Van Dyke R W
Department of Medicine, University of California Medical Center, San Francisco 94143.
J Biol Chem. 1988 Feb 25;263(6):2603-11.
Endocytic vesicles possess an electrogenic proton pump, and measurements of ATPase activity suggest that Cl- may stimulate proton pump activity. This study was undertaken to measure the steady-state pH, potential (delta psi), and total proton electrochemical gradients established by the rat liver multivesicular body (MVB) proton pump and to examine the effects of Cl- (0.5-140 mM) on these gradients. Radiolabeled [( 14C] methylamine and 36Cl-) and fluorescent (fluorescein isothiocyanate-conjugated low density lipoproteins) probes were used to assess internal pH (pHi) and delta psi. In the absence of ATP, pHi averaged 7.37 +/- 0.05 (extracellular pH 7.31 +/- 0.02), and delta psi ranged from -32 to -71 mV; but neither pHi nor delta psi varied consistently with [Cl-]. In the presence of ATP, pHi decreased progressively with increasing [Cl-] to a plateau value of about 5.89 at greater than or equal to 25 mM Cl-, and MVB exhibited an interior positive delta psi that was maximal at the lowest Cl- concentration (+65.5 mV) and decreased as medium Cl- increased. The total ATP-dependent proton electrochemical gradient (proton-motive force (delta p] averaged 118.0 +/- 4.3 mV and did not change in any consistent manner as [Cl-] varied almost 300-fold. However, initial rates of MVB acidification increased with increasing [Cl-]. These studies indicate that: (a) in the absence of ATP, isolated MVB exhibited a negative delta psi, probably a Donnan potential; (b) in the presence of ATP and at a [Cl-] similar to that in hepatocyte cytoplasm (25 mM), MVB pHi was 5.89, and delta psi was +9.6 mV; and (c) over the range of [Cl-] tested, the magnitudes of delta pH and delta psi were inversely related, apparently related to Cl- availability, but the ATP-dependent delta p did not vary. Therefore, it is concluded that Cl- increases the initial rate of vesicle acidification in MVB and also affects the relative chemical and electrical contributions of the steady-state proton pump-determined delta p. Cl-, however, does not alter steady-state delta p.
内吞小泡具有一种生电质子泵，对ATP酶活性的测量表明Cl-可能刺激质子泵活性。本研究旨在测量大鼠肝脏多囊泡体（MVB）质子泵建立的稳态pH、电位（Δψ）和总质子电化学梯度，并研究Cl-（0.5 - 140 mM）对这些梯度的影响。使用放射性标记的[14C]甲胺和36Cl-以及荧光（异硫氰酸荧光素偶联的低密度脂蛋白）探针来评估内部pH（pHi）和Δψ。在没有ATP的情况下，pHi平均为7.37±0.05（细胞外pH 7.31±0.02），Δψ范围为-32至-71 mV；但pHi和Δψ均未随[Cl-]一致变化。在有ATP的情况下，随着[Cl-]增加，pHi逐渐降低，在Cl-浓度大于或等于25 mM时达到约5.89的平台值，并且MVB表现出内部正Δψ，在最低Cl-浓度时最大（+65.5 mV），并随着介质Cl-增加而降低。总的ATP依赖性质子电化学梯度（质子动力势（Δp））平均为118.0±4.3 mV，并且在[Cl-]变化近300倍时没有以任何一致的方式改变。然而，MVB酸化的初始速率随着[Cl-]增加而增加。这些研究表明：（a）在没有ATP的情况下，分离的MVB表现出负Δψ，可能是唐南电位；（b）在有ATP且[Cl-]与肝细胞质中的[Cl-]相似（25 mM）时，MVB的pHi为5.89，Δψ为+9.6 mV；（c）在所测试的[Cl-]范围内，ΔpH和Δψ的大小呈负相关，显然与Cl-的可用性有关，但ATP依赖性Δp没有变化。因此，得出结论，Cl-增加了MVB中小泡酸化的初始速率，并且还影响了稳态质子泵确定的Δp的相对化学和电贡献。然而，Cl-不会改变稳态Δp。
