STUDIES ON ACTIVE TRANSPORT CARRIER OF TETRACYCLINE IN RESISTANT ESCHERICHIA COLI USING RECONSTITUTED MEMBRANE CONTAINING BACTERIORHODOPSI

首先，利用从抗性细胞中制备的内外膜囊泡(ISO囊泡)研究了四环素(TC)主动外排在抗性细胞中的能量耦合。TC被ISO囊泡摄取主要受pH的驱动，因为pH破坏剂黑素抑制了TC的摄取，而瓦林霉素不抑制TC的摄取(1)。TC和质子反向转运的化学计量比为1：1。然而，随后的研究表明，即使在没有pH的情况下，TC的吸收也可以由人工驱动。当使用&lt；DELTAPSI&>作为驱动力时，观察到&lt；DELTAPSI&GT；的阈值(17 MV)。另一方面，当pH<50 mV时，转运系统对TC的亲和力显著降低。然后，用&lt；^(35)S&>蛋氨酸标记鉴定了一种TC载体蛋白(Tet A)。将&lt；tc；r&gt；基因克隆到多拷贝质粒中，制备Maxicell。用~lt；^(35)S和~(35)-蛋氨酸特异性标记了编码在该质粒上的蛋白质。Tet A分子量为35,000，可被辛基葡萄糖苷增溶，合成了含14个氨基酸的Tet A蛋白C端多肽，并制备了抗体，该抗体可识别Tet A为抗原。用纯化的抗体用亲和柱层析法纯化Tet A，并将纯化的Tet A掺入含有细菌视紫红质的脂质体中。

At first, the energy coupling of the active efflux of tetracycline (TC) in resistant cells was studied using inside-out membrane vesicles (ISO vesicle) prepared from resistant cells. The uptake of TC into the ISO vesicle was mainly driven by <DELTA> pH because nigericin, a <DELTA> pH destroyer, inhibited the TC uptake although valinomycin, a <DELTAPSI> destroyer, did not inhibit the uptake (1). The stoichiometry of the counter transport of TC and proton was 1:1. Subsequent studies, however, revealed that the TC uptake could be driven by artificial <DELTAPSI> even in the absence of <DELTA> pH. When <DELTAPSI> was used as a driving force, a threshold value of <DELTAPSI> (17 mV) was observed. On the other hand, <DELTA> pH showed no such a threshold, but the affinity of the transport system to TC was significantly reduced when <DELTA> pH was less than 50 mV.Then, a TC carrier protein (Tet A) was identified using <^(35)S> -methionin labelling. <TC^r> gene was cloned in multicopy plasmid and then maxicell was prepared. Proteins encoded on the plasmid was specifically labelled with <^(35)S> -methionin. Tet A had a molecular weight of 35,000 and could be solubilized by octylglucoside.A C-terminal peptide of Tet A protein containing 14 amino acids was synthesized and an antibody against this peptide was prepared, The anibody could recognize the Tet A as an antigen. Tet A was purified by an affinity column chromatography using the purified antibody.Incorporation of purified Tet A into bacteriorhodopsin-containing liposomes is now under study.