Kunori S, Katoh T, Mogi Y, Morita F
Department of Chemistry, Hokkaido University, Sapporo.
J Biochem. 1995 Dec;118(6):1239-47. doi: 10.1093/oxfordjournals.jbchem.a125013.
The N-terminal 28-residue peptide of actin whose Cys10 was labeled with 5-iodoacetamido-fluorescein (F3K peptide) was isolated from the fluorescently labeled thrombin digest of actin. The effect of myosin subfragment 1 (S1) on the fluorescence of F3K peptide was examined in the absence of ATP. With increasing concentration of S1 added, the fluorescence intensity of F3K peptide increased by maximally 7.3% with an apparent dissociation constant of 5.7 microM, suggesting a role of this peptide region of actin in acto-S1 binding in rigor. F3K peptide was crosslinked with S1 at 10 mM NaCl using a zero-length crosslinker by the method of Grabarek and Gergely [Anal. Biochem. 185, 131-135 (1990)]. The crosslinking was greatly inhibited by the presence of either 0.2 M NaCl or 5 mM MgATP. The analyses of amino acid compositions and sequences of the fluorescent peptides isolated from a lysylendopeptidase digest of the crosslinked S1 indicated that F3K peptide was mainly crosslinked to residues 637-642 of the S1 heavy chain. The crosslinked S1 was isolated by selectively pelleting the uncrosslinked S1 with F-actin. ATPase activity of the isolated crosslinked S1 alone was twice as high as that of control S1. The actin-activated ATPase activity of the crosslinked S1 was much lower than that of uncrosslinked S1. The estimated Vm and Km values were 1.72 s-1 and 125 microM, respectively. The Vm decreased to less than 1/8, while Km increased only twofold. The results suggest that the N-terminal 28-residue segment of actin may be implicated in the rigor binding of actomyosin and in the actin-activation of myosin ATPase, but may not be the main determinant of actomyosin binding in the presence of ATP.
从肌动蛋白的荧光标记凝血酶消化物中分离出肌动蛋白的N端28个残基的肽段，其Cys10用5-碘乙酰胺荧光素标记（F3K肽）。在无ATP的情况下，检测了肌球蛋白亚片段1（S1）对F3K肽荧光的影响。随着添加的S1浓度增加，F3K肽的荧光强度最大增加7.3%，表观解离常数为5.7 microM，这表明肌动蛋白的该肽段区域在僵直状态下的肌动蛋白-S1结合中起作用。按照Grabarek和Gergely的方法[《分析生物化学》185, 131 - 135 (1990)]，使用零长度交联剂在10 mM NaCl条件下将F3K肽与S1交联。0.2 M NaCl或5 mM MgATP的存在会极大地抑制交联反应。对从交联S1的赖氨酰内肽酶消化物中分离出的荧光肽的氨基酸组成和序列分析表明，F3K肽主要与S1重链的637 - 642位残基交联。通过用F-肌动蛋白选择性沉淀未交联的S1来分离交联的S1。单独分离出的交联S1的ATP酶活性是对照S1的两倍。交联S1的肌动蛋白激活的ATP酶活性远低于未交联的S1。估计的Vm和Km值分别为1.72 s-1和125 microM。Vm降至不到1/8，而Km仅增加两倍。结果表明，肌动蛋白的N端28个残基片段可能与肌动球蛋白的僵直结合以及肌球蛋白ATP酶的肌动蛋白激活有关，但在有ATP存在时可能不是肌动球蛋白结合的主要决定因素。
