Tamir A, Gill D M
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts.
J Neurochem. 1988 Jun;50(6):1791-7. doi: 10.1111/j.1471-4159.1988.tb02480.x.
We have developed a method to ADP-ribosylate the stimulatory guanine nucleotide-binding protein of adenylate cyclase (GS) in brain membranes by using cholera toxin. In particular, we used isonicotinic acid hydrazide and 3-acetylpyridine adenine dinucleotide to inhibit the potent NAD-glycohydrolase activity of brain membranes, and we used the detergent Triton X-100 (at 0.1%) to improve the accessibility of the toxin and guanine nucleotides used for supporting the ADP-ribosylation. This method reveals that GS is a very abundant protein in membranes derived from calf brain (approximately 30 pmol/mg of protein). In brain, GS exists in large excess over the previously reported amount of the adenylate cyclase catalytic subunit. The modification of GS with an ADP-ribosyl residue (a) elicits a four- to fivefold activation of adenylate cyclase by GTP, (b) increases the stabilization of adenylate cyclase by GTP, and (c) reduces adenylate cyclase activation by fluoride but does not change basal activity, activation by guanosine 5'-(beta, gamma-imido)triphosphate, or the sensitivity of adenylate cyclase to heat-induced denaturation. A correlation between ADP-ribosylation and the alterations in the activation of adenylate cyclase by guanine nucleotides and by fluoride is presented.
我们已开发出一种方法，通过使用霍乱毒素对脑膜中腺苷酸环化酶的刺激性鸟嘌呤核苷酸结合蛋白（GS）进行ADP核糖基化修饰。具体而言，我们使用异烟肼和3-乙酰吡啶腺嘌呤二核苷酸来抑制脑膜强大的NAD-糖水解酶活性，并使用去污剂Triton X-100（0.1%）来提高毒素和用于支持ADP核糖基化的鸟嘌呤核苷酸的可及性。该方法表明，GS是小牛脑膜中一种非常丰富的蛋白质（约30 pmol/mg蛋白质）。在脑中，GS的存在量大大超过先前报道的腺苷酸环化酶催化亚基的量。用ADP-核糖基残基对GS进行修饰：（a）可使GTP对腺苷酸环化酶的激活作用增强4至5倍；（b）增加GTP对腺苷酸环化酶的稳定作用；（c）降低氟化物对腺苷酸环化酶的激活作用，但不改变基础活性、5'-（β，γ-亚氨基）三磷酸鸟苷对腺苷酸环化酶的激活作用或腺苷酸环化酶对热诱导变性的敏感性。本文还阐述了ADP核糖基化与鸟嘌呤核苷酸和氟化物对腺苷酸环化酶激活作用改变之间的相关性。
