MECHANISM OF LACTOSE TRANSLOCATION IN PROTEOLIPOSOMES RECONSTITUTED WITH LAC CARRIER PROTEIN PURIFIED FROM ESCHERICHIA-COLI .1. EFFECT OF PH AND IMPOSED MEMBRANE-POTENTIAL ON EFFLUX, EXCHANGE, AND COUNTERFLOW
MECHANISM OF LACTOSE TRANSLOCATION IN PROTEOLIPOSOMES RECONSTITUTED WITH LAC CARRIER PROTEIN PURIFIED FROM ESCHERICHIA-COLI .1. EFFECT OF PH AND IMPOSED MEMBRANE-POTENTIAL ON EFFLUX, EXCHANGE, AND COUNTERFLOW
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DOI：
10.1021/bi00279a033
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发表时间：
1983-01-01
期刊：
影响因子：
2.9
通讯作者：
KABACK, HR
中科院分区：
文献类型：
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作者：
GARCIA, ML;VIITANEN, P;KABACK, HR
用纯化的紫胶载体经辛基葡萄糖稀释和冻融/超声重组的蛋白脂质体是单层的，直径50- 150nm，不渗透离子。利用该制剂，利用载体介导的乳糖沿浓度梯度的外排来探讨- β的机制。半乳糖苷易位。最大外排速率与pH有关，从pH 5.5到7.5增加约20倍。在相同条件下用等摩尔乳糖在外部介质中进行的实验（即在交换条件下）表明，交换反应对pH值不敏感，相对于外排非常快。在乳糖外排过程中发生质子同位，导致膜电位（. delta…psi）的短暂形成。（内部为负）。离子载体valinomycin和羰基氰化物间氯苯腙提高了外排速率，蛋白脂质体在valinomycin存在下表现出外排依赖性的铷积累。施加。delta…psi。（内部负）将最大流出率延迟了10倍，而对表观Km没有影响。外排反应和交换反应的比较表明，外排的速率决定步骤对应于一个反应，涉及到卸载载流子返回到膜的内表面，并且从载流子中丢失对称质子或卸载载流子的易位可能是有限的。在不同pH值下进行的逆流实验表明，外部乳糖会影响载体的质子损失。当外部乳糖的浓度低于载体的表观Km时，逆流是pH依赖性的，从pH 5.5降低到7.5，表明在这些条件下，载体的去质子化经常发生，以限制逆流。当外部乳糖饱和时，初始逆流速率和超调幅度基本上不受ph的影响。在这些条件下，乳糖外排被消除。这一结果证实并扩展了早期的右向外膜囊泡的观察结果，后者提出了乳糖外排是一种有序的机制，并且在逆流和交换过程中，负载载体以质子化形式再循环。
Proteoliposomes reconstituted with purified lac carrier by octyl glucose dilution and freeze-thaw/sonication are unilamellar, 50-150 nm in diameter and impermeable to ions. By use of this preparation, carrier-mediated lactose efflux down a concentration gradient was used to probe the mechanism of .beta.-galactoside translocation. The maximum rate of efflux is pH dependent, increasing about 20-fold from pH 5.5 to 7.5. Experiments performed under identical conditions with equimolar lactose in the external medium (i.e., under exchange conditions) demonstrate that the exchange reaction is insensitive to pH and extremely fast relative to efflux. Proton symport occurs during lactose efflux, resulting in transient formation of a membrane potential (.DELTA..psi., interior negative). The ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone enhance the rate of efflux and the proteoliposomes exhibit efflux-dependent accumulation of rubidium in the presence of valinomycin. Imposition of a .DELTA..psi. (interior negative) retards the maximum rate of efflux by as much as 10-fold with no effect on apparent Km. Comparison of efflux and exchange reactions suggests that the rate-determining step for efflux corresponds to a reaction involving return of the unloaded carrier to the inner surface of the membrane and that either loss of the symported proton from the carrier or translocation of the unloaded carrier may be limiting. Counterflow experiments conducted at various pH reveal that external lactose affects proton loss from the carrier. When external lactose is present at concentrations below the apparent Km of the carrier, counterflow is pH dependent and decreases from pH 5.5 to 7.5, indicating that deprotonation of the carrier occurs frequently under these conditions to limit counterflow. When external lactose is saturating, the initial rate of counterflow and the magnitude of the overshoot are essentially unaffected by pH. The transient formation of .DELTA..psi. observed during lactose efflux is abolished under these conditions. The results confirm and extend earlier observations with right-side-out membrane vesicles which led to the suggestion that lactose efflux is an ordered mechanism and that the loaded carrier recycles in the protonated form during counterflow and exchange.