Activation of actin-cardiac myosin subfragment 1 MgATPase rate by Ca2+ shows cooperativity intrinsic to the thin filament.
Activation of actin-cardiac myosin subfragment 1 MgATPase rate by Ca2+ shows cooperativity intrinsic to the thin filament.
复制标题
Ca2+ 激活肌动蛋白-心肌肌球蛋白亚片段 1 MgATPase 速率显示细丝固有的协同作用。
DOI：
10.1021/bi00376a022
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Tobacman,LS
中科院分区：
文献类型：
--
作者：
Tobacman,LS
摘要：为了研究钙离子协同诱导心肌收缩的机制，我们研究了心肌肌球蛋白亚片段1（S-1）的镁腺苷三磷酸酶（MgATP酶）在调节肌动蛋白存在下的速率。MgATP酶的速率增加协同Ca 2+，表现出希尔系数为1.8和50%的活化pCa 5.75。尽管实验设计排除了几个潜在的合作性来源，这种合作反应还是发生了。首先，为了排除由于在低离子强度下错误计算pCa而导致的虚假协同性，通过使用Quin 2的新方法测量Ca2+和Mg2+对[亚乙基双（氧乙烯基次氮基）]四乙酸（EGTA）的亲和力。在pH 7.06、25 ℃和μ = 30 mM时，CaEGTA的KD为140 nM，MgEGTA的KD为2.7 mM。其次，肌动蛋白-肌球蛋白S1结合并不产生协同性;肌球蛋白S1仅与每300个肌动蛋白启动子中的1个结合，并且早期的工作[Tobacman，L.美国，& Adelstein，R. S.（1986）Biochemistry 25，798 - 802]已经表明，心肌肌球蛋白S1在非常低的Ca2+浓度和饱和Ca2+浓度下以相等的亲和力与细丝结合。此外，在低、中和饱和Ca~（2+）浓度下，肌球蛋白S-1的腺苷5'-三磷酸周转率与酶浓度无关。最后，由于心肌肌钙蛋白只有一个调节Ca2+特异性位点，因此这些位点之间不会发生协同作用。这些数据表明，由相邻肌钙蛋白-原肌球蛋白复合物之间的相互作用赋予的部分协同性是细丝固有的，并且不依赖于肌球蛋白。C/a2+诱导骨骼肌和心肌中的协同反应，如肌肉张力的研究所证明的（Hellam & Podolsky，1969; Julian，1971; Fabiato & Fabiato，1978; Brandt等人，1980; Kerrick等人，1980; Moss等人，1986）、肌原纤维腺苷三磷酸酶（ATP酶）1（Solaro & Shiner，1976; Murray & Weber，1980）和肌动球蛋白ATP酶（Murray & Weber，1980; Grabarek等人，1983年）。越来越多的实验证据表明，这种现象不需要与肌钙蛋白上的多个位点合作的Ca2+结合。协同性不仅发生在肌钙蛋白含有四个Ca2+结合位点的肌肉中，而且发生在具有三个肌钙蛋白位点的心脏中（Holroyde et al.，1980）和具有一个肌钙蛋白位点的小龙虾（Wnuk等人，1984年）。
Revised Manuscript Received October I, 1986 abstract: The magnesium adenosinetriphosphatase (MgATPase) rate of cardiac myosin subfragment 1 (S-1) was studied in the presence of regulated actin in order to investigate the mechanism by which Ca2+ cooperatively induces cardiac muscle contraction. The MgATPase rate increased cooperatively with Ca2+, exhibiting a Hill coefficient of 1.8 and 50% activation at pCa 5.75. This cooperative response occurred despite an experimental design excluding several potential sources of cooperativity. First, to exclude spurious cooperativity due to erroneous calculation of pCa at low ionic strength, the affinities of Ca2+ and Mg2+ for [ethylenebis (oxyethylenenitrilo)] tetraacetic acid (EGTA) were measured by a novel method using Quin 2. At pH 7.06, 25 C, and µ= 30 mM, the KD was 140 nM for CaEGTA and 2.7 mM for MgEGTA. Second, the cooperativity was not produced by actin-myosin Sl binding; myosin Sl was bound to only 1 of every 300 actin promoters, and earlier work [Tobacman, L. S., & Adelstein, R. S.(1986) Biochemistry 25, 798-802] had shown that cardiacmyosin Sl binds with equal affinity to the thin filament at very low Ca2+ and at saturating Ca2+ concentrations. Furthermore, the adenosine 5'-triphosphate turnover rate of the myosin S-1 was independent of enzyme concentration at low, intermediate, and saturating Ca2+ con-centrations. Finally, since cardiac troponin has only one regulatory Ca2+-specific site, cooperative interactions between such sites could not occur. These data suggest that part of the cooperativity conferred by interaction between adjacent troponin-tropomyosin complexes is intrinsic to the thin filament and independent of myosin.C/a2+ induces a cooperative response in both skeletal muscle and cardiac muscle as demonstrated by studies of muscle tension (Hellam & Podolsky, 1969; Julian, 1971; Fabiato & Fabiato, 1978; Brandt et al., 1980; Kerrick et al., 1980; Moss et al., 1986), myofibrillar adenosinetriphosphatase (ATPase) 1 (Solaro & Shiner, 1976; Murray & Weber, 1980), and ac-tomyosin ATPase (Murray & Weber, 1980; Grabarek et al., 1983). Increasing experimental evidence indicates that co-operative Ca2+ binding to multiple sites on troponin is not required for this phenomenon. Cooperativity occurs not only in muscles where troponin contains four Ca2+ binding sites but also in the heart with three troponin sites (Holroyde et al., 1980) and incrayfish with one troponin site (Wnuk et al., 1984).