。假定通过酶或化学方法去除所有寡糖导致出现单个亚基（Mr为37，150 ± 1200）。基于该单体分子量，碳水化合物将分别占非去糖基化重亚基和轻亚基的相对分子量的16%和11%。因此，非去糖基化rbTeBG亚基的大小异质性是其差异糖基化的结果。除了大小异质性，rbTeBG亚基由多电荷变体组成。虽然酶和化学方法的聚糖去除改变了同工型的等电点，没有一个治疗产生一个单一的同工型。因此，有可能是寡糖以外的部分对rbTeBG的等电变体贡献电荷。
Affinity-purified rabbit testosterone-binding globulin (rbTeBG) is a homodimer with a molecular weight (Mr) of about 92,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the chemically cross-linked protein. When noncross-linked rbTeBG is subjected to SDS-PAGE, individual protomers (Mr≅ 44,400 ± 400 and Mr≅ 42,000 ± 1300) are resolved. The protomers are present in a ratio of approximately 2 (heavy):1 (light). Enzymatic deglycosylation of native rbTeBG or of rbTeBG that had been photoaffinity-labeled with [1,2-3H]17β-hydroxy-4,6-androstadien-3-one was conducted. The products were then identified on immunoblots using a monoclonal antibody that cross-reacts with rbTeBG, or by fluorography. These analyses indicated that rbTeBG contained sialic acid and asparagine (Asn)-linked oligosaccharides and provided evidence for the presence of serine/threonine (O)-linked glycans on the molecule. The presumptive removal of all oligosaccharides by enzymatic or chemical means resulted in the appearance of a single subunit (Mr≅ 37,150 ± 1200). On the basis of this monomeric molecular weight, carbohydrate would contribute 16% and 11% to the relative molecular mass of the nondeglycosylated heavy and light subunits, respectively. Therefore, the size heterogeneity of the nondeglycosylated rbTeBG subunits is a result of their differential glycosylation. In addition to size heterogeneity, the rbTeBG subunits are composed of multiple-charge variants. Although enzymatic and chemical methods of glycan removal altered the isoelectric points of the isoforms, none of the treatments yielded a single isoform. Thus, it is possible that moieties other than oligosaccharides are contributing charge to the isoelectric variants of rbTeBG.