Coso O A, Díaz Añel A, Martinetto H, Muschietti J P, Kazanietz M, Fraidenraich D, Torres H N, Flawia M M
Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Buenos Aires, Argentina.
Biochem J. 1992 Oct 15;287 ( Pt 2)(Pt 2):443-6. doi: 10.1042/bj2870443.
A guanosine 5'-[gamma-[35S]thio]triphosphate-binding activity was detergent-extracted from Trypanosoma cruzi membranes. This binding activity was co-eluted from gel-filtration columns with a factor which, in a heterologous reconstitution system, blocks glucagon stimulation of adenylate cyclase activity in liver membranes. ADP-ribosylation of these membranes by pertussis toxin eliminated this blocking capacity. Incubation of T. cruzi membranes with activated pertussis toxin and [adenylate-32P]NAD+ led to the incorporation of radioactivity into a labelled product with an apparent M(r) of approx. 43,000. Crude membranes were electrophoresed on SDS/polyacrylamide gels and analysed, by Western blotting, with GA/1 anti-alpha common, AS/7 anti-alpha t, anti-alpha i1 and anti-alpha i2 polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 anti-beta antibody, of about 30 kDa, was also detected in the membrane fraction.
从克氏锥虫膜中用去污剂提取了一种鸟苷 5'-[γ-[³⁵S]硫代]三磷酸结合活性。这种结合活性在凝胶过滤柱上与一种因子共洗脱，该因子在异源重组系统中可阻断肝膜中胰高血糖素对腺苷酸环化酶活性的刺激。百日咳毒素对这些膜进行 ADP - 核糖基化消除了这种阻断能力。将克氏锥虫膜与活化的百日咳毒素和[腺苷酸 - ³²P]NAD⁺一起孵育，导致放射性掺入到一种表观分子量约为 43,000 的标记产物中。将粗膜在 SDS/聚丙烯酰胺凝胶上进行电泳，并用 GA/1 抗α通用、AS/7 抗αt、抗αi1 和抗αi2 多克隆抗体通过 Western 印迹法进行分析。这些步骤导致鉴定出一条约 43 kDa 的特异性多肽带。在膜组分中还检测到另一条与 SW/1 抗β抗体反应的约 30 kDa 的多肽。
