Pfister K K, Haley B E, Witman G B
J Biol Chem. 1984 Jul 10;259(13):8499-504.
Chlamydomonas 12 S dynein, which makes up part of the outer arm of the flagellar axoneme, consists of three polypeptides of 330,000, 22,000, and 18,000 daltons. We have used 8-azidoadenosine 5'-triphosphate (8-N3ATP), a photoaffinity analog of ATP, to investigate which of the dynein polypeptides contains the site of ATP hydrolysis. 8-N3ATP is a competitive inhibitor of the hydrolysis of ATP by 12 S dynein and is hydrolyzed by 12 S dynein in an ATP- and vanadate-sensitive fashion, indicating that it binds to the 12 S dynein hydrolytic site in the same way as ATP. When dynein was incubated with [gamma-32P]- or [alpha-32P]8-N3ATP in the presence of UV light to activate the azido moiety, the analog was incorporated into 12 S dynein's heavy polypeptide chain, but not its light chains. The incorporation was UV-dependent, was blocked by addition of ATP or vanadate plus ADP to the reaction mixture, and did not occur in heat-denatured dynein. These results strongly suggest that the hydrolytic site of 12 S dynein is contained in its heavy chain.
衣藻12 S动力蛋白是鞭毛轴丝外臂的组成部分，由分子量分别为330,000、22,000和18,000道尔顿的三种多肽组成。我们使用了8-叠氮腺苷5'-三磷酸（8-N3ATP），一种ATP的光亲和类似物，来研究动力蛋白的哪种多肽含有ATP水解位点。8-N3ATP是12 S动力蛋白水解ATP的竞争性抑制剂，并且以对ATP和钒酸盐敏感的方式被12 S动力蛋白水解，这表明它以与ATP相同的方式结合到12 S动力蛋白的水解位点。当在紫外光存在下将动力蛋白与[γ-32P]-或[α-32P]8-N3ATP一起孵育以激活叠氮部分时，该类似物被掺入12 S动力蛋白的重多肽链中，而不是其轻链中。这种掺入依赖于紫外光，通过向反应混合物中加入ATP或钒酸盐加ADP而被阻断，并且在热变性的动力蛋白中不发生。这些结果强烈表明12 S动力蛋白的水解位点包含在其重链中。
