Lipid bilayer surface association of lung surfactant protein SP-B, amphipathic segment detected by flow immunofluorescence.
Lung surfactant protein, SP-B, and synthetic amphipathic peptides derived from SP-B were studied in model lung surfactant lipid bilayers by immunofluorescent labeling. Liposomes were formed by hydrating a lipid film on the glass viewing port of a temperature controlled flow chamber. Membrane associated peptides were detected by epifluorescence optical microscopy of the binding of anti-peptide polyclonal monospecific antibodies and FITC-conjugated secondary antibodies added to buffer contained in the flow chamber. Liposomes were bound by antibody to residues 1-25 of SP-B if formed from lipid films containing the 1-25 peptide, (SP-B(1-25)), or if SP-B(1-25) was added to already formed liposomes in buffer solution. The distribution of antigen-antibody complex was temperature dependent with aggregation occurring at greater than or equal to 30 degrees C. Surface association was not detected in liposomes formed from lipid films containing the 49-66 peptides (SP-B(49-66)), using an antibody to the 49-66 peptide, or to a synthetic version of the SP-B protein, (SP-B(1-78)), using both antibodies to the 49-66 peptide and the 1-25 peptide. The detection of SP-B(1-78) with antibody to the 49-66 sequence was only possible after reducing SP-B(1-78) with dithiothreitol, suggesting that the COOH-terminus of the full monomer protein is accessible to the bulk aqueous environment unlike the COOH-terminal peptide. The size, number of layers, and fluidity of the liposomes were not altered by protein or peptides, although they were affected by lipid composition and temperature.
Longo
M L
ML
Department of Chemical and Nuclear Engineering, University of California, Santa Barbara 93106.
Waring
A
A
Zasadzinski
J A
JA
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D005455:Fluorescent Antibody Technique; D008051:Lipid Bilayers / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010715:Phosphatidylglycerols / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011510:Proteolipids / Q000276:immunology / Q000378:metabolism*; D011663:Pulmonary Surfactants / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits / Q000276:immunology
D000906:Antibodies; D008051:Lipid Bilayers; D010455:Peptides; D010715:Phosphatidylglycerols; D011510:Proteolipids; D011663:Pulmonary Surfactants; D015060:1,2-Dipalmitoylphosphatidylcholine