Toutant M, Barhanin J, Bockaert J, Rouot B
Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France.
Biochem J. 1988 Sep 1;254(2):405-9. doi: 10.1042/bj2540405.
In muscle, it has been established that guanosine 5'-[gamma-thio]triphosphate (GTP[S]), a non-hydrolysable GTP analogue, elicits a rise in tension in chemically skinned fibres, and that pretreatment with Bordetella pertussis toxin (PTX) decreases GTP[S]-induced tension development [Di Virgilio, Salviati, Pozzan & Volpe (1986) EMBO J. 5, 259-262]. In the present study, G-proteins were analysed by PTX-catalysed ADP-ribosylation and by immunoblotting experiments at cellular and subcellular levels. First, the nature of the G-proteins present in neural and aneural zones of rat diaphragm muscle was investigated. PTX, known to catalyse the ADP-ribosylation of the alpha subunit of several G-proteins, was used to detect G-proteins. Three sequential extractions (low-salt-soluble, detergent-soluble and high-salt-soluble) were performed, and PTX was found to label two substrates of 41 and 40 kDa only in the detergent-soluble fraction. The addition of pure beta gamma subunits of G-proteins to the low-salt-soluble extract did not provide a way to detect PTX-catalysed ADP-ribosylation of G-protein alpha subunits in this hydrophilic fraction. In neural as well as in aneural zones, the 39 kDa PTX substrate, very abundant in the nervous system (Go alpha), was not observed. We then studied the nature of the G alpha subunits present in membranes from transverse tubules (T-tubules) purified from rabbit skeletal muscle. Only one 40 kDa PTX substrate was found in T-tubules, known to be the key element of excitation-contraction coupling. The presence of a G-protein in T-tubule membranes was further confirmed by the immunoreactivity detected with an anti-beta-subunit antiserum. A 40 kDa protein was also detected in T-tubule membranes with an antiserum raised against a purified bovine brain Go alpha. The presence of two PTX substrates (41 and 40 kDa) in equal amounts in total muscle extracts, compared with only one (40 kDa) found in purified T-tubule membranes, suggests that this 40 kDa PTX substrate might be involved in excitation-contraction coupling.
在肌肉中，已经证实，鸟苷5'-[γ-硫代]三磷酸（GTP[S]），一种不可水解的GTP类似物，可引起化学去皮纤维中的张力升高，并且用百日咳博德特氏菌毒素（PTX）预处理可降低GTP[S]诱导的张力发展[迪·维尔吉利奥、萨尔维亚蒂、波赞和沃尔佩（1986年）《欧洲分子生物学组织杂志》5，259 - 262]。在本研究中，通过PTX催化的ADP核糖基化以及在细胞和亚细胞水平的免疫印迹实验对G蛋白进行了分析。首先，研究了大鼠膈肌神经区和非神经区中存在的G蛋白的性质。已知PTX可催化几种G蛋白α亚基的ADP核糖基化，用于检测G蛋白。进行了三次连续提取（低盐可溶性、去污剂可溶性和高盐可溶性），发现PTX仅在去污剂可溶性部分标记了41 kDa和40 kDa的两种底物。向低盐可溶性提取物中添加G蛋白的纯βγ亚基并不能提供一种方法来检测该亲水性部分中G蛋白α亚基的PTX催化的ADP核糖基化。在神经区和非神经区，均未观察到在神经系统中非常丰富的39 kDa PTX底物（Goα）。然后，我们研究了从兔骨骼肌纯化的横管（T管）膜中存在的Gα亚基的性质。在已知为兴奋 - 收缩偶联关键元件的T管中仅发现一种40 kDa的PTX底物。用抗β亚基抗血清检测到的免疫反应性进一步证实了T管膜中存在G蛋白。用针对纯化的牛脑Goα产生的抗血清在T管膜中也检测到了一种40 kDa的蛋白质。与在纯化的T管膜中仅发现一种（40 kDa）相比，在总肌肉提取物中等量存在两种PTX底物（41 kDa和40 kDa），这表明这种40 kDa的PTX底物可能参与兴奋 - 收缩偶联。
