Keeling D J, Malcolm R C, Laing S M, Ife R J, Leach C A
SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Hertfordshire, U.K.
Biochem Pharmacol. 1991 Jun 21;42(1):123-30. doi: 10.1016/0006-2952(91)90690-7.
SK&F 96067 [3-butyryl-4-(2-methylphenylamino)-8-methoxyquinoline] has been identified, from a novel class of 4-aminoquinolines, as a reversible inhibitor of the gastric (H+ + K+)-ATPase. This compound has been studied in gastric membrane vesicle preparations enriched in the (H+ + K+)-ATPase. At pH 7.0, SK&F 96067 inhibited K(+)-stimulated ATPase activity competitively with respect to the activating cation K+, with a Ki value of 0.39 +/- 0.05 microM. Under comparable conditions, SK&F 96067 was 32 times more potent as an inhibitor of the gastric (H+ + K+)-ATPase relative to the closely related (Na+ + K+)-ATPase. Studies in intact gastric vesicles showed that SK&F 96067 also inhibited hydrogen ion transport. Using the initial rate of acridine orange quenching as the index of acidification, an IC50 of 0.84 +/- 0.24 microM was observed. Steady state acidification, as measured by aminopyrine accumulation, was inhibited with greater potency (IC50 = 0.06 +/- 0.01 microM) consistent with the accumulation of this inhibitor into the intravesicular acidic space to a site of action on the inside (lumenal) face of the enzyme. Inhibition of ATPase activity in the presence of both SK&F 96067 and the K(+)-competitive (H+ + K+)-ATPase inhibitor, SCH 28080, indicated that their binding was mutually exclusive, consistent with SK&F 96067 acting at the same lumenal binding site as does SCH 28080. The steady-state inhibition kinetics of SK&F 96067 against K(+)-stimulated ATPase activity were followed as a function of pH. At pH 6.6 and 7.0 the inhibition was competitive with respect to the activating cation K+. At pH 7.5 and 8.1 a mixed pattern of inhibition was detected. Thus, at alkaline pH values, the binding of SK&F 96067 and K+ were no longer mutually exclusive. The potency of SK&F 96067 decreased as pH rose, consistent with the protonated form of the inhibitor being the preferred inhibitory species. A kinetic model is discussed, in which, at acidic pH, the protonated form of SK&F 96067 binds to the enzyme competitively with respect to K+, whereas, at alkaline pH, the neutral form of SK&F 96067 can bind simultaneously with K+.
SK&F 96067（3-丁酰基-4-（2-甲基苯基氨基）-8-甲氧基喹啉）已从一类新型4-氨基喹啉中被鉴定为胃（H⁺+K⁺）-ATP酶的可逆抑制剂。该化合物已在富含（H⁺+K⁺）-ATP酶的胃膜囊泡制剂中进行了研究。在pH 7.0时，SK&F 96067相对于激活阳离子K⁺竞争性抑制K⁺刺激的ATP酶活性，Ki值为0.39±0.05微摩尔。在可比条件下，相对于密切相关的（Na⁺+K⁺）-ATP酶，SK&F 96067作为胃（H⁺+K⁺）-ATP酶抑制剂的效力高32倍。在完整胃囊泡中的研究表明，SK&F 96067也抑制氢离子转运。以吖啶橙淬灭的初始速率作为酸化指标，观察到IC50为0.84±0.24微摩尔。通过氨基比林积累测量的稳态酸化受到更强的抑制（IC50 = 0.06±0.01微摩尔），这与该抑制剂在囊泡内酸性空间中积累到酶的内侧（腔面）作用位点一致。在SK&F 96067和K⁺竞争性（H⁺+K⁺）-ATP酶抑制剂SCH 28080同时存在的情况下对ATP酶活性的抑制表明它们的结合是相互排斥的，这与SK&F 96067与SCH 28080作用于相同的腔面结合位点一致。跟踪了SK&F 96067对K⁺刺激的ATP酶活性的稳态抑制动力学随pH的变化。在pH 6.6和7.0时，抑制相对于激活阳离子K⁺是竞争性的。在pH 7.5和8.1时检测到混合抑制模式。因此，在碱性pH值下，SK&F 96067和K⁺的结合不再相互排斥。SK&F 96067的效力随pH升高而降低，这与抑制剂的质子化形式是优选的抑制物种一致。讨论了一个动力学模型，其中在酸性pH下，SK&F 96067的质子化形式相对于K⁺竞争性地与酶结合，而在碱性pH下，SK&F 96067的中性形式可以与K⁺同时结合。
