Takashi R
Cardiovascular Research Institute, University of California, San Francisco 94143.
Biochemistry. 1988 Feb 9;27(3):938-43. doi: 10.1021/bi00403a015.
By peptide isolation and analysis, it has been shown that the dansyl fluorophore of dansylcadaverine [N-(5-aminopentyl)-5-(dimethylamino)naphthalene-1-sulfonamide] transfers to Gln-41 of actin from rabbit skeletal muscle when the reaction is catalyzed by guinea pig liver transglutaminase. As a function of time, the degree of labeling asymptotically approaches 1 mol of dansyl/l mol of actin. About 80-85% of the attached dansyl fluorophore was found at Gln-41. Such labeled G-actin polymerizes to the same extent as control actin, but the polymerization rate is greater and the critical concentration is less than for control actin. Complete polymerization is accompanied by a 1.5-2.0-fold increase in the emission intensity of the attached fluorophore. Labeled F-actin thus obtained activates myosin subfragment 1 (S-1) Mg2+-ATPase activity with the same Kapp, and to the same Vmax, as control actin; moreover, when such labeled F-actin is cross-linked to S-1 by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, the resulting superactivation of Mg2+-ATPase is the same as that attained with control actin. The attributes of this label thus make it an ideal reporter of events in the N-terminal 10-kilodalton region of actin, and a new topological point for proximity mapping.
通过肽的分离和分析表明，当反应由豚鼠肝脏转谷氨酰胺酶催化时，丹磺酰尸胺[N-(5-氨基戊基)-5-(二甲基氨基)萘-1-磺酰胺]的丹磺酰荧光团转移至兔骨骼肌肌动蛋白的Gln-41。作为时间的函数，标记程度渐近地接近1摩尔丹磺酰/1摩尔肌动蛋白。在Gln-41处发现约80 - 85%的附着丹磺酰荧光团。这种标记的G-肌动蛋白与对照肌动蛋白聚合程度相同，但聚合速率更高，临界浓度低于对照肌动蛋白。完全聚合伴随着附着荧光团发射强度增加1.5 - 2.0倍。由此获得的标记F-肌动蛋白与对照肌动蛋白一样，以相同的Kapp和相同的Vmax激活肌球蛋白亚片段1(S-1)的Mg2 + -ATP酶活性；此外，当这种标记的F-肌动蛋白通过1-乙基-3-[3-(二甲基氨基)丙基]碳二亚胺与S-1交联时，产生的Mg2 + -ATP酶超激活与对照肌动蛋白相同。因此，这种标记的特性使其成为肌动蛋白N端10千道尔顿区域事件的理想报告分子，以及近距离作图的新拓扑点。
