INDO-1 BINDING TO PROTEIN IN PERMEABILIZED VENTRICULAR MYOCYTES ALTERS ITS SPECTRAL AND CA-BINDING PROPERTIES
INDO-1 BINDING TO PROTEIN IN PERMEABILIZED VENTRICULAR MYOCYTES ALTERS ITS SPECTRAL AND CA-BINDING PROPERTIES
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DOI：
10.1016/s0006-3495(92)81597-7
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发表时间：
1992-07-01
影响因子：
3.4
通讯作者：
BERS, DM
中科院分区：
文献类型：
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作者：
HOVEMADSEN, L;BERS, DM
我们已经检测了荧光钙指示剂indo-1与渗透性心室肌细胞的细胞蛋白以及可溶性和颗粒性肌细胞蛋白的结合。使用过滤技术或平衡透析，以及类似于在贴片移液管中夹住100 μ m的indo-1的心肌细胞贴片的条件，我们发现在100mg /ml的蛋白质浓度下，总indo-1的72%与心肌细胞蛋白结合。这对应于3.8 +/- 0.5 nmol indo-1/mg蛋白的结合。将肌细胞蛋白分离成可溶性和颗粒状，结果表明，63%的结合indo-1与可溶性蛋白结合，对应于3.22 +/- 0.99 nmol/mg的结合，而37%的结合indo-1与颗粒蛋白结合（0.85 +/- 0.14 nmol/mg）在低[Ca]下（pCa类似于9）。在饱和Ca浓度下（pCa = 3），渗透性肌细胞中吲哚-1的结合比无Ca条件下（1 mM EGTA）高约60%。用Ca选择电极和吲哚-1荧光同时测量游离[Ca]表明，与在体外获得的值相比，在渗透性肌细胞存在下，Ca的解离常数（K(d)）增加了4-5倍。与结合实验一致，我们估计Ca与indo-1结合的真K(d)和表观K(d)（使用比率测量法）在肌细胞蛋白浓度为100 mg/ml时增加了大约四倍。
We have examined the binding of the fluorescent Ca indicator indo-1 to cellular protein in permeabilized ventricular myocytes and also to soluble and particulate myocyte protein. Using either a filtration technique or equilibrium dialysis, and conditions similar to those in a cardiac myocyte patch clamped with 100-mu-M indo-1 in the patch pipette, we found that 72% of the total indo-1 was bound to myocyte protein at a protein concentration of 100 mg/ml. This corresponds to a binding of 3.8 +/- 0.5 nmol indo-1/mg protein. Separation of the myocyte protein into a soluble and a particulate fraction showed that 63% of the bound indo-1 was bound to soluble protein, corresponding to a binding of 3.22 +/- 0.99 nmol/mg, whereas 37% of the bound indo-1 was bound to particulate protein (0.85 +/- 0.14 nmol/mg) at a low [Ca] (pCa is similar to 9). Binding of indo-1 in permeabilized myocytes was approximately 60% higher at a saturating Ca concentration (pCa = 3), than under Ca free conditions (1 mM EGTA). Simultaneous measurements of free [Ca] with a Ca selective electrode and indo-1 fluorescence showed that, the dissociation constant (K(d)) for Ca was increased 4-5 fold in the presence of permeabilized myocytes as compared to the value obtained in vitro. In agreement with the binding experiments we estimate that the true K(d) and the apparent K(d) (using ratiometric measurements) for Ca binding to indo-1 are increased approximately four fold, at a myocyte protein concentration of 100 mg/ml.