Harhammer R, Nürnberg B, Spicher K, Schultz G
Institut für Pharmakologie, Universitätsklinikum Rudolf Virchow, Freie Universität Berlin, Germany.
Biochem Biophys Res Commun. 1994 Oct 28;204(2):835-40. doi: 10.1006/bbrc.1994.2535.
G13 belongs to the G12-subfamily of heterotrimeric regulatory G-proteins. Employing specific antibodies, we isolated G alpha 13 from bovine brain by a four-step purification protocol combining conventional and affinity chromatography. The use of ethylene glycol as a protective agent influenced the elution properties of G alpha 13 markedly. Only in the presence of ethylene glycol (30% v/v) a clear separation of G alpha 13 from other G-proteins was achieved during the initial anion exchange chromatography. This allowed isolation of G alpha 13 by subunit exchange chromatography on beta gamma-agarose. G alpha 13 was only released from immobilized beta gamma-dimers via activation by AMF but not by GTP gamma S, pointing to a poor basal nucleotide exchange of this protein. In contrast, N-terminally truncated G alpha 13 did not bind to immobilized beta gamma-dimers.
G13属于异源三聚体调节型G蛋白的G12亚家族。我们使用特异性抗体，通过结合传统色谱法和亲和色谱法的四步纯化方案，从牛脑中分离出Gα13。使用乙二醇作为保护剂对Gα13的洗脱特性有显著影响。仅在存在乙二醇（30% v/v）的情况下，在初始阴离子交换色谱过程中才能实现Gα13与其他G蛋白的清晰分离。这使得能够通过在βγ-琼脂糖上进行亚基交换色谱法分离Gα13。Gα13仅通过AMF激活而非GTPγS从固定化的βγ二聚体中释放出来，这表明该蛋白的基础核苷酸交换能力较差。相比之下，N端截短的Gα13不与固定化的βγ二聚体结合。
