Kurokawa T, Dan'ura T, Ishibashi S
J Pharmacobiodyn. 1984 Sep;7(9):665-70. doi: 10.1248/bpb1978.7.665.
Rat brain adenylate cyclase was solubilized with a combination of 0.7% sodium cholate and 0.6 M ammonium sulfate, and fractionated by addition of solid ammonium sulfate. The precipitate at 35% ammonium sulfate saturation contained neither guanine nucleotide-binding regulatory protein (G protein) nor calmodulin, and was used as the catalytic unit of the enzyme system. This catalytic unit was activated synergistically by forskolin and Mn2+. An apparent Km value for Mg-adenosine triphosphate (ATP) of the catalytic unit was about 80 microM in the basal state, while it increased in concurrence with the increase in the enzyme activity when forskolin was added to the assay system. The increase in the Km value depended on the forskolin concentration up to 1 microM, above which the value converged on ca. 200 microM. Furthermore, activation of the catalytic unit by forskolin was more marked at higher concentration of Mg-ATP. On the other hand, Mn2+ suppressed the increase in the Km value for Mg-ATP by forskolin, though the value in the basal state was not changed by Mn2+ alone. These findings indicate that the activation of the catalytic unit by forskolin is accompanied by the change in the affinity for Mg-ATP and Mn2+ modifies the change.
用0.7%胆酸钠和0.6 M硫酸铵的组合溶解大鼠脑腺苷酸环化酶，并通过添加固体硫酸铵进行分级分离。硫酸铵饱和度为35%时的沉淀物既不包含鸟嘌呤核苷酸结合调节蛋白（G蛋白）也不包含钙调蛋白，并被用作酶系统的催化单元。该催化单元被福斯高林和Mn2+协同激活。在基础状态下，催化单元对Mg-三磷酸腺苷（ATP）的表观Km值约为80 μM，而当向测定系统中添加福斯高林时，它随酶活性的增加而增加。Km值的增加取决于福斯高林浓度，直至1 μM，高于该浓度时该值趋于约200 μM。此外，在较高浓度的Mg-ATP下，福斯高林对催化单元的激活更为显著。另一方面，Mn2+抑制了福斯高林引起的Mg-ATP的Km值增加，尽管基础状态下的值不受单独的Mn2+影响。这些发现表明，福斯高林对催化单元的激活伴随着对Mg-ATP亲和力的变化，而Mn2+改变了这种变化。
