McVey M, Hill J, Howlett A, Klein C
Departments of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA.
J Biol Chem. 1999 Jul 2;274(27):18887-92. doi: 10.1074/jbc.274.27.18887.
Nitric oxide (NO) donors inhibit hormone- and forskolin-stimulated adenylyl cyclase activity in purified plasma membrane preparations from N18TG2 neuroblastoma cells. Northern blot analyses indicate that the predominant isoform of adenylyl cyclase in N18TG2 cells is the type VI. Our experiments eliminate all the known regulatory proteins for this isoform as possible targets of NO. NO decreases the Vmax of the enzyme without altering the Km for ATP. Occupancy of the substrate-binding site protects the enzyme from the inhibitory effects of NO, suggesting that the conformation of the enzyme determines its sensitivity. The inhibition is reversed by reducing agents, implicating a Cys residue(s) as the target for nitric oxide and an S-nitrosylation as the underlying modification. These findings implicate NO as a novel cellular regulator of the type VI isoform of adenylyl cyclase.
一氧化氮（NO）供体可抑制来自N18TG2神经母细胞瘤细胞的纯化质膜制剂中激素和福斯可林刺激的腺苷酸环化酶活性。Northern印迹分析表明，N18TG2细胞中腺苷酸环化酶的主要同工型是VI型。我们的实验排除了该同工型的所有已知调节蛋白作为NO的可能靶点。NO降低了酶的Vmax，而不改变ATP的Km。底物结合位点的占据可保护酶免受NO的抑制作用，这表明酶的构象决定了其敏感性。还原剂可逆转这种抑制作用，这表明半胱氨酸残基是一氧化氮的作用靶点，而S-亚硝基化是潜在的修饰方式。这些发现表明NO是腺苷酸环化酶VI型同工型的一种新型细胞调节剂。
