Biophys J, November 2000, p. 2657-2666, Vol. 79, No. 5

Pulmonary Surfactant Protein A Interacts with Gel-Like Regions in Monolayers of Pulmonary Surfactant Lipid Extract

Lynn-Ann D. Worthman,^* Kaushik Nag,^* Nathan Rich, Miguel L. F. Ruano, Cristina Casals, Jesus Pérez-Gil, and Kevin M. W. Keough^*§

Departments of  ^*Biochemistry and  Physics, and  ^§Discipline of Pediatrics, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3X9, Canada; and the  Departmento de Bioquimica y Biologia Molecular I, Facultad de Biologia, Universidad Complutense, 28040 Madrid, Spain

Epifluorescence microscopy was used to investigate the interaction of pulmonary surfactant protein A (SP-A) with spread monolayers of porcine surfactant lipid extract (PSLE) containing 1 mol % fluorescent probe (NBD-PC) spread on a saline subphase (145 mM NaCl, 5 mM Tris-HCl, pH 6.9) containing 0, 0.13, or 0.16 µg/ml SP-A and 0, 1.64, or 5 mM CaCl₂. In the absence of SP-A, no differences were noted in PSLE monolayers in the absence or presence of Ca²⁺. Circular probe-excluded (dark) domains were observed against a fluorescent background at low surface pressures ( ~5 mN/m) and the domains grew in size with increasing . Above 25 mN/m, the domain size decreased with increasing . The amount of observable dark phase was maximal at 18% of the total film area at  ~25 mN/m, then decreased to ~3% at  ~40 mN/m. The addition of 0.16 µg/ml SP-A with 0 or 1.64 mM Ca²⁺ in the subphase caused an aggregation of dark domains into a loose network, and the total amount of dark phase was increased to ~25% between  of 10-28 mN/m. Monolayer features in the presence of 5 mM Ca²⁺ and SP-A were not substantially different from those spread in the absence of SP-A, likely due to a self-association and aggregation of SP-A in the presence of higher concentrations of Ca²⁺. PSLE films were spread on a subphase containing 0.16 µg/ml SP-A with covalently bound Texas Red (TR-SP-A). In the absence of Ca²⁺, TR-SP-A associated with the reorganized dark phase (as seen with the lipid probe). The presence of 5 mM Ca²⁺ resulted in an appearance of TR-SP-A in the fluid phase and of aggregates at the fluid/gel phase boundaries of the monolayers. This study suggests that SP-A associates with PSLE monolayers, particularly with condensed or solid phase lipid, and results in some reorganization of rigid phase lipid in surfactant monolayers.

Biophys J, November 2000, p. 2657-2666, Vol. 79, No. 5
© 2000 by the Biophysical Society   0006-3495/00/11/2657/10  $2.00

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