Andreev O A, Takashi R, Borejdo J
Baylor Research Institute, Baylor University Medical Center, Dallas, TX 75226, USA.
J Muscle Res Cell Motil. 1995 Aug;16(4):353-67. doi: 10.1007/BF00114501.
A serine residue located in the active site of myosin head (S1) was labelled by 9-anthroylnitrile, an amino group located in the central domain of S1 was labelled by 7-diethylamino-3-(4'-isothio-cyanato-phenyl)-4-methylcoumari n, a cysteine residue located near the C-terminus of S1 was labelled by 5-[2-((iodoacetyl)-amino)ethyl]-amino-naphthalene-1-sulfonic acid (1,5-IAEDANS) and a cysteine residue located near the C-terminus of the alkali light chain 1 was labelled with iodoacetamido-tetramethyl-rhodamine. Polarization of fluorescence of S1 was measured in solution (where it indicated the mobility of actin-bound S1) and in myofibrils (where it indicated orientation of probes) to check whether the anisotropy of S1 labelled at different positions depended on the molar ratio S1:actin. In solution, when increasing amounts of actin were added to a fixed amount of labelled S1 (i.e. when myosin heads were initially in excess over actin), anisotropy saturated at 1 mol of S1 per 1 mol of actin. When increasing amounts of S1 were added to a fixed amount of F-actin (i.e. when actin was initially in excess over S1), the anisotropy saturated at 1 mol of S1 per 2 mols of actin. In myofibrils, orientation of S1 was different when S1 was added at nanomolar concentration (intrinsic actin was in excess over extrinsic S1) then when it was added at micromolar concentration (excess of S1 over actin). The fact that the anisotropy of S1 labelled at different positions depended on the molar ratio excluded the possibility that changes were confined to one part of the cross-bridge and supports our earlier proposal that the two rigor complexes which S1 can form with F-actin differ globally in conformation.
肌球蛋白头部（S1）活性位点中的一个丝氨酸残基用9-蒽腈标记，S1中央结构域中的一个氨基用7-二乙氨基-3-（4'-异硫氰酸根合苯基）-4-甲基香豆素标记，S1 C末端附近的一个半胱氨酸残基用5-[2-（（碘乙酰基）氨基）乙基]-氨基萘-1-磺酸（1,5-IAEDANS）标记，碱轻链1 C末端附近的一个半胱氨酸残基用碘乙酰胺基四甲基罗丹明标记。在溶液中（其中它指示肌动蛋白结合的S1的迁移率）和肌原纤维中（其中它指示探针的取向）测量S1荧光的偏振，以检查在不同位置标记的S1的各向异性是否取决于S1：肌动蛋白的摩尔比。在溶液中，当向固定量的标记S1中添加越来越多的肌动蛋白时（即当肌球蛋白头部最初比肌动蛋白过量时），各向异性在每1摩尔肌动蛋白1摩尔S1时达到饱和。当向固定量的F-肌动蛋白中添加越来越多的S1时（即当肌动蛋白最初比S1过量时），各向异性在每2摩尔肌动蛋白1摩尔S1时达到饱和。在肌原纤维中，当以纳摩尔浓度添加S1时（内在肌动蛋白比外在S1过量），S1的取向与以微摩尔浓度添加时（S1比肌动蛋白过量）不同。在不同位置标记的S1的各向异性取决于摩尔比这一事实排除了变化局限于横桥一部分的可能性，并支持了我们早期的提议，即S1可以与F-肌动蛋白形成的两种僵直复合物在构象上整体不同。
