Fusion of phospholipid vesicles mediated by cytochrome c and apocytochrome cCytochrome c 와 apocytochrome c 에 의해 유도되는 인산지방질막의 융합

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Fusion of phosphatidylserine/phosphatidylethanolamine (1:1) vesicles induced by cytochrome c is studied at a wide range of pH values. A pH profile for the fusion with maximum values at pH 5 and pH 8 is obtained and this is found to be similar to the profile for cytochrome c binding to the vesicles. The binding property of apocytochrome c to the same phospholipid vesicles is found to be about the same as that of cytochrome c at low ionic strength, but very different at high salt concentrations. No appreciable fusion of vesicles by apocytochrome c is observed. A segment of each protein is found to be resistant to the proteolytic digestion when incubated with the vesicles. Hydrophobic labeling with dansyl chloride supports that this fragment indeed penetrates into the hydrophobic interior of bilayer. The molecular weights of penetrated fragments of cytochrome c and apocytochrome c determined by SDS-PAGE are found to be approximately 3,000 and 5,500, respectively, for each pH value studied. The amino acid compositions of these segments were determined, and from these the sequences of fragments, which appear to be inserted into the bilayer, are deduced. The residues from 80(Met) to 99(Lys) of cytochrome c, and from 55(Lys) to 104(Glu) of apocytochrome c appear to penetrate the bilyaer. In order to obtain more information about the topology of the penetrating fragments, several hydrophobic and hydrophilic labelings were used. Hydrophobic labeling by [$^{125}I$]TID confirms that these fragments hydrophobically interact with bilayer. Studies with photoreactive label positioned near the surface and also at the interior of bilayer demonstrate that apocytochrome c penetrate deep into the bilayer while cytochrome c interact mostly with the surface region of the bilayer. Comparative studies with a hydrophobic label PITC in the presence and absence of its hydrophilic analogue p-sulfo-PITC supports the above topology. It is also found that the apocytochrome c placed outside o...
Kim, Hyoung-Man김형만
한국과학기술원 : 생물공학과,
Issue Date
61315/325007 / 000845226

학위논문(박사) - 한국과학기술원 : 생물공학과, 1989.2, [ x, 125 p. ]

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