Defective conformational response in a selectively trypsinized (Na+ + K+)-ATPase studied with tryptophan fluorescence.
Defective conformational response in a selectively trypsinized (Na+ + K+)-ATPase studied with tryptophan fluorescence.
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用色氨酸荧光研究选择性胰蛋白酶化 (Na K )-ATP 酶的构象反应缺陷。
DOI：
10.1016/0005-2736(80)90108-x
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
S. Karlish
中科院分区：
文献类型：
--
作者：
P. L. Jørgensen;S. Karlish
1.用内源荧光监测纯化的（Na++ K+）-ATP酶的蛋白质构象，我们已经检查了在选择性修饰的“无效”（Na++ K+）-ATP酶中是否伴随有缺陷的催化和运输过程而改变的构象响应，所述选择性修饰的“无效”（Na++ K+）-ATP酶通过分级胰蛋白酶消化Na+形式的蛋白质而获得。2. 2.对照和无效（Na++ K+）-ATP酶的K+型（E2 K）蛋白荧光强度比Na+型（E1 Na）高2-3%。通过改变NaCl浓度，我们发现两种磷酸酶的荧光强度不同，E2 P与E2 K的荧光强度相同，E1 P与E1 Na的荧光强度相似。因此，以E2 P形式存在的磷酸酶的分数可以确定为在不存在K+的情况下伴随磷酸化的荧光变化的幅度除以对K+的完全响应的幅度。3. 3.无效的（Na++ K+）-ATP酶的荧光响应的滴定表明，胰蛋白酶裂解改变了阳离子结合构象之间的平衡，E1 Na和E2 K，和磷酸化，E1 P和E2 P，在E1形式的方向。4. 4.钒酸盐与Mg 2+结合形式的E 2 K结合，阻止蛋白质荧光强度的进一步变化。无效的（Na++ K+）-ATP酶的构象反应对钒酸盐不敏感，这与该酶的钒酸盐结合亲和力降低一致。5. 5.无效的（Na++ K+）-ATP酶的构象反应缺陷与其催化缺陷、Na+转运减少和对钒酸盐不敏感有关，这表明蛋白质的Na+形式（E1）和K+形式（E2）之间的转换与（Na++ K+）-泵的催化和转运反应耦合。
1. Monitoring protein conformations of purified (Na++ K+)-ATPase with intrinsic fluorescence we have examined if altered conformational responses accompany the defective catalytic and transport processes in selectively modified ‘invalid’(Na++ K+)-ATPase which is obtained by graded tryptic digestion of the Na+ form of the protein. 2. 2. The protein fluorescence intensity of the K+ form (E 2 K) of both control and invalid (Na++ K+)-ATPase is 2–3% higher than that of the Na+ form (E 1 Na). By varying the NaCl concentration we found evidence for different fluorescence intensities of the two phosphoenzymes; E 2 P has the same fluorescence intensity as E 2 K and the intensity of E 1 P is similar to that of E 1 Na. The fraction of phosphoenzyme present as E 2 P can therefore be determined as the amplitude of the fluorescence change accompanying phosphorylation in the absence of K+ divided by the amplitude of the full response to K+. 3. 3. Titration of the fluorescence responses of the invalid (Na++ K+)-ATPase shows that the tryptic split alters the poise of the equilibria between the cation-bound conformations, E 1 Na and E 2 K, and between the phosphoforms, E 1 P and E 2 P, in the direction of the E 1 forms. 4. 4. Vanadate binds to the Mg 2+-bound form of E 2 K and prevents further changes in fluorescence intensity of the protein. The conformative responses of invalid (Na++ K+)-ATPase are insensitive to vanadate in agreement with the reduced vanadate binding affinity of this enzyme. 5. 5. The defective conformative response of the invalid (Na++ K+)-ATPase in relation to its catalytic defects, reduced Na+ transport, and insensitivity to vanadate suggest that the transitions between Na+ forms (E 1) and K+ forms (E 2) of the protein are coupled to the catalytic and transport reactions of the (Na++ K+)-pump.