Inaba K, Okuno M, Mohri H
Department of Biology, College of Arts and Sciences, University of Tokyo, Japan.
Arch Biochem Biophys. 1989 Oct;274(1):209-15. doi: 10.1016/0003-9861(89)90432-3.
We synthesized an anthraniloyl ATP (ant-ATP), which has a fluorescent anthraniloyl moiety at the OH group of ribose, to elucidate the mechanism of flagellar bend formation and its propagation in relation to the mechanochemical cycle of dynein ATPase. This fluorescent analog of ATP was efficiently hydrolyzed by 21 S dynein from sea urchin sperm flagella with Km = 7.6 microM, whereas the Km was 12 microM when ATP was used as the substrate. Similar Vmax values were obtained with both ATP and ant-ATP. Inhibition of the hydrolysis of ant-ATP by vanadate was a little smaller than that with ATP. Photosensitized cleavage of 21 S dynein heavy chains in the presence of ant-ATP and vanadate was also a little less efficient than that in the presence of ATP and vanadate. Ant-ATP also induced the disintegration of the trypsin-treated axoneme and the motility of demembranated sperm in a manner similar to ATP. When ATP was used as a substrate for the demembranated sperm, the apparent Michaelis constant for beat frequency (Km f) was 0.22 mM and the maximum frequency (fmax) was 36 Hz, whereas Km f) was 0.14 mM and fmax was 20 Hz for ant-ATP. Thus ant-ATP could be an efficient fluorescent analog of ATP for studying dynein ATPase and the mechanisms of flagellar motility.
我们合成了一种邻氨基苯甲酰基ATP（ant-ATP），其在核糖的OH基团处具有荧光邻氨基苯甲酰基部分，以阐明鞭毛弯曲形成及其与动力蛋白ATP酶的机械化学循环相关的传播机制。这种ATP的荧光类似物被海胆精子鞭毛中的21S动力蛋白有效水解，Km = 7.6 microM，而以ATP为底物时Km为12 microM。使用ATP和ant-ATP获得的Vmax值相似。钒酸盐对ant-ATP水解的抑制作用略小于对ATP的抑制作用。在ant-ATP和钒酸盐存在下，21S动力蛋白重链的光敏切割效率也略低于在ATP和钒酸盐存在下的效率。Ant-ATP还以类似于ATP的方式诱导胰蛋白酶处理的轴丝解体和去膜精子的运动。当ATP用作去膜精子的底物时，搏动频率的表观米氏常数（Km f）为0.22 mM，最大频率（fmax）为36 Hz，而ant-ATP的Km f为0.14 mM，fmax为20 Hz。因此，ant-ATP可能是用于研究动力蛋白ATP酶和鞭毛运动机制的一种有效的ATP荧光类似物。
