H+ ATPase of chromaffin granules. Kinetics, regulation, and stoichiometry.
H+ ATPase of chromaffin granules. Kinetics, regulation, and stoichiometry.
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DOI：
10.1016/s0021-9258(18)33879-1
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发表时间：
1982-09
期刊：
影响因子：
--
通讯作者：
R. Johnson;M. Beers;A. Scarpa
中科院分区：
文献类型：
--
作者：
R. Johnson;M. Beers;A. Scarpa
嗜铬颗粒ATP酶介导H+逆浓度梯度的向内定向运输，从而形成并维持电化学跨膜H+梯度。这ATP酶的动力学，其活性调制的电化学H+梯度的变化，和H+运输和ATP水解之间的化学计量进行了研究，在完整的牛嗜铬颗粒，resealed嗜铬颗粒鬼，和高度纯化的碎片嗜铬颗粒膜。在破碎的膜中，H+ ATP酶对ATP的KM为69 μ M，在pH 7.3时活性最大，在20 ℃时Vmax为111 nmol/min/mg蛋白质。三甲基锡抑制ATP酶在低得多的浓度比二环己基碳二亚胺，而寡霉素，利血平，和其他抑制剂没有效果。在完整的嗜铬颗粒，ATP酶活性刺激高达300%的崩溃的H+跨膜梯度。H+/ATP的化学计量测定在resealed嗜铬鬼缺乏ATP和儿茶酚胺的条件下，没有净pH值的变化发生在ATP水解。加入ATP后，血影中H+的积累速率与ATP的水解速率在60-100 s内呈线性关系，H+与ATP的比值为1.71。这些数据表明，嗜铬颗粒的H+ ATP酶既有动力学的相似性和不同之处与其他已知的H+ ATP酶。该ATP酶的H+梯度变化和固定H+/ATP比的调节进一步证明了其在嗜铬颗粒中建立电致H+移位和H+梯度中的主要作用。
The chromaffin granule ATPase mediates an inwardly directed transport of H+ against concentration gradients, thereby forming and maintaining an electrochemical transmembrane H+ gradient. The kinetics of this ATPase, its activity modulation by changes in electrochemical H+ gradients, and the stoichiometry between H+ transport and ATP hydrolysis were studied in intact bovine chromaffin granules, resealed chromaffin granule ghosts, and highly purified fragmented chromaffin granule membranes. In fragmented membranes the H+ ATPase has a KM for ATP of 69 microM, a maximum of activity at pH 7.3, and a Vmax of 111 nmol/min/mg of protein at 20 degrees C. Trimethyl tin inhibits the ATPase at much lower concentrations than dicyclohexylcarbodiimide, whereas oligomycin, reserpine, and other inhibitors were without effect. In intact chromaffin granules, the ATPase activity was stimulated up to 300% by collapsing the H+ transmembrane gradients. H+/ATP stoichiometry was measured in resealed chromaffin ghosts devoid of ATP and catecholamines under conditions where no net pH changes occur upon ATP hydrolysis. After addition of ATP, the rates of H+ accumulation in the ghosts and ATP hydrolysis were both linear for about 60-100 s, and the ratio of H+ to ATP was 1.71. These data indicate that the H+ ATPase of chromaffin granules has both kinetic similarities and dissimilarities with other known H+ ATPases. The regulation by changes in H+ gradients and the fixed H+/ATP ratio of this ATPase is further evidence of its primary role in establishing electrogenic H+ translocation and H+ gradients in chromaffin granules.