Characterization of the Na+/H+ antiporter of alkalophilic bacilli in vivo: delta psi-dependent 22Na+ efflux from whole cells.
Characterization of the Na+/H+ antiporter of alkalophilic bacilli in vivo: delta psi-dependent 22Na+ efflux from whole cells.
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体内嗜碱杆菌 Na /H 逆向转运蛋白的表征：全细胞中 δ psi 依赖性 22Na 流出。
DOI：
10.1128/jb.156.3.1151-1157.1983
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发表时间：
1983
影响因子：
3.2
通讯作者：
Krulwich,TA
中科院分区：
文献类型：
--
作者：
Garcia,ML;Guffanti,AA;Krulwich,TA
本文研究嗜碱芽孢杆菌的Na~+/H~+逆向转运蛋白，方法是通过测量饥饿的氰化物抑制的细胞的~(22)Na~+外流，这些细胞是由呋喃霉素诱导的钾扩散电位，正向输出(Δpsi)。在没有βpsi的情况下，pH为9.0时~(22)Na~+外流缓慢，N-乙基马来酰亚胺对~(22)Na~+外流有明显的抑制作用。在施加增量psi后，出现了非常快的22Na+外流速率。这一快速的~(22)Na~+流出速率被Li~+竞争性抑制，并与βpsi的大小成正比。与嗜碱芽孢杆菌和嗜碱硬芽孢杆菌RAb的动力学实验表明，Delta psi导致22Na+外流的Vmax显著增加。Na+的Km值不受增量psi的影响。当施加pH为7.0的βPsi时，pH为7.0的缓慢的22Na+外流速率是由βPsi引起的。这表明，Na+/H+逆向转运蛋白在pH值为7.0时的失活并不次于在该pH值下呼吸产生的低增量psi。事实上，相对较高的内部质子浓度似乎抑制了22Na+的外流活动。相反，在一定的内部pH下，当外部pH为7.0~9.0时，酶活性变化不大，当外部pH为10.0时，22Na+外流速率下降。在典型pH值下生长的这种下降可能是由于质子不足，而扩散势而不是呼吸作用是驱动力。嗜碱芽孢杆菌和坚硬芽孢杆菌RAb的非嗜碱突变株的外流速度较慢，在pH 7.0或9.0时，增量psi均不能促进其外流。
The Na+/H+ antiporter of Bacillus alcalophilus was studied by measuring 22Na+ efflux from starved, cyanide-inhibited cells which were energized by means of a valinomycin-induced potassium diffusion potential, positive out (delta psi). In the absence of a delta psi, 22Na+ efflux at pH 9.0 was slow and appreciably inhibited by N-ethylmaleimide. Upon imposition of a delta psi, a very rapid rate of 22Na+ efflux occurred. This rapid rate of 22Na+ efflux was competitively inhibited by Li+ and varied directly with the magnitude of the delta psi. Kinetic experiments with B. alcalophilus and alkalophilic Bacillus firmus RAB indicated that the delta psi caused a pronounced increase in the Vmax for 22Na+ efflux. The Km values for Na+ were unaffected by the delta psi. Upon imposition of a delta psi at pH 7.0, a retardation of the slow 22Na+ efflux rate at pH 7.0 was caused by the delta psi. This showed that inactivity of the Na+/H+ antiporter at pH 7.0 was not secondary to a low delta psi generated by respiration at this pH. Indeed, 22Na+ efflux activity appeared to be inhibited by a relatively high internal proton concentration. By contrast, at a constant internal pH, there was little variation in the activity at external pH values from 7.0 to 9.0; at an external pH of 10.0, the rate of 22Na+ efflux declined. This decline at typical pH values for growth may be due to an insufficiency of protons when a diffusion potential rather than respiration is the driving force. Non-alkalophilic mutant strains of B. alcalophilus and B. firmus RAB exhibited a slow rate of 22Na+ efflux which was not enhanced by a delta psi at either pH 7.0 or 9.0.