Homareda H, Matsui H
J Biochem. 1982 Jul;92(1):219-31. doi: 10.1093/oxfordjournals.jbchem.a133917.
General properties of ouabain-sensitive K+ binding to purified Na+,K+-ATPase [EC 3.6.1.3] were studied by a centrifugation method with 42K+. 1) The affinity for K+ was constant at pH values higher than 6.4, and decreased at pH values lower than 6.4. 2) Mg2+ competitively inhibited the K+ binding. The dissociation constant (Kd) for Mg2+ of the enzyme was estimated to be about 1 mM, and the ratio of Kd for Mg2+ to Kd for K+ was 120 : 1. The order of inhibitory efficiency of divalent cations toward the K+ binding was Ba2+ congruent to Ca2+ greater than Zn2+ congruent to Mn2+ greater than Sr2+ greater than Co2+ greater than Ni2+ greater than Mg2+. 3) The order of displacement efficiency of monovalent cations toward the K+ binding in the presence or absence of Mg2+ was Tl+ greater than Rb+ greater than or equal to (K+) greater than NH4+ greater than or equal to Cs+ greater than Na+ greater than Li+. The inhibition patterns of Na+ and Li+ were different from those of other monovalent cations, which competitively inhibited the K+ binding. 4) The K+ binding was not influenced by different anions, such as Cl-, SO4(2-), NO3-, acetate, and glycylglycine, which were used for preparing imidazole buffers. 5) Gramicidin D and valinomycin did not affect the K+ binding, though the former (10 micrograms/ml) inhibited the Na+,K+-ATPase activity by about half. Among various inhibitors of the ATPase, 0.1 mM p-chloromercuribenzoate and 0.1 mM tri-n-butyltin chloride completely inhibited the K+ binding. Oligomycin (10 micrograms/ml) and 10 mM N-ethylmaleimide had no effect on the K+ binding. In the presence of Na+, however, oligomycin decreased the K+ binding by increasing the inhibitory effect of Na+, whether Mg2+ was present or not. 6) ATP, adenylylimido diphosphate and ADP each at 0.2 mM decreased the K+ binding to about one-fourth of the original level at 10 microM K+ without MgCl2 and at 60 microM K+ with 5 mM MgCl2. On the other hand, AMP, Pi, and p-nitrophenylphosphate each at 0.2 mM had little effect on the K+ binding.
采用含(^{42}K^+)的离心法研究了哇巴因敏感的(K^+)与纯化的(Na^+)，(K^+)-ATP酶[EC 3.6.1.3]结合的一般特性。1）在pH值高于6.4时，对(K^+)的亲和力恒定，在pH值低于6.4时降低。2）(Mg^{2+})竞争性抑制(K^+)结合。该酶对(Mg^{2+})的解离常数（(K_d)）估计约为1 mM，(Mg^{2+})的(K_d)与(K^+)的(K_d)之比为120 : 1。二价阳离子对(K^+)结合的抑制效率顺序为(Ba^{2+})≈(Ca^{2+})＞(Zn^{2+})≈(Mn^{2+})＞(Sr^{2+})＞(Co^{2+})＞(Ni^{2+})＞(Mg^{2+})。3）在有或无(Mg^{2+})存在时，单价阳离子对(K^+)结合的置换效率顺序为(Tl^+)＞(Rb^+)≥((K^+))＞(NH_4^+)≥(Cs^+)＞(Na^+)＞(Li^+)。(Na^+)和(Li^+)的抑制模式与其他单价阳离子不同，其他单价阳离子竞争性抑制(K^+)结合。4）(K^+)结合不受用于制备咪唑缓冲液的不同阴离子如(Cl^-)、(SO_4^{2 - })、(NO_3^-)、乙酸盐和甘氨酰甘氨酸的影响。5）短杆菌肽D和缬氨霉素不影响(K^+)结合，尽管前者（10微克/毫升）使(Na^+)，(K^+)-ATP酶活性抑制约一半。在各种ATP酶抑制剂中，0.1 mM对氯汞苯甲酸和0.1 mM三正丁基氯化锡完全抑制(K^+)结合。寡霉素（10微克/毫升）和10 mM N - 乙基马来酰亚胺对(K^+)结合无影响。然而，在有(Na^+)存在时，无论有无(Mg^{2+})，寡霉素都会通过增强(Na^+)的抑制作用而降低(K^+)结合。6）在无(MgCl_2)时10微摩尔/升(K^+)以及有5 mM (MgCl_2)时60微摩尔/升(K^+)条件下，0.2 mM的ATP、腺苷酰亚胺二磷酸和ADP各自都使(K^+)结合降低至约原始水平的四分之一。另一方面，0.2 mM的AMP、磷酸和对硝基苯磷酸对(K^+)结合几乎没有影响。
Zotero 插件