HORMONAL MODULATION OF BONE ADENYLATE CYCLASE

这项研究的长期目标是了解 糖皮质激素过量的机制，甲状腺 激素过多和雌激素缺乏导致骨量减少和 镁缺乏导致骨骼对吸收的抵抗 刺激物。在拟议的研究中，我们将检验这一假设 这些情况导致了对 骨腺苷环化酶。其中约三分之二 调查将涉及到对 腺苷环化酶存在于从骨骼中获得的膜中 正常成年豚鼠。这将需要：1)确定 甲状旁腺素、异丙肾上腺素和前列腺素E_2激动剂的作用 激活骨腺苷环化酶，在KA上为镁离子等 KI对高、低亲和力钙离子的抑制作用 先前在酶复合体中确定的位点；2) 酶复合体中镁离子结合部位的鉴定 发生结合激活；以及3)识别其中的位点 钙离子结合抑制腺苷酸的酶复合体 环化酶活性及其作用机制的研究 发生了钙离子抑制。重组方法论采用 火鸡红细胞和S49细胞系小鼠的质膜 淋巴瘤细胞和骨膜中的胆酸盐提取物 在后两项研究中使用。可能起到的作用 抑制性鸟嘌呤核苷酸调节蛋白，Ni，在 还将评估钙对腺苷环化酶的抑制作用。 骨膜将用百日咳毒素进行预处理，以 失活镍与钙抑制参数的关系 处理膜中腺苷环化酶活性的比较 控制膜。一旦获得了该基线数据， 腺苷酸环化酶的调节特性 对镁离子和激动剂的激活和钙离子的抑制作用 在接触过的动物的骨膜中进行检查 体内：1)糖皮质激素过多，2)甲状腺激素过多，3) 雌激素缺乏；4)镁缺乏。此外, 糖皮质激素或甲状腺激素过多和 雌激素对缺镁大鼠的相对量影响 刺激性和抑制性鸟嘌呤核苷酸调节蛋白 将测定骨膜中的NS和Ni。

The long-term objectives of this research is to understand the mechanisms by which glucocorticoid hormone excess, thyroid hormone excess and estrogen deficiency lead to osteopenia and magnesium deficiency leads to skeletal resistance to resorptive stimuli. In the proposed study we will test the hypothesis that these conditions induce changes in the regulatory properties of bone adenylate cyclase. Approximately two-thirds of this investigation will involve the detailed characterization of the adenylate cyclase present in membranes obtained from bones of normal adult guinea pigs. This will entail: 1) determination of the effects of PTH, isoproterenol, and prostaglandin E2, agonists which activate bone adenylate cyclase, on the Ka for Mg++ and on the Ki for calcium of the high and low affinity calcium inhibition sites previously identified in the enzyme complex; 2) identification of the sites within the enzyme complex where Mg++ binding activation occurs; and 3) identification of the sites within the enzyme complex where Ca++ binding inhibits adenylate cyclase activity and investigation of the mechanism by which Ca++ inhibition occurs. Reconstitution methodology employing plasma membranes from turkey erythrocytes and S49 cyc-murine lymphoma cells and cholate extracts from bone membranes will be used in these latter two studies. The possible role of the inhibitory guanine nucleotide regulatory protein, Ni, in the calcium inhibition of the adenylate cyclase will also be evaluated. Bone membranes will be pretreated with pertussis toxin to inactivate Ni and the parameters of calcium inhibition of the adenylate cyclase activity in the treated membranes compared to control membranes. Once this baseline data has been obtained, the regulatory characteristics of adenylate cyclase with respect to Mg++ and agonist activation and Ca++ inhibition will be examined in bone membranes obtained from animals exposed in vivo to: 1) glucocorticoid excess, 2) thyroid hormone excess, 3) estrogen deficiency, and 4) magnesium deficiency. In addition, the effects of glucocorticoid or thyroid hormone excess and estrogen on magnesium deficiency on the relative amounts of the stimulatory and inhibitory guanine nucleotide regulatory proteins, Ns and Ni, in the bone membranes will be determined.