Surface properties of membrane systems. Transport of staphylococcal delta-toxin from aqueous to membrane phase.

Hemolytic delta-toxin from Staphylococcus aureus was soluble in either water, methanol or chloroform/methanol (2 : 1, v/v). The toxin spread readily from distilled water into films with pressures (pi) of 10 dynes/cm on water and 30 dynes/cm on 6 M urea; from chloroform/methanol it produced 40 dynes/cm pressure on distilled water. The toxin adsorbed barely from water (pi = 1 dyne/ cm) but it did rapidly from 6 M urea (pi = 35 dynes/cm). The protein films had unusually high surface potentials, which increased with the film pressure and decreased with increasing both pH and urea concentration in the aqueous phase. The fluorescence of 1-aniline 8-naphthalene sulfonate with delta-toxin was much greater than that with RNAase and dipalmitoyl phosphatidylcholine itself, indicating probably a marked lipid-binding character of the toxin. By circular dichroism the alpha-helix content of delta-toxin was 42% in water, 45% in methanol, 24% in 6 M urea. Infrared spectroscopy showed predominant alpha-helix in both 2H2O and deuterated chloroform/methanol as well as in films spread from either solvent on 2H2O. In spreading from 6 M [2H]urea, in which the major infrared absorption was that of [2H]urea with peaks at 1600 and 1480 cm(-1), the delta-toxin film showed prevalently non-alpha-helix structures with major peak intensities at 1633 cm(-1) > 1680 cm(-1), indicating the appearance of new beta-aggregated and beta-antiparallel pleated sheet structures in the film. The data prove that (1) high pressure protein films can consist of alpha-helix as well as non-alpha-helix structures and, differently from another cytolytic protein, melittin, delta-toxin does not resume the alpha-helix conformation in going into the film phase from the extended chain in 6 M urea; (2) conformational changes are important in the transport of proteins from aqueous to lipid or membrane phase; (3) delta-toxin is by far more versatile in structural dynamics and more surface active than alpha-toxin.

Isolation of secretory IgA from a lung surfactant fraction.

Although dipalmitoyl lecithin, is the essential component of the pulmonary surfactant system that is invoked for alveolar stability, there is no explanation as yet for the origin and role of certain proteins that are found with the phospholipid in pulmonary washings. Aqueous lavages obtained from rabbit lung contain three major proteins, two of which are serum proteins (albumin 60%, gamma-globulin 10%), and the third, protein "T" (20%), is described here as pulmonary secretory immunoglobulin A (sIgA1. The protein is first recovered quantitatively in the surface active lipid-protein fraction from filtration of pulmonary lavage on Sephadex G-200. The protein is then isolated from the lipid either by filtration on SDS-Sephadex G-200 or by ethanol-ether precipitation. After reductive cleavage with either mercaptoethanol or dithiothreitol and alkylation with iodoacetamide, three protein peaks are obtained from SDS-sephadex G-200 separation. The products of reductive cleavage are analogous to the secretory component (MW approximately 60,000), H-chain (MW approximately 50,000), L-chain (MW approximately 25,000), and J-piece (MW approximately 28,000) of secretory IgA from colostrum. In immunodiffusion the lung protein and colostrum sIgA show striking identity lines, as do antibodies to rabbit colostrum and to the lung protein. Amino acid and carbohydrate analyses reveal some differences between the two secretory immunoglobulins. Although this protein has been found together with the phospholipid surfactant of the lung in vitro, the present structural study concludes that the protein is sIgA. Although concurrent immunofluorescence studies showed that this protein is in the alveolar lining layer, we cannot as yet conclude that it belongs to the surfactant system of the lung.