Kinosian H J, Selden L A, Gershman L C, Estes J E
Center for Molecular Biology and Cancer Research and Department of Medicine, Albany Medical College, Albany, New York 12208, USA.
Biochemistry. 2000 Oct 31;39(43):13176-88. doi: 10.1021/bi001520+.
The interaction of profilin and non-muscle beta,gamma-actin prepared from bovine spleen has been investigated under physiologic ionic conditions. Profilin binding to actin decreases the affinity of actin for MgADP and MgATP by about 65- and 13-fold, respectively. Kinetic measurements indicate that profilin binding to actin weakens the affinity of actin for nucleotides primarily due to an increased nucleotide dissociation rate constant, but the nucleotide association rate constant is also increased about 2-fold. Removal of the actin-bound nucleotide and divalent cation produces the labile intermediate species in the nucleotide exchange reaction, nucleotide free actin (NF-actin), and increases the affinity of actin for profilin about 10-fold. Profilin binds NF-actin with high affinity, K(D) = 0.013 microM, and slows the observed denaturation rate of NF-actin. Addition of ATP to NF-actin weakens the affinity for profilin and addition of Mg(2+) to ATP-actin further weakens the affinity for profilin. The high-affinity Mg(2+) of actin regulates binding of both nucleotide and profilin to actin and is important for actin interdomain coupling. The data suggest that profilin binding to actin weakens nucleotide binding to actin by disrupting Mg(2+) coordination in the actin central cleft.
在生理离子条件下，研究了来自牛脾脏的丝切蛋白与非肌肉β，γ - 肌动蛋白之间的相互作用。丝切蛋白与肌动蛋白的结合分别使肌动蛋白对MgADP和MgATP的亲和力降低约65倍和13倍。动力学测量表明，丝切蛋白与肌动蛋白的结合削弱了肌动蛋白对核苷酸的亲和力，主要是由于核苷酸解离速率常数增加，但核苷酸结合速率常数也增加了约2倍。去除与肌动蛋白结合的核苷酸和二价阳离子会在核苷酸交换反应中产生不稳定的中间物种，即无核苷酸肌动蛋白（NF - 肌动蛋白），并使肌动蛋白对丝切蛋白的亲和力增加约10倍。丝切蛋白以高亲和力结合NF - 肌动蛋白，K(D)=0.013 microM，并减缓观察到的NF - 肌动蛋白的变性速率。向NF - 肌动蛋白中添加ATP会削弱对丝切蛋白的亲和力，向ATP - 肌动蛋白中添加Mg(2+)会进一步削弱对丝切蛋白的亲和力。肌动蛋白的高亲和力Mg(2+)调节核苷酸和丝切蛋白与肌动蛋白的结合，对肌动蛋白结构域间偶联很重要。数据表明，丝切蛋白与肌动蛋白的结合通过破坏肌动蛋白中央裂隙中的Mg(2+)配位来削弱核苷酸与肌动蛋白的结合。
