Pulmonary surfactant contains two hydrophobic polypeptides, SP-B and SP-C, with known amino acid sequences and with truncated subforms lacking the N-terminal residues. Treatment of SP-C with KOH releases fatty acids (palmitic acid to more than 85%) in molar ratios of 1.8-2.0 relative to the polypeptide. Furthermore, plasma-desorption mass spectrometry shows native SP-C of both the intact and truncated types to be monomers with masses about 500 units higher than those expected for the polypeptide chains. After treatment with KOH, trimethylamine, or dithioerythritol, the polypeptide masses are obtained. These results prove that native SP-C is a lipopeptide with two palmitoyl groups covalently linked to the polypeptide chain. The deacylation conditions, the presence of two cysteine residues in the polypeptide, and the absence of other possible attachment sites establish that the palmitoyl groups are thioester-linked to the two adjacent cysteine residues. In contrast, the major form of porcine SP-B is a dimer without fatty acid components. That SP-C is a true lipopeptide with covalently bound palmitoyl groups suggests possibilities for functional interactions. It gives a direct physical link between SP-C and surfactant phospholipid components. Long-chain acylation may constitute a means for association of proteins with membranes and could conceivably modulate the stability and biological activity of surfactant fims.polypeptides in different subforms constituting N-terminally truncated molecules (20,21).Nevertheless, the primary structures of the polypeptides have been determined (18)(19)(20), showing SP-B to be a 79-residue polypeptide with a high content of cysteine residues, and SP-C to be a 35-residue polypeptide with two adjacent cysteine residues in a "palindromic" sequence (-Ile-Pro- Surfactant is essential for pulmonary function. It reduces the alveolar surface tension, preventing alveolar collapse, and is a prerequisite for normal respiration (1). The capacity for surfactant synthesis is limited in premature infants, and low amounts of surfactant are associated with neonatal respiratory distress syndrome. This serious disease can be treated effectively by replacement therapy through instillation of surfactant in the airways (2-7). Natural surfactant is composed of phospholipids and small amounts of specific polypeptides. In particular, two highly hydrophobic, low molecular weight polypeptides named SP-B and SP-C (8) appear to be essential components of surfactant preparations for replacement therapy (2, 3, 5-7). Both are derived from larger precursor polypeptides and have been structurally analyzed at the cDNA (9-16) and protein (17)(18)(19)(20) levels. Protein studies are essential for characterization ofthe mature polypeptides but are complicated by the extreme peptide hydrophobicities. SP-C in particular has essentially no aqueous solubility and is largely inert to conventional hydrolysis and other analytical steps. Furthermore, native preparations are heterogeneous, with the presence of the two M...

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