Mac Neil S, Walker S W, Senior H J, Pollock A, Brown B L, Bleehen S S, Munro D S, Tomlinson S
Biochem J. 1984 Dec 1;224(2):453-60. doi: 10.1042/bj2240453.
Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.
钙调蛋白拮抗剂可抑制培养的小鼠B16黑色素瘤细胞及其细胞裂解物中激素刺激的环磷酸腺苷（cAMP）积累。B16黑色素瘤的微粒体制剂含有细胞总钙调蛋白的34%-45%，无论缓冲液中是否存在乙二醇双四乙酸（EGTA），通过大量洗涤都无法使其解离。这种制剂中的腺苷酸环化酶活性不受外源钙调蛋白添加的影响。然而，在许多系统中可以模拟或替代钙离子（Ca2+）的稀土金属离子镧离子（La3+）可立即抑制激动剂刺激的腺苷酸环化酶活性，微粒体制剂用La3+预孵育，然后用不含La3+的缓冲液洗涤，可使钙调蛋白从微粒体制剂中解离（损失96%）。钙调蛋白从微粒体制剂中的丢失与激动剂反应性降低（74%）以及该酶对Ca2+敏感性的显著变化有关，低浓度的钙（约10 nM）现在无法刺激酶活性，高浓度的钙（大于或等于100 nM）对酶活性产生比正常情况更大的抑制作用。在这种钙调蛋白耗尽的微粒体制剂中，现在可以证明通过添加纯钙调蛋白直接激活腺苷酸环化酶。在存在10 μM游离Ca2+的情况下，激动剂反应性腺苷酸环化酶的半最大刺激发生在80 nM钙调蛋白时。钙调蛋白（在300-600 nM时）的最大刺激使酶活性恢复到未处理的、钙调蛋白完整的制剂中活性的89±5%（平均值±标准误，n = 7）。
