Mullaney I, Magee A I, Unson C G, Milligan G
Department of Biochemistry, University of Glasgow, Scotland, U.K.
Biochem J. 1988 Dec 1;256(2):649-56. doi: 10.1042/bj2560649.
Incubation of the neuroblastoma x glioma hybrid cell line NG108-15 in tissue culture with dibutyryl cyclic AMP (1 mM) for up to 8 days produced a morphological differentiation of the cells, during which they extended neurite-like processes. Pertussis-toxin-catalysed ADP-ribosylation indicated that amounts of guanine-nucleotide-binding proteins (G-proteins), which are substrates for this toxin, were approximately doubled in membranes from the 'differentiated' cells in comparison with the control cells. Immunoblotting of membranes derived from either untreated or dibutyryl cyclic AMP-treated cells with anti-peptide antisera specific for the alpha subunits of the pertussis-toxin-sensitive G-proteins Gi and Go demonstrated that amounts of these G-proteins were reciprocally modulated during the differentiation process. In comparison with the untreated cells, the amount of Gi in the 'differentiated' cells was decreased, whereas the amount of Go was substantially increased. Stimulation of high-affinity GTPase activity in response to opioid peptides, which in this cell line interact with an opioid receptor of the delta subclass, was much decreased, and inhibition of adenylate cyclase activity was almost entirely attenuated in the 'differentiated'-cell membranes in comparison with membranes of untreated cells. Opioid receptor number was also decreased in membranes of the dibutyryl cyclic AMP-treated cells in comparison with the control cells. These data demonstrate that relatively small changes in the observed pattern of pertussis-toxin-catalysed ADP-ribosylation of membranes can mask more dramatic alterations in amounts of the individual pertussis-toxin-sensitive G-proteins, and further demonstrate the importance of methodologies able to discriminate between the different gene products.
将神经母细胞瘤x胶质瘤杂交细胞系NG108 - 15在组织培养中与二丁酰环磷腺苷（1 mM）孵育长达8天，可使细胞发生形态分化，在此期间细胞伸出神经突样突起。百日咳毒素催化的ADP核糖基化表明，作为该毒素底物的鸟嘌呤核苷酸结合蛋白（G蛋白）的量，与对照细胞相比，“分化”细胞的膜中大约增加了一倍。用针对百日咳毒素敏感的G蛋白Gi和Go的α亚基的抗肽抗血清对未处理或经二丁酰环磷腺苷处理的细胞衍生的膜进行免疫印迹分析表明，这些G蛋白的量在分化过程中相互调节。与未处理的细胞相比，“分化”细胞中Gi的量减少，而Go的量显著增加。在该细胞系中，与δ亚类阿片受体相互作用的阿片肽刺激的高亲和力GTP酶活性大大降低，与未处理细胞的膜相比，“分化”细胞膜中腺苷酸环化酶活性的抑制几乎完全减弱。与对照细胞相比，经二丁酰环磷腺苷处理的细胞的膜中阿片受体数量也减少。这些数据表明，膜上百日咳毒素催化的ADP核糖基化观察模式中相对较小的变化可以掩盖单个百日咳毒素敏感G蛋白量的更显著变化，并进一步证明了能够区分不同基因产物的方法的重要性。
