Palmer T M, Houslay M D
Molecular Pharmacology Group, University of Glasgow, U.K.
Biochim Biophys Acta. 1991 Oct 21;1097(3):193-204. doi: 10.1016/0925-4439(91)90035-8.
Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of Gi alpha-2, Gi alpha-3 and G-protein beta-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of Gs alpha-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of Gs alpha-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 microM), GTP (100 microM), p[NH]ppG (100 microM), NaF (10 mM) and glucagon (10 microM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 microM) was lower by about 22%. Lower concentrations (EC50-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC50-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional Gi activity. The lower levels of expression of G-protein beta-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.
从遗传性糖尿病（db/db）小鼠制备的肝细胞膜中，Giα-2、Giα-3和G蛋白β亚基的表达水平与瘦小鼠膜中的水平相比，分别降低了约75%、63%和73%。相比之下，42 kDa和45 kDa形式的Gsα亚基的表达没有显著差异。百日咳毒素催化的瘦小鼠膜的ADP核糖基化鉴定出一条单一的41 kDa条带，其在糖尿病小鼠膜中的标记减少了约86%。霍乱毒素催化的ADP核糖基化鉴定出两种形式的Gsα亚基，其在糖尿病小鼠膜中的标记比瘦小鼠膜中的标记大约高4倍。福斯高林（100 μM）、GTP（100 μM）、p[NH]ppG（100 μM）、NaF（10 mM）和胰高血糖素（10 μM）对腺苷酸环化酶活性的最大刺激在瘦小鼠和糖尿病小鼠的膜中相似，而异丙肾上腺素（100 μM）的刺激降低了约22%。与瘦小鼠膜相比，糖尿病小鼠膜中激活腺苷酸环化酶需要更低浓度（EC50 - 60 nM）的p[NH]ppG（瘦小鼠膜的EC50 - 158 nM）。除了引起激活外，p[NH]ppG还能够对瘦小鼠和糖尿病小鼠膜中福斯高林刺激的腺苷酸环化酶活性产生百日咳毒素敏感的抑制作用。然而，糖尿病小鼠膜中腺苷酸环化酶活性的最大抑制降低到瘦小鼠膜的约60%。体内百日咳毒素处理通过一种被认为是由功能性Gi活性的消除介导的过程增强了胰高血糖素、异丙肾上腺素和p[NH]ppG对腺苷酸环化酶的最大刺激。与瘦小鼠相比，糖尿病小鼠膜中G蛋白β亚基的表达水平较低，这被认为会扰乱全聚体和解离的G蛋白亚基之间的平衡。我们认为，这可能解释了糖尿病小鼠膜中腺苷酸环化酶对p[NH]ppG刺激的敏感性增强，以及百日咳毒素和霍乱毒素催化这两种动物膜中G蛋白ADP核糖基化的能力改变。
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