Margossian S S, White H D, Lefford J, Holt J C, Malhotra A, Stafford W F, Slayter H S
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10467.
J Muscle Res Cell Motil. 1993 Feb;14(1):3-14. doi: 10.1007/BF00132175.
The effect of LC1 on cardiac myosin structure and activity was investigated using as a model S1 prepared by papain digestion in the presence of Mg2+. The resulting S1 contained LC2 but a part of the N-terminal region of LC1 was cleaved. Sequencing the N-terminal part of the band migrating below LC1 on SDS gels revealed it to consist of alternating alanyl and prolyl residues thus establishing LC1 as the origin of this band. However, Western blots did not reveal any LC1 while radioimmunoassays indicated it to be present at the 5% level suggesting the anti-LC1 antibody used in these experiments did not recognize the C-terminal portion of LC1 still attached to Mg.S1. Mixing a 10-15 M excess of isolated light chains with Mg.S1 in the presence of 10 mM ATP, 12 mM MgCl2, 4.7 M NH4Cl allowed LC1 to recombine with LC1-deficient Mg.S1. Equilibrium ultracentrifugation analysis revealed a highly heterogeneous LC1-deficient S1 which upon recombination with intact LC1 became monodisperse as indicated by the superimposition of molecular weight averages all across the centrifuge cell. LC1-deficient Mg.S1 had a Vm of 0.4 s-1, Ka of 30 microM and a Kbind of 28 microM. In the presence of intact LC1, Vm rose to 0.8 s-1 while Ka and Kbind were reduced to 7.5 and 12 microM, respectively. The fourfold decrease in Ka strongly indicated an increased affinity for actin by Mg.S1 in the presence of uncleaved LC1. Also, Ca(2+)-regulation of dog heart myofibrils was suppressed when Ca(2+)-activated MgATPase assays, as a function of Ca2+, were performed in the presence of anti-LC1 antibodies. These observations suggest the presence of intact, uncleaved LC1 in S1 is required for the stability of S1 heavy chains and proper Ca(2+)-regulation.
以在Mg2+存在下经木瓜蛋白酶消化制备的S1为模型，研究了LC1对心肌肌球蛋白结构和活性的影响。所得的S1含有LC2，但LC1的一部分N端区域被切割。对SDS凝胶上迁移至LC1下方的条带的N端部分进行测序，结果显示其由丙氨酰和脯氨酰残基交替组成，从而确定该条带起源于LC1。然而，蛋白质免疫印迹未显示任何LC1，而放射免疫分析表明其含量为5%，这表明这些实验中使用的抗LC1抗体未识别仍与Mg.S1相连的LC1的C端部分。在10 mM ATP、12 mM MgCl2、4.7 M NH4Cl存在的情况下，将10 - 15 M过量的分离轻链与Mg.S1混合，可使LC1与缺乏LC1的Mg.S1重新结合。平衡超速离心分析显示，缺乏LC1的S1高度不均一，与完整的LC1重组后变得单分散，这通过整个离心池分子量平均值的叠加得以表明。缺乏LC1的Mg.S1的Vm为0.4 s-1，Ka为30 μM，Kbind为28 μM。在存在完整LC1的情况下，Vm升至0.8 s-1，而Ka和Kbind分别降至7. five和12 μM。Ka降低四倍强烈表明，在未切割的LC1存在下Mg.S对肌动蛋白的亲和力增加。此外，当在抗LC1抗体存在的情况下进行Ca(2+)激活的MgATPase分析（作为Ca2+的函数）时，犬心肌肌原纤维的Ca(2+)调节受到抑制。这些观察结果表明，S1中存在完整、未切割的LC1是S1重链稳定性和适当Ca(2+)调节所必需的。
