两种强心苷对重组于蛋白脂质体中的Na⁺/K⁺-ATP酶的调节作用

Modulatory effect of two cardioglycosides on reconstituted Na+/K(+)-ATPase in proteoliposomes.

Cavaletto M, Giunta C, Pessione E, Pergola L

Dipartimento di Biologia Animale, Universita di Torino, Italia.

Int J Biochem. 1991;23(11):1267-75. doi: 10.1016/0020-711x(91)90227-e.

Abstract

Na,K-ATPase was extracted from Cavia cobaya kidneys, solubilized with nonionic detergent C12E8 (octaethyleneglycol dodecyl monoether) in mixed lipid-detergent-protein micelles. The Na,K-ATPase specific activity was 30-35 IU/mg protein. 2. The enzyme was reconstituted in vesicles, made of phosphatidylethanolamine and cholesterol: an enhancement of +60% in specific activity was obtained. 3. Two different vesicle-types were carried out: open liposomes (partially organized membranes) and closed liposomes. 4. Proteoliposomes were employed for measuring the modulatory effect of two cardioglycosides: ouabain and digoxin. 5. Inhibition of the Na,K-ATPase activity revealed apparent Ki of 1.25 microM for ouabain and 0.25 microM for digoxin in open liposomes, and apparent Ki of 0.75 microM for ouabain and of 1.75 microM for digoxin in closed liposomes. 6. Maximum enhancement of enzymatic activity was found at concentrations of 5-0.5 nM for ouabain and 5-1 nM for digoxin in open liposomes, and 25-1 nM for both digoxin and ouabain in closed liposomes.

摘要

钠钾ATP酶从豚鼠肾脏中提取，用非离子洗涤剂C12E8（八乙二醇十二烷基单醚）溶解在脂质 - 洗涤剂 - 蛋白质混合胶束中。钠钾ATP酶的比活性为30 - 35 IU/mg蛋白质。2. 该酶在由磷脂酰乙醇胺和胆固醇制成的囊泡中重构：比活性提高了60%。3. 制备了两种不同类型的囊泡：开放脂质体（部分有序膜）和封闭脂质体。4. 蛋白脂质体用于测量两种强心苷：哇巴因和地高辛的调节作用。5. 钠钾ATP酶活性的抑制显示，在开放脂质体中，哇巴因的表观抑制常数Ki为1.25 microM，地高辛为0.25 microM；在封闭脂质体中，哇巴因的表观抑制常数Ki为0.75 microM，地高辛为1.75 microM。6. 在开放脂质体中，哇巴因浓度为5 - 0.5 nM、地高辛浓度为5 - 1 nM时，酶活性达到最大增强；在封闭脂质体中，地高辛和哇巴因浓度为25 - 1 nM时，酶活性达到最大增强。