Schnefel S, Pröfrock A, Hinsch K D, Schulz I
Max Planck Institut für Biophysik, Frankfurt am Main, Federal Republic of Germany.
Biochem J. 1990 Jul 15;269(2):483-8. doi: 10.1042/bj2690483.
On separation of rat pancreatic plasma membrane proteins by two-dimensional gel electrophoresis, 15 GTP-binding protein (G-protein) alpha-subunits could be detected immunochemically using an alpha common antibody. These consisted of five 48 kDa proteins (pI 5.70, 5.80, 5.90, 6.10 and 6.25) and five 45 kDa proteins (pI 5.90, 6.05, 6.25, 6.30 and 6.70), presumably corresponding to low- and high-molecular mass forms of the Gs-protein, as well as three 40/41 kDa proteins (pI 5.50, 5.70 and 6.00) and two 39 kDa proteins (pI 5.50 and 6.00). All of these proteins except for the more acidic 39 kDa protein were ADP-ribosylated by cholera toxin (CT). In addition, the three 40/41 kDa proteins and the more alkaline 39 kDa protein were also ADP-ribosylated by pertussis toxin (PT). CT- and PT-induced ADP-ribosylation changed the pI values of G-protein alpha-subunits by 0.2 pI units to more acidic values. Preincubation of isolated pancreatic membranes with cholecystokinin octapeptide (CCK-OP), which stimulates phospholipase C in acinar cells, decreased CT-induced as well as PT-induced ADP-ribosylation of the three 40/41 kDa proteins, whereas CT-induced ADP-ribosylation of one 45 kDa (pI 5.80) and all 48 kDa proteins was enhanced in the presence of CCK. Carbachol, another stimulant of phospholipase C, had no effect. The three 40/41 kDa proteins and one 48 kDa protein could be labelled with the GTP analogue [alpha-32P]GTP-gamma-azidoanilide. CCK, but not carbachol, stimulated incorporation of the GTP analogue into all of these four proteins. Using different anti-peptide antisera specific for alpha-subunits of G-proteins we identified the three 40/41 kDa Gi-proteins as Gi1 (pI 6.00), Gi2 (pI 5.50) and Gi3 (pI 5.70). The Gi3-protein was found to be the major Gi-protein of pancreatic plasma membranes. One of the 39 kDa proteins (pI 6.0) was identified as Go. These results indicate that CCK receptors functionally interact with six Gs-proteins and with Gi1, Gi2 and Gi3-proteins. Since evidence suggests that a 40/41 kDa CT substrate is involved in the stimulation of phospholipase C in pancreatic acinar cells, it is likely that one, two or all three 40/41 kDa Gi-proteins are involved in the coupling of CCK receptors with phospholipase C.
用二维凝胶电泳分离大鼠胰腺质膜蛋白时，使用α通用抗体可通过免疫化学方法检测到15种GTP结合蛋白（G蛋白）α亚基。这些亚基包括5种48 kDa的蛋白（等电点分别为5.70、5.80、5.90、6.10和6.25）和5种45 kDa的蛋白（等电点分别为5.90、6.05、6.25、6.30和6.70），推测分别对应于Gs蛋白的低分子量和高分子量形式，还有3种40/41 kDa的蛋白（等电点分别为5.50、5.70和6.00）和2种39 kDa的蛋白（等电点分别为5.50和6.00）。除酸性更强的39 kDa蛋白外，所有这些蛋白都能被霍乱毒素（CT）进行ADP核糖基化。此外，3种40/41 kDa的蛋白和碱性更强的39 kDa蛋白也能被百日咳毒素（PT）进行ADP核糖基化。CT和PT诱导的ADP核糖基化使G蛋白α亚基的等电点值向更酸性方向改变0.2个等电点单位。用能刺激腺泡细胞中磷脂酶C的八肽胆囊收缩素（CCK-OP）对分离的胰腺膜进行预孵育，可降低3种40/41 kDa蛋白的CT诱导以及PT诱导的ADP核糖基化，而在CCK存在的情况下，一种45 kDa（等电点5.80）和所有48 kDa蛋白的CT诱导的ADP核糖基化增强。另一种磷脂酶C的刺激剂卡巴胆碱则无此作用。3种40/41 kDa的蛋白和一种48 kDa的蛋白可用GTP类似物[α-32P]GTP-γ-叠氮苯胺进行标记。CCK能刺激GTP类似物掺入所有这4种蛋白，而卡巴胆碱则不能。使用针对G蛋白α亚基的不同抗肽抗血清，我们鉴定出3种40/41 kDa的Gi蛋白分别为Gi1（等电点6.00）、Gi2（等电点5.50）和Gi3（等电点5.70）。发现Gi3蛋白是胰腺质膜的主要Gi蛋白。其中一种39 kDa的蛋白（等电点6.0）被鉴定为Go。这些结果表明CCK受体在功能上与6种Gs蛋白以及Gi1、Gi2和Gi3蛋白相互作用。由于有证据表明一种40/41 kDa的CT底物参与胰腺腺泡细胞中磷脂酶C的刺激，很可能1种、2种或所有3种40/41 kDa的Gi蛋白都参与CCK受体与磷脂酶C的偶联。
