Erythrosin isothiocyanate selectively labels lysine464 within an ATP-protectable binding site on the Ca-ATPase in skeletal sarcoplasmic reticulum membranes.
Erythrosin isothiocyanate selectively labels lysine464 within an ATP-protectable binding site on the Ca-ATPase in skeletal sarcoplasmic reticulum membranes.
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Erythrosin isothiocyanate 选择性标记骨骼肌质网膜 Ca-ATP 酶上 ATP 可保护结合位点内的赖氨酸 464。
DOI：
10.1021/bi980275f
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Squier,TC
中科院分区：
文献类型：
--
作者：
Huang,S;Negash,S;Squier,TC
利用异硫氰酸红素(er-ITC)对骨骼肌浆网(SR)膜上的Ca-ATPase上的ATP保护部位进行选择性修饰的条件已经确定。对修饰后的Ca-ATPase进行彻底的胰酶消化后，用反相高效液相色谱和正反相Fab-MS确定了主要的标记位点。用er-ITC修饰M452NVFNTEVRNLSK464VER467的三磷酸腺苷保护肽，其平均质量为2830.1±0.3Da。赖氨酸残基的排他性修饰表明Lys464是Er-ITC修饰的位点。用Er-ITC衍生化可以减少稳定状态的ATPase活性的二次激活和毫摩尔浓度的ATP的去磷酸化速度。相反，在存在微摩尔浓度的ATP时，er-ITC对Ca-ATPase的修饰并不影响(I)ATP的表观亲和力，(Ii)ATP形成磷酸酶的最大程度，(Iii)稳态ATP水解的速度，或(Iv)Ca-ATPase的去磷酸化速度。此外，无论ER-ITC修饰与否，Ca-ATPase对ATP的利用都不受洗涤剂增溶作用的影响，这表明ATP水解酶的二次激活涉及到单一的Ca-ATPase多肽链。因此，Er-ITC不干扰与磷酸酶衰变相关的正常结构转变。相反，这些结果表明，er-ITC与Lys464结合会干扰ATP与低亲和力位点的结合，或者干扰相关的结构转变，从而调节酶的去磷酸化速度。
Conditions that permit the selective modification of an ATP-protectable site on the Ca-ATPase in skeletal sarcoplasmic reticulum (SR) membranes using erythrosin isothiocyanate (Er-ITC) have been identified. The major labeling site for Er-ITC has been identified using reversed-phase HPLC and positive FAB mass spectrometry after exhaustive tryptic digestion of the Er-ITC-modified Ca-ATPase. An ATP-protectable peptide corresponding to M452NVFNTEVRNLSK464VER467is modified by Er-ITC, the average mass of which is 2830.1 ± 0.3 Da. The exclusive modification of lysine residues indicates Lys464as the site of Er-ITC modification. Derivatization with Er-ITC diminishes the secondary activation of steady-state ATPase activity and the rate of dephosphorylation by millimolar concentrations of ATP. In contrast, in the presence of micromolar ATP concentrations Er-ITC modification of the Ca-ATPase does not affect (i) the apparent affinity of ATP, (ii) the maximal extent of phosphoenzyme formation by ATP, (iii) the rate of steady-state ATP hydrolysis, or (iv) the rate of dephosphorylation of the Ca-ATPase. Furthermore, ATP utilization by the Ca-ATPase is unaffected by detergent solubilization, irrespective of Er-ITC modification, indicating that the secondary activation of ATP hydrolysis involves a single Ca-ATPase polypeptide chain. Therefore, Er-ITC does not interfere with the normal structural transitions associated with phosphoenzyme decay. Rather, these results indicate that Er-ITC bound to Lys464interferes with either ATP binding to a low-affinity site or the associated structural transitions that modulate the rate of enzyme dephosphorylation.