α-Lactalbumin (α-LA) is found to induce fusion of phosphatidylserine/phosphatidylethanolamine (1:1) vesicles at low pH. The fusogenic behavior and the binding to phospholipid vesicles of α-LA are studied at a wide range of conditions. The initial rate of α-LA-induced fusion increases with decreasing pH. The binding of α-LA to phospholipid vesicles also increases with increasing acidity below pH 6, but there is practically no binding at pH 6 and pH 7. By determining the amount of α-LA which had been incubated with the α-LA at acidic pH (pH 2, 3 and 4), released after neutralization of the vesicles to pH 7, the binding reaction is found to be partially irreversible. The amount of irreversively bound protein also increases with the decreasing pH. As the ionic strength is increased from 0.1 to 0.5, the initial rate of fusion value decreases gradually, but it approaches a finite value for each α-LA concentration. A segment of α-LA is found to be resistant to the proteolytic digestion when initially incubated with the vesicles at low pH. Hydrophobic labeling with dansyl chloride renders support that this fragment indeed penetrates into the hydrophobic interior of bilayer. The molecular weight of this segment as determined by SDS-PAGE is found to be approximately 4000 for each pH value studied. The amino acid composition of this segment was determined, and from this the sequence of α-LA fragment, which appears to be inserted into the bilayer, is deduced. This is the segment from 80 (Phe) to 108 (Lys) of α-LA. Since both the N-terminal and C-terminal of this vesicle-bound α-LA are accessible to the externally added proteolytic enzymes, it is concluded that a loop of the polypeptide segment goes into the bilayer. The α-LA segment which penetrates into PS/PE (1:1) vesicle bilayer under acidic condition was photoactively labeled with 3-(trifluoromethyl)-3-(m-(125I)iodophenyl)diazirine ((125I)TID) which had been partitioned into the hydrophobic interior of the bilayer. The hydrophobically labeled amino acid residues are identified by trypsin digestion of α-LA/vesicle complex, extraction and Edman degradation of the membrane embedded fragment. The results suggest that the segment exists in the membrane as α-helix and only one surface of this α-helix is exposed to the hydrophobic interior of the bilayer. The time-course of penetration, as determined by time-dependent (125I)TID labeling was found to coincide with the fusion kinetics.
Lysozyme is also found to induce fusion of phospholipid vesicles at a wide range of pH. The irreversible binding of lysozyme to the vesicles appears to be closely related to the lysozyme-induced fusion. An identical 6000 molecular weight segment of lysozyme is found to be protected from the tryptic digestion when initially incubated with vesicles at several pH values. Only this segment is labeled by dansyl chloride which was partitioned into the bilayer. These results suggest that a segment of lysozyme penetrates into the bilayer. The amino acid composition indicates that this is the segment from 1 (Lys) to 45 (Arg). Photoactivable labeling of the membrane-penetrating segment of lysozyme with (125I)TID and subsequent identification of the labeled residues by Edman degradation and $\gamma$-ray counting indicate, however, that the amino acids from 1 to 17 and from 32 to C-terminal are located outside of the hydrophobic core of the bilayer. Although the treatment of the membrane embedded segment with aminopeptidase failed to cleave any amino acid from the N-terminal, it appears that a loop of lysozyme segment near the N-terminal penetrates into bilayer at acidic pH. The helical wheel diagram showed that the labeling is done mainly on one surface of the α-helix. The kinetics of lysozyme penetration was also found to coincide with the kinetics.
Possible topologies of the segments of α-LA and lysozyme which penetrate into the bilayer are discussed.
α-LA과 lysozyme에 의해 유도되는 인산지방질막의 융합 현상은 이들 단백질이 인산지방질막에 결합하는 현상과 밀접한 관련이 있다. 융합곡선과 결합곡선이 같은 현상을 보인다. 또한, 이들 단백질과 인산지방질 막의 비가역적인 결합인 단백질의 일부가 막 내의 소수성 부분으로 뚫고 들어가는 정도가 일치한다. 막의 이중층 내로 뚫고 들어가는 단백질 부분은 모든 pH 에 따라 들어 가는 부분의 양에 차이가 있다. 이 사실은 SDS-PAGE 의 결과 에서 보여 준다. 인산지방질 막의 이중층 내로 뚫고 들어가는 단백질 부분의 아미노산 조성과 그 부분의 구조를 살펴 보았다. α-LA 의 경우에는 여러가지 proteolytic digestion 에 보호되는 부분의 아미노산 배열을 살펴 볼때 81 번 잔기부터 108 번 잔기까지가 막 내부로 들어가며, lysozyme 은 1번 잔기에서 45번 잔기 까지의 N-말단이 이중층내의 소수성 부분으로 뚫고 들어가는 것 같다. 실제로 이 부분들이 막의 이중층 내로 들어가는 것과 막 융합 현상 사이에 직접적인 관련이 있는 지를 살피기 위하여 kinetic study 가 hydrophobic labeling 을 이용하였다. 이 결과 α-LA 은 81-106 번 사이의 아미노산 배열이, lysozyme은 8-31 번 사이의 아미노산 배열이 이중층 내로 뚫고 들어가는 정보를 시간별로 측정하였을때 2분 이내에 정상치에 도달하였다. 2분 이전에 뚫고 들어가는 현상을 살핀 결과 가운데 부분이 먼저 뚫고 들어가며 주위 부분이 딸려 들어 가는 것 같다. 막의 이중층 내로 들어 간 부분의 각 아미노산의 hydrophobic labeling을 조사한 결과 2-3 개의 아미노산 간격으로 표지 되었다. 이 사실로 보아 막의 이중층 내부로 들어가는 단백질 부분은 나선형을 이루는 것 같다. 이 나선형은 amphiphathic 한 성격을 띤다. 융합 곡선과 시간에 따른 뚫고 들어가는 정도의 곡선은 서로 일치한 결과를 보였다. 이 사실로 보아 막내부로 뚫고 들어 간 단백질 부분이 막 융합에 필수적인 역할을 하는 것으로 추측된다.
