Ambudkar I S, Baum B J
Clinical Investigations and Patient Care Branch, National Institute of Dental Research, Bethesda, Maryland 20892.
J Membr Biol. 1988 Apr;102(1):59-69. doi: 10.1007/BF01875353.
The ATP-dependent Ca2+ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum, Biochem. J. 227:239-245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg2+. Phosphate (5 mM), but not oxalate (5 mM), increased maximum Ca2+ accumulation by 50%. Half-maximal Ca2+ transport was achieved at approximately 70 nM Ca2+ in EGTA-buffered medium while maximal activity required greater than 1 microM Ca2+ (Vmax = 54 nmol/mg protein/min). Optimal rates of Ca2+ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) approximately 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca2+ transport could be significantly altered by preimposed membrane potentials generated by K+ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by approximately 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca2+ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca2+ transport in the absence of a pH gradient, pHi = 7.5/pHo = 7.5; the activity was approximately 60% higher in the presence of an outwardly directed pH gradient, pHi = 7.5/pHo = 8.5; while it was approximately 80% lower when an inwardly directed pH gradient was imposed, pHi = 7.5/pHo = 6.2. The data show that the ATP-dependent Ca2+ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca2+ uptake, which could be compensated by other ion movements.
对从大鼠腮腺分离得到的基底外侧膜翻转囊泡所表现出的ATP依赖的Ca2+转运活性（T. 泷间、B.L. 库亚特和B.J. 鲍姆，《生物化学杂志》227:239 - 245，1985年）进行了进一步表征。该活性依赖于Mg2+。5 mM的磷酸盐可使最大Ca2+积累量增加50%，而5 mM的草酸盐则无此作用。在EGTA缓冲介质中，约70 nM的Ca2+可实现半数最大Ca2+转运，而最大活性需要大于1 μM的Ca2+（Vmax = 54 nmol/mg蛋白质/分钟）。在KCl存在的情况下可获得最佳的Ca2+转运速率，而在无KCl的介质（甘露醇或蔗糖）中，可达到总活性的约40%，且该活性不受FCCP刺激。在缬氨霉素存在的情况下，由K+梯度产生的预施加膜电位可显著改变Ca2+转运的初始速率。与无膜电位时的转运速率相比，负（内）电位刺激摄取增加约30%，而正（内）电位则抑制摄取。在无KCl的情况下，施加pH梯度也可改变Ca2+摄取的初始速率。与无pH梯度时Ca2+转运的初始速率相比，pHi = 7.5/pHo = 7.5；在存在外向pH梯度pHi = 7.5/pHo = 8.5时，活性约高60%；而在施加内向pH梯度pHi = 7.5/pHo = 6.2时，活性约低80%。数据表明，基底外侧膜翻转囊泡中ATP依赖的Ca2+转运可被膜电位调节，因此表明在Ca2+摄取过程中有电荷转移到囊泡中，这可由其他离子移动来补偿。
