Ho R J, Shi Q H, Ruiz J
Arch Biochem Biophys. 1986 Nov 15;251(1):148-55. doi: 10.1016/0003-9861(86)90061-5.
Forskolin-activated adenylate cyclases (AC) in intact membranes, solubilized with Lubrol or eluted following adsorption on a forskolin-Sepharose column, were examined for inhibition by GDP and GDP beta S. AC in intact membranes of rat or rabbit adipocytes was activated by 100 microM forskolin and further potentiated by 10 microM Gpp(NH)p in combination with either 230 microM epinephrine or 50 mU X ml-1 ACTH. GDP (0-1 mM) or GDP beta S (0-500 microM) inhibited activation in a dose-dependent manner to a level similar to or slightly below that produced by 100 microM forskolin alone. Forskolin at 100 microM stimulated solubilized AC of rabbit adipocytes and rat liver membranes for 10 +/- 4 to 160 +/- 10 and from 26 +/- 2 to 274 +/- 21 pmol(mg X min)-1, respectively, in the absence of GDP beta S; forskolin-activated activity decreased from 160 +/- 10 to 157 +/- 6 and from 274 +/- 21 to 238 +/- 14 pmol(mg X min)-1 in the presence of 500 microM GDP beta S. Forskolin-activated solubilized enzyme was further potentiated by 10 microM Gpp(NH)p from 160 +/- 10 to 289 +/- 52 and from 274 +/- 21 to 702 +/- 50 pmol(mg X min)-1. GDP beta S at 500 microM inhibited 93 and 103% of the Gpp(NH)p-potentiated activity. AC of rat adipocytes eluted from forskolin-Sepharose affinity column with 500 mM NaCl and 100 microM forskolin was not significantly activated by Gpp(NH)p nor inhibited by GDP beta S. However, it was activated by forskolin. The lack of inhibition of unmodified forskolin-activated activity by GDP or GDP beta S in contrast to the inhibition of Gpp(NH)p-activated enzyme or Gpp(NH)p-potentiated forskolin-activated enzyme may be a general phenomenon descriptive of the action of forskolin on AC. Furthermore, inhibition of forskolin-activated AC by GDP and its analog may be a useful index in analyzing the degree of guanine nucleotide potentiation of this enzyme.
用Lubrol溶解或吸附在福斯高林-琼脂糖柱上洗脱后的完整膜中，对福斯高林激活的腺苷酸环化酶（AC）进行了GDP和GDPβS抑制作用的检测。大鼠或兔脂肪细胞完整膜中的AC被100μM福斯高林激活，并在10μM Gpp(NH)p与230μM肾上腺素或50mU/ml促肾上腺皮质激素（ACTH）联合使用时进一步增强。GDP（0 - 1mM）或GDPβS（0 - 500μM）以剂量依赖性方式抑制激活，抑制水平与单独使用100μM福斯高林产生的水平相似或略低。在不存在GDPβS的情况下，100μM福斯高林分别刺激兔脂肪细胞和大鼠肝膜的可溶性AC，活性分别为10±4至160±10和26±2至274±21 pmol/（mg·min）-1；在存在500μM GDPβS的情况下，福斯高林激活的活性从160±10降至157±6，从274±21降至238±14 pmol/（mg·min）-1。10μM Gpp(NH)p使福斯高林激活的可溶性酶进一步增强，从160±10升至289±52，从274±21升至702±50 pmol/（mg·min）-1。500μM GDPβS抑制了93%和103%的Gpp(NH)p增强的活性。用500mM NaCl和100μM福斯高林从福斯高林-琼脂糖亲和柱上洗脱的大鼠脂肪细胞的AC，未被Gpp(NH)p显著激活，也未被GDPβS抑制。然而，它被福斯高林激活。与对Gpp(NH)p激活的酶或Gpp(NH)p增强的福斯高林激活的酶的抑制相反，GDP或GDPβS对未修饰的福斯高林激活的活性缺乏抑制作用，这可能是描述福斯高林对AC作用的普遍现象。此外，GDP及其类似物对福斯高林激活的AC的抑制作用可能是分析该酶鸟嘌呤核苷酸增强程度的有用指标。
