磷酸甘油酸激酶与人红细胞膜的相互作用。

Interaction of phosphoglycerate kinase with human erythrocyte membranes.

作者信息

De B K, Kirtley M E

出版信息

J Biol Chem. 1977 Oct 10;252(19):6715-20.

Abstract

The purpose of this study was to investigate the interaction of phosphoglycerate kinase with the human erythrocyte ghost membrane. Ghosts prepared in 0.1 mM EDTA and 17 mM Tris buffer (pH 7.5) have about 230 molecules of phosphoglycerate kinase/ghost. No additional binding is observed after incubating soluble enzyme with these leaky ghosts. This binding is tight but reversible with Kd = 7.1 X 10(-10) M. The enzyme can be eluted significantly from the membrane by incubation with 0.15 M NaCl and it rebinds to the membrane when the depleted ghosts are incubated with rabbit muscle phosphoglycerate kinase. Ligand binding studies show that NADH and NAD have opposite effects on the binding of the enzyme to the membrane; NAD (1.0 MM) favors binding while NADH (0.25 MM) does not. Similarly, ADP (0.2 mM) favors binding while ATP does not. ATP elutes the membrane-bound enzyme with Kd = 0.058 mM. MgSO4 also stimulates dissociation of the membrane-bound phosphoglycerate kinase (Kd = 0.36 mM), an effect which appears to be due to the magnesium ion. ADP (0.2 mM) can counteract the negative effect of MgSO4 (1.0 mM) on binding of phosphoglycerate kinase to the membrane. We have been unable so far to find tight coupling of the (Na+-K+)-ATPase with the membrane-bound phosphoglycerate kinase.

摘要

本研究的目的是调查磷酸甘油酸激酶与人红细胞血影膜的相互作用。在0.1 mM乙二胺四乙酸（EDTA）和17 mM Tris缓冲液（pH 7.5）中制备的血影，每个血影约有230个磷酸甘油酸激酶分子。将可溶性酶与这些有渗漏的血影孵育后，未观察到额外的结合。这种结合紧密但可逆，解离常数（Kd）= 7.1×10⁻¹⁰ M。通过与0.15 M氯化钠孵育，酶可从膜上显著洗脱，当耗尽磷酸甘油酸激酶的血影与兔肌肉磷酸甘油酸激酶孵育时，酶会重新结合到膜上。配体结合研究表明，还原型辅酶Ⅰ（NADH）和辅酶Ⅰ（NAD）对酶与膜结合有相反的影响；NAD（1.0 mM）促进结合，而NADH（0.25 mM）则不然。同样，二磷酸腺苷（ADP，0.2 mM）促进结合，而三磷酸腺苷（ATP）则不然。ATP以Kd = 0.058 mM的浓度洗脱膜结合的酶。硫酸镁也会刺激膜结合的磷酸甘油酸激酶解离（Kd = 0.36 mM），这种效应似乎是由镁离子引起的。ADP（0.2 mM）可以抵消硫酸镁（1.0 mM）对磷酸甘油酸激酶与膜结合的负面影响。到目前为止，我们尚未发现（钠⁺-钾⁺）-ATP酶与膜结合的磷酸甘油酸激酶之间存在紧密偶联。