| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1986: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1985: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
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.
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