Fanning A S, Wolenski J S, Mooseker M S, Izant J G
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06521-8019.
Cell Motil Cytoskeleton. 1994;29(1):29-45. doi: 10.1002/cm.970290104.
In this report, we have compared the physical properties and actin-binding characteristics of several bacterially produced nonmuscle and striated muscle tropomyosins, and we have examined the effects of these isoforms on the interactions of actin with two structurally distinct classes of myosin: striated muscle myosin-II and brush border (BB) myosin-I. All of the bacterially produced nonmuscle tropomyosins bind to F-actin with the expected stoichiometry and with affinities comparable to that of a tissue produced alpha-tropomyosin, although the striated muscle tropomyosin CTm7 has a lower affinity for F-actin than a tissue-purified striated muscle alpha tropomyosin. The bacterially produced isoforms also protect F-actin from severing by villin as effectively as tissue-purified striated muscle alpha-tropomyosin. The bacterially produced 284 amino acid striated muscle tropomyosin isoform CTm7, the 284 amino acid nonmuscle tropomyosin isoform CTm4, and two chimeric tropomyosins (CTm47 and CTm74) all inhibit the actin-activated MgATPase activity of muscle myosin S1 by approximately 70-85%, comparable to the inhibition seen with tissue-purified striated muscle alpha tropomyosin. The 248 amino acid tropomyosin XTm4 stimulated the actin-activated MgATPase activity of muscle myosin S1 approximately two- to threefold. The in vitro sliding of actin filaments translocated by muscle myosin-II (2.4 microns/sec at 19 degrees C, 5.0 microns/s at 24 degrees C) increased 25-65% in the presence of XTm4. Tropomyosins CTm4, CTm7, CTm47, and CTm74 had no detectable effect on myosin-II motility. The actin-activated MgATPase activity of BB myosin-I was inhibited 75-90% by all of the tropomyosin isoforms tested, including the 248 amino acid tropomyosin XTm4. BB myosin-I motility (50 nm/s) was completely inhibited by both the 248 and 284 amino acid tropomyosins. These results demonstrate that bacterially produced tropomyosins can differentially regulate myosin enzymology and mechanochemistry, and suggest a role for tropomyosin in the coordinated regulation of myosin isoforms in vivo.
在本报告中，我们比较了几种细菌产生的非肌肉和横纹肌原肌球蛋白的物理性质及肌动蛋白结合特性，并研究了这些同工型对肌动蛋白与两类结构不同的肌球蛋白相互作用的影响：横纹肌肌球蛋白-II和刷状缘（BB）肌球蛋白-I。所有细菌产生的非肌肉原肌球蛋白均以预期的化学计量比与F-肌动蛋白结合，其亲和力与组织产生的α-原肌球蛋白相当，不过横纹肌原肌球蛋白CTm7对F-肌动蛋白的亲和力低于组织纯化的横纹肌α-原肌球蛋白。细菌产生的同工型对F-肌动蛋白的保护作用，使其免受绒毛蛋白切断的效果与组织纯化后的横纹肌α-原肌球蛋白一样有效。细菌产生的含284个氨基酸的横纹肌原肌球蛋白同工型CTm7、含284个氨基酸的非肌肉原肌球蛋白同工型CTm4以及两种嵌合原肌球蛋白（CTm47和CTm74）均能抑制肌肉肌球蛋白S1的肌动蛋白激活的MgATP酶活性约70 - 85%，这与组织纯化的横纹肌α-原肌球蛋白的抑制效果相当。含248个氨基酸的原肌球蛋白XTm4使肌肉肌球蛋白S1的肌动蛋白激活的MgATP酶活性提高了约两到三倍。在XTm4存在的情况下，由肌肉肌球蛋白-II驱动的肌动蛋白丝的体外滑动速度（19℃时为2.4微米/秒，24℃时为5.0微米/秒）提高了25 - 65%。原肌球蛋白CTm4、CTm7、CTm47和CTm74对肌球蛋白-II的运动性没有可检测到的影响。所测试的所有原肌球蛋白同工型，包括含248个氨基酸的原肌球蛋白XTm4，均能抑制BB肌球蛋白-I的肌动蛋白激活的MgATP酶活性75 - 90%。含248和284个氨基酸的原肌球蛋白均完全抑制BB肌球蛋白-I的运动性（50纳米/秒）。这些结果表明，细菌产生的原肌球蛋白能够差异性地调节肌球蛋白的酶学和机械化学性质，并提示原肌球蛋白在体内对肌球蛋白同工型的协同调节中发挥作用。
