Gould A R, Norton R S
School of Biochemistry, University of New South Wales, Kensington, Australia.
Toxicon. 1995 Feb;33(2):187-99. doi: 10.1016/0041-0101(94)00142-u.
Chemical modification studies have been carried out on the sea anemone polypeptide anthopleurin-A in order to clarify the role of Arg-14 in its cardiac stimulatory activity. Reaction with 1,2-cyclohexanedione at 37 degrees C produced a range of protein products, including some with amino group modifications. These side-reactions were eliminated by prior citraconylation of the amino groups, which, following reaction with cyclohexanedione, could be reversed under conditions which preserved the cyclohexanedione adduct. Citraconylation of the three amino groups, one from the N-terminus and two from Lys-37 and Lys-48, destroyed the cardiac stimulatory activity of the molecule, but this was fully recoverable upon reversal of this reaction. It appears that one or more of the amino groups is essential for activity. Anthopleurin-A contains only one arginine residue, and this was confirmed as the site of modification by cyclohexanedione by showing that the product was refractory to proteolysis by trypsin, which normally cleaves the molecule at this residue. The positive inotropic activity of the cyclohexanedione adduct on isolated guinea-pig atria was identical to that of unmodified anthopleurin-A, indicating that the side-chain of Arg-14 is not required for cardiotonic activity.
为了阐明精氨酸-14在海葵多肽刺海葵素A的心脏刺激活性中的作用，已对其进行了化学修饰研究。在37℃下与1,2-环己二酮反应产生了一系列蛋白质产物，包括一些氨基被修饰的产物。通过氨基预先柠康酰化消除了这些副反应，柠康酰化后的产物在与环己二酮反应后，在保留环己二酮加合物的条件下可以逆转。对三个氨基（一个来自N端，两个来自赖氨酸-37和赖氨酸-48）进行柠康酰化，破坏了该分子的心脏刺激活性，但此反应逆转后活性可完全恢复。似乎一个或多个氨基对活性至关重要。刺海葵素A仅含有一个精氨酸残基，通过显示该产物对通常在此残基处切割分子的胰蛋白酶的蛋白水解具有抗性，证实该残基是环己二酮修饰的位点。环己二酮加合物对离体豚鼠心房的正性肌力活性与未修饰的刺海葵素A相同，表明心脏兴奋活性不需要精氨酸-14的侧链。
