Saari L L, Pope M R, Murrell S A, Ludden P W
J Biol Chem. 1986 Apr 15;261(11):4973-7.
Removal of ADP-ribose from the iron protein of nitrogenase by activating enzyme resulted in the activation of the inactive iron protein. A radioassay that directly measured the initial velocity of the activation was developed using iron protein radiolabeled with either [8-3H]- or [G-32P]ADP-ribose. The release of radiolabeled ADP-ribose by activating enzyme was linearly correlated with the increase in the specific activity of the iron protein as measured by acetylene reduction. Both ATP and MnCl2 were required for the activation of inactive iron protein. The optimal ratio of [MnCl2]/[ATP] in the radioassay was 2:1, and the optimal concentrations were 4 mM and 2 mM for [MnCl2] and [ATP], respectively. The Km for inactive iron protein was 74 microM and the Vmax was 628 pmol of [32P] ADP-ribose released min-1 microgram of activating enzyme-1. Adenosine, cytidine, guanosine, or uridine mono-, di-, or triphosphates did not substitute for ATP in the activation of native iron protein. Activating enzyme removed ADP-ribose from oxygen-denatured iron protein in the absence of ATP. ADP, ADP-ribose, pyrophosphate, and high concentrations of NaCl inhibited activating enzyme activity.
通过激活酶从固氮酶的铁蛋白中去除ADP-核糖，导致无活性的铁蛋白被激活。利用用[8-3H]-或[G-32P]ADP-核糖放射性标记的铁蛋白，开发了一种直接测量激活初始速度的放射性测定法。激活酶释放放射性标记的ADP-核糖与通过乙炔还原测量的铁蛋白比活性的增加呈线性相关。无活性铁蛋白的激活需要ATP和MnCl2。放射性测定中[MnCl2]/[ATP]的最佳比例为2:1，[MnCl2]和[ATP]的最佳浓度分别为4 mM和2 mM。无活性铁蛋白的Km为74 microM，Vmax为每分钟每微克激活酶释放628 pmol的[32P]ADP-核糖。腺苷、胞苷、鸟苷或尿苷的单磷酸、二磷酸或三磷酸在天然铁蛋白的激活中不能替代ATP。在没有ATP的情况下，激活酶从氧变性的铁蛋白中去除ADP-核糖。ADP、ADP-核糖、焦磷酸和高浓度的NaCl抑制激活酶活性。
