Shpetner H S, Paschal B M, Vallee R B
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
J Cell Biol. 1988 Sep;107(3):1001-9. doi: 10.1083/jcb.107.3.1001.
We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro in the direction corresponding to retrograde transport. (Paschal, B. M., H. S. Shpetner, and R. B. Vallee. 1987b. J. Cell Biol. 105:1273-1282; Paschal, B. M., and R. B. Vallee. 1987. Nature [Lond.]. 330:181-183.). Biochemical analysis of this protein (op. cit.) as well as scanning transmission electron microscopy revealed that MAP 1C is a brain cytoplasmic form of the ciliary and flagellar ATPase dynein (Vallee, R. B., J. S. Wall, B. M. Paschal, and H. S. Shpetner. 1988. Nature [Lond.]. 332:561-563). We have now characterized the ATPase activity of the brain enzyme in detail. We found that microtubule activation required polymeric tubulin and saturated with increasing tubulin concentration. The maximum activity at saturating tubulin (Vmax) varied from 186 to 239 nmol/min per mg. At low ionic strength, the Km for microtubules was 0.16 mg/ml tubulin, substantially lower than that previously reported for axonemal dynein. The microtubule-stimulated activity was extremely sensitive to changes in ionic strength and sulfhydryl oxidation state, both of which primarily affected the microtubule concentrations required for half-maximal activation. In a number of respects the brain dynein was enzymatically similar to both axonemal and egg dyneins. Thus, the ATPase required divalent cations, calcium stimulating activity less effectively than magnesium. The MgATPase was inhibited by metavandate (Ki = 5-10 microM for the microtubule-stimulated activity), 1 mM NEM, and 1 mM EHNA. In contrast to other dyneins, the brain enzyme hydrolyzed CTP, TTP, and GTP at higher rates than ATP. Thus, the enzymological properties of the brain cytoplasmic dynein are clearly related to those of other dyneins, though the brain enzyme is unique in its substrate specificity and in its high sensitivity to stimulation by microtubules.
我们最近发现，脑胞质微管相关蛋白1C（MAP 1C）是一种微管激活的ATP酶，能够在体外使微管沿与逆向运输相对应的方向移位。（帕斯卡，B.M.，H.S.施佩特纳，和R.B.瓦利。1987b。《细胞生物学杂志》105:1273 - 1282；帕斯卡，B.M.，和R.B.瓦利。1987。《自然》[伦敦]。330:181 - 183。）对该蛋白的生化分析（同上）以及扫描透射电子显微镜显示，MAP 1C是纤毛和鞭毛ATP酶动力蛋白的脑细胞质形式（瓦利，R.B.，J.S.沃尔，B.M.帕斯卡，和H.S.施佩特纳。1988。《自然》[伦敦]。332:561 - 563）。我们现在已详细表征了脑酶的ATP酶活性。我们发现微管激活需要聚合微管蛋白，并随着微管蛋白浓度增加而饱和。微管蛋白饱和时的最大活性（Vmax）为每毫克186至239纳摩尔/分钟。在低离子强度下，微管的Km为0.16毫克/毫升微管蛋白，大大低于先前报道的轴丝动力蛋白的值。微管刺激的活性对离子强度和巯基氧化态的变化极为敏感，这两者主要影响半最大激活所需的微管浓度。在许多方面，脑动力蛋白在酶学上与轴丝动力蛋白和卵动力蛋白相似。因此，该ATP酶需要二价阳离子，钙刺激活性的效果不如镁。MgATP酶受到偏钒酸盐（微管刺激活性的Ki = 5 - 10微摩尔）、1毫摩尔N - 乙基马来酰亚胺和1毫摩尔EHNA的抑制。与其他动力蛋白不同，脑酶水解CTP、TTP和GTP的速率高于ATP。因此，脑细胞质动力蛋白的酶学性质显然与其他动力蛋白相关，尽管脑酶在底物特异性和对微管刺激的高敏感性方面是独特的。
