Lachaal M, Berenski C J, Kim J, Jung C Y
Biophysical Laboratory and Medical Service, Veterans Administration Medical Center, Buffalo, New York.
J Biol Chem. 1990 Sep 15;265(26):15449-54.
Glyceraldehyde-3-phosphate dehydrogenase was found to bind in vitro to purified, human erythrocyte glucose transporter reconstituted into vesicles. Mild tryptic digestion of the glucose transporter totally inactivated the binding, suggesting that the cytoplasmic domain of the transporter is involved in the binding to glyceraldehyde-3-phosphate dehydrogenase. The binding was abolished in the presence of antisera raised against the purified glucose transporter, further supporting specificity of this interaction. The binding was reversible with a dissociation constant (Kd) of 3.3 x 10(-6) M and a total capacity (Bt) of approximately 30 nmol/mg of protein indicating a stoichiometry of one enzyme-tetramer per accessible transporter. The binding was sensitive to changes in pH showing an optimum at around pH 7.0. KCl and NaCl inhibited the binding in a simple dose-dependent manner with Ki of 40 and 20 mM, respectively. The binding was also inhibited by NAD+ with an estimated Ki of 3 mM. ATP, on the other hand, enhanced the binding by up to 3-fold in a dose-dependent manner with an apparent Ka of approximately 6 mM. The binding was not affected by D-glucose or cytochalasin B. The binding did not affect either the glucose or cytochalasin B in binding affinities or the transport activity of the transporter. However, the enzyme was inactivated totally upon binding to the transporter. Based on these findings, we suggest that a significant portion of glyceraldehyde-3-phosphate dehydrogenase in human erythrocytes exists as an inactive form via an ATP-dependent, reversible association with glucose transporter, and that this association may exert regulatory intervention on nucleotide metabolism in vitro.
研究发现，3-磷酸甘油醛脱氢酶在体外可与重构到脂质体中的纯化人红细胞葡萄糖转运体结合。对葡萄糖转运体进行温和的胰蛋白酶消化可完全消除这种结合，这表明转运体的胞质结构域参与了与3-磷酸甘油醛脱氢酶的结合。在存在针对纯化葡萄糖转运体产生的抗血清时，这种结合被消除，进一步支持了这种相互作用的特异性。这种结合是可逆的，解离常数（Kd）为3.3×10⁻⁶ M，总容量（Bt）约为30 nmol/mg蛋白质，表明每个可及转运体的酶四聚体化学计量比为1。这种结合对pH变化敏感，在pH约7.0时显示最佳状态。KCl和NaCl以简单的剂量依赖性方式抑制结合，Ki分别为40和20 mM。NAD⁺也抑制这种结合，估计Ki为3 mM。另一方面，ATP以剂量依赖性方式使结合增强高达3倍，表观Ka约为6 mM。这种结合不受D-葡萄糖或细胞松弛素B的影响。这种结合对转运体的葡萄糖或细胞松弛素B结合亲和力或转运活性均无影响。然而，酶在与转运体结合后完全失活。基于这些发现，我们认为人红细胞中很大一部分3-磷酸甘油醛脱氢酶以无活性形式存在，通过与葡萄糖转运体的ATP依赖性可逆结合，并且这种结合可能在体外对核苷酸代谢产生调节作用。
