Albanesi J P, Lynch T J, Fujisaki H, Bowers B, Korn E D
J Biol Chem. 1987 Mar 5;262(7):3404-8.
A protein which cross-links actin filaments in a nucleotide-sensitive manner has been purified to homogeneity from Acanthamoeba castellanii. This protein, GF-210, is a slightly asymmetric molecule composed of six subunits, each with an apparent mass of 35,000 Da. As determined by the method of falling ball vicometry, GF-210 was shown to cross-link actin filaments at hexamer:actin molar ratios of 1:500, with gelation occurring at molar ratios of 1:300 and higher. Actin gels did not form in the presence of 10 microM ATP, and filament cross-linking was completely inhibited by 100 microM ATP. Although ATP was the most effective inhibitor of actin filament cross-linking, other phospho-compounds including ADP, GTP, sodium phosphate, and sodium pyrophosphate prevented gelation at concentrations lower than 1.5 mM. In contrast, 50 mM KCl was required to inhibit the formation of actin networks. Direct binding studies showed that GF-210 binds to F-actin with a KD of 1.2 microM in the absence of ATP but with a KD of 72.8 microM in the presence of 2 mM ATP. This weakening of the interaction between F-actin and GF-210 may explain the inhibition of GF-210-induced actin cross-linking by nucleotides and other phospho-compounds.
一种能以核苷酸敏感方式交联肌动蛋白丝的蛋白质已从卡氏棘阿米巴中纯化至同质。这种蛋白质GF - 210是一种略微不对称的分子，由六个亚基组成，每个亚基的表观质量为35,000道尔顿。通过落球粘度测定法确定，GF - 210在六聚体与肌动蛋白的摩尔比为1:500时能交联肌动蛋白丝，在摩尔比为1:300及更高时发生凝胶化。在10微摩尔ATP存在下不形成肌动蛋白凝胶，100微摩尔ATP完全抑制丝的交联。虽然ATP是肌动蛋白丝交联最有效的抑制剂，但其他磷化合物，包括ADP、GTP、磷酸钠和焦磷酸钠，在浓度低于1.5毫摩尔时可阻止凝胶化。相比之下，需要50毫摩尔氯化钾来抑制肌动蛋白网络的形成。直接结合研究表明，在没有ATP的情况下，GF - 210与F -肌动蛋白结合的解离常数为1.2微摩尔，但在2毫摩尔ATP存在下解离常数为72.8微摩尔。F -肌动蛋白与GF - 210之间相互作用的这种减弱可能解释了核苷酸和其他磷化合物对GF - 210诱导的肌动蛋白交联的抑制作用。
