Kutchai H, Geddis L M, Otero A S
Department of Physiology, University of Virginia, Charlottesville.
Biochim Biophys Acta. 1993 Jan 17;1175(2):140-6. doi: 10.1016/0167-4889(93)90016-i.
Direct photoaffinity labeling of proteins of longitudinal sarcoplasmic reticulum (LSR) of rabbit skeletal muscle with [32P]GTP revealed GTP-binding proteins of about 52, 45 and 30 kDa. ADP-ribosylation with [32P]NAD in the presence of cholera toxin (CTX) or pertussis toxin (PTX) indicates the existence of a CTX substrate (about 45 kDa); no PTX substrates were observed. Western blots of LSR probed with RM/1, an antiserum against a decapeptide from the C-terminus of Gs alpha, showed an immunoreactive band at about 45 kDa. [32P]GTP overlays of Western blots of LSR showed a heavily-labeled protein of about 29 kDa and one or more additional slightly smaller proteins that were more weakly labeled. When LSR was subjected to mild trypsin hydrolysis, the Western blot overlay revealed three bands at about 23, 25 and 29 kDa. Western blots of LSR proteins showed no significant immunoreactivity with the anti-(pan)-ras monoclonal antibodies 142-24E05 and Ras 11. ADP-ribosylation of LSR with [32P]NAD in the presence of C3 exoenzyme of Clostridium botulinum yielded a labeled band at about 23 kDa. Our results indicate the presence in rabbit LSR of a Gs alpha, the absence of Gi and G(o), and the presence of several low molecular weight GTP-binding proteins, distinct from p21 ras, one of which belongs to the rho or rac family.
用[32P]GTP对兔骨骼肌纵行肌浆网（LSR）的蛋白质进行直接光亲和标记，揭示了约52、45和30 kDa的GTP结合蛋白。在霍乱毒素（CTX）或百日咳毒素（PTX）存在下用[32P]NAD进行ADP核糖基化表明存在一种CTX底物（约45 kDa）；未观察到PTX底物。用RM/1（一种针对Gsα C末端十肽的抗血清）探测LSR的蛋白质免疫印迹显示在约45 kDa处有一条免疫反应带。LSR蛋白质免疫印迹的[32P]GTP覆盖显示一条约29 kDa的强标记蛋白和一条或多条稍小的、标记较弱的额外蛋白。当LSR进行轻度胰蛋白酶水解时，蛋白质免疫印迹覆盖显示在约23、25和29 kDa处有三条带。LSR蛋白质的免疫印迹与抗（泛）ras单克隆抗体142 - 24E05和Ras 11没有显著免疫反应性。在肉毒梭菌C3外切酶存在下用[32P]NAD对LSR进行ADP核糖基化产生一条约23 kDa的标记带。我们的结果表明兔LSR中存在Gsα，不存在Gi和G(o)，并且存在几种与p21 ras不同的低分子量GTP结合蛋白，其中一种属于rho或rac家族。
