Properties of the separated catalytic and regulatory units of brain adenylate cyclase.
Properties of the separated catalytic and regulatory units of brain adenylate cyclase.
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脑腺苷酸环化酶分离催化和调节单元的特性。
DOI：
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发表时间：
1980
影响因子：
11.1
通讯作者：
E. Neer
中科院分区：
文献类型：
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作者：
S. Strittmatter;E. Neer
用14 mM胆酸盐和1M(NH4)2SO4增溶牛脑皮质腺苷环化酶。Sepharose6B柱上的凝胶过滤将催化单元(CU)从给予5‘-鸟氨酰亚胺磷酸盐(p[NH]ppg)或氟化物的反应因子(G/F)中分离出来。分离出来的铜对p[NH]ppg或氟化物不敏感，当镁离子时相对不活跃，其Kav为0.48+/-0.01(n=5)。ATP是底物，但被Mn2+激活了8-15倍。分离的G/F洗脱峰面积为0.70+/-0.02(n=4)。它恢复了CU对p[NH]ppg和氟化物的反应性。在增溶前用p[NH]ppg激活该酶不会减少G/F的洗脱量，其Kav为0.7。因此，G/F在大脑皮层中的存在可能超过了CU。P[NH]PPG稳定G/F，但不稳定CU，表明它与G/F相互作用，而不与CU相互作用。在加入铜之前将G/F与p[NH]ppg孵育，显著提高了p[NH]ppg对重组酶的激活速度。因此，我们认为腺苷环化酶激活的限速步骤是一个单独在G/F中的过程，而不是CU的缓慢构象变化或G/F与CU的缓慢结合。P[NH]ppg与分离的G/F的结合似乎是可逆的；如果在CU之前加入GDP，则完全激活的G/F刺激CU的能力可以被阻断。相反，在CU通过与G/F的交互作用被激活后，GDP无法逆转这种激活。这表明与CU的缔合增加了G/F对p[NH]ppg的亲和力。
Adenylate cyclase from bovine brain cortex was solubilized with 14 mM cholate and 1 M (NH4)2SO4. Gel filtration over a column of Sepharose 6B separated the catalytic unit (CU) from a factor (G/F) that confers responsiveness to 5'-guanylyl imidophosphate (p[NH]ppG) or fluoride. The separated CU, which elutes with a Kav, of 0.48 +/- 0.01 (n=5), is not responsive to p[NH]ppG or fluoride and is relatively inactive when Mg . ATP is the substrate but activated 8-15-fold by Mn2+. The separated G/F elutes with a Kav of 0.70 +/- 0.02 (n=4). It restores the responsiveness of the CU to p[NH]ppG and fluoride. Activation of the enzyme by p[NH]ppG before solubilization does not decrease the amount of G/F eluting with a Kav of 0.7. Therefore, the G/F is probably present in brain cortex in excess over the CU. p[NH]ppG stabilizes the G/F but not the CU against thermal inactivation, suggesting that it interacts with G/F and not with CU. Incubation of the G/F with p[NH]ppG before addition of CU markedly increases the rate of activation of the reconstituted enzyme by p[NH]ppG. We propose, therefore, that the rate-limiting step in adenylate cyclase activation is a process in G/F alone and not a slow conformational change in CU or a slow association of G/F with CU. Binding of p[NH]ppG to the isolated G/F appears to be readily reversible; the ability of fully activated G/F to stimulate CU can be blocked if GDP is added before CU. In contrast, after the CU has been activated by interaction with G/F, GDP cannot reverse the activation. This suggests that association with the CU increases the affinity of G/F for p[NH]ppG.