Pulmonary surfactant is a lipid-rich material that promotes alveolar stability by lowering the surface tension at the air-fluid interface in the peripheral air spaces. The turnover of surfactant phospholipids in the alveolar space is fast, and several lines of evidence suggest there is rapid formation and replenishment of the phospholipid surface film during normal respiration. Specific proteins may regulate these dynamic surface properties. The predominant surfactant protein is a well-characterized, lipid-associated glycoprotein, SP [28][29][30][31][32][33][34][35][36] Pulmonary surfactant is a lipid-rich material secreted as tightly packed lamellae into the extracellular alveolar fluid layer (1). Within the alveolar space, surfactant lipids are found in a number of different structural forms, including lamellar bodies, tubular myelin, and various vesicular structures (2). Although the lipid compositions of the surfactant structures are similar, the physical properties, particularly the ability ofthe lipoprotein complexes to form a surface film, are quite different (3). Specific proteins appear to influence the structure and surface activity of surfactant-lipid complexes (4-10).The predominant surfactant-associated protein is the glycoprotein SP 28-36 (28-36 kDa) characterized by a collagenlike NH2-terminal domain and variable N-linked glycosylation of the COOH-terminal region (11-13). SP 28-36 is water soluble but readily associates with phospholipids (PLs) (14). In the presence of calcium, SP 28-36 causes PL aggregation and increases the rate of adsorption of surfactant lipids to an air-fluid interface (6,8,14). A second group of very hydrophobic proteins has been identified in lamellar bodies isolated from lung homogenate and in surfactant isolated from the bronchoalveolar wash (4, 9, 10, 15-17). Very little is known about the homogeneity, structure, or function ofthis group of hydrophobic proteins, but it has been reported that at least one of these proteins enhances PL surface film formation (9,10,18 (20). The surfactant in water (-16 mg of PL per ml, 2 mg of protein per ml) was extracted in 1-butanol (1:50, vol/vol) at room temperature (21). The surfactant/butanol mixture was spun twice at 10,000 X gav for 20 min to sediment the butanolinsoluble protein (94% of the total). The butanol supernatant was dried by rotary evaporation and the residue was resuspended in chloroform/methanol/0.1 M HCl, 1:1:0.5, vol/vol). A small amount of insoluble material was removed by centrifugation and the supernatant, containing 30 mg ofPL in 1 ml, was applied to a 1 cm x 45 cm column of Sephadex and eluted at 4 ml/hr with the same solvent at 40C. The eluted fractions, 0.5 ml, were assayed for protein (22) in the presence of 1% NaDodSO4, phosphorus (23) for the calculation of the PL content, and cholesterol (24). An aliquot of each fraction containing protein was analyzed by NaDodSO4/PAGE (25) and silver staining (26). 66The publication costs of this article were defrayed in part by page charge payment. This article...

Pulmonary surfactant is a phospholipid-protein complex which serves to lower the surface tension at the air-liquid interface in the alveoli of the mammalian lung and is essential for normal respiration. Inadequate levels of surfactant at birth, a frequent situation in premature infants, results in respiratory failure. In all species examined, surfactant is composed primarily of dipalmitoylphosphatidylcholine and two major protein species of relative molecular mass (Mr) 32,000 (32K) and 10K (refs 2-5). Reconstitution in vitro of purified 32K pulmonary surfactant apoprotein (PSAP) with synthetic lipids forms a lipoprotein complex that lowers surface tension by spreading to create a thin interfacial film. Here we describe the cloning of the human PSAP gene and complementary DNA, and discuss features of the unusual encoded protein.

The adsorptive properties of phospholipids of pulmonary surfactant are markedly influenced by the presence of three related proteins (26-38 KD, reduced) found in purified surfactant. Whether these proteins are pre-assembled with lipids before secretion is uncertain but would be expected for a lipoprotein secretion. We performed indirect immunocytochemistry on frozen thin sections of rat lung to identify cells and intracellular organelles that contain these proteins. The three proteins, purified from lavaged surfactant, were used to generate antisera in rabbits. Immunoblotting of rat surfactant showed that the IgG reacted with the three proteins and a 55-60 KD band which may be a polymer of the lower MW species. Specific gold labeling occurred over alveolar type II cells, bronchiolar Clara cells, alveolar macrophages, and tubular myelin. In type II cells labeling occurred in synthetic organelles and lamellar bodies, which contain surfactant lipids. Lamellar body labeling was increased fivefold by pre-treating tissue sections with a detergent. Multivesicular bodies and some small apical vesicles in type II cells were also labeled. Secondary lysosomes of alveolar macrophages were immunoreactive. Labeling in Clara cells exceeded that of type II cells, with prominent labeling in secretory granules, Golgi apparatus, and endoplasmic reticulum. These observations clarify the organelles and pathways utilized in the elaboration of surfactant. After synthesis, the proteins move, probably via multivesicular bodies, to lamellar bodies. Both lipids and proteins are present in tubular myelin. Immunologically identical or closely similar proteins are synthesized by Clara cells and secreted from granules which appear not to contain lipid. The role of these proteins in bronchiolar function is unknown.

Pulmonary surfactant is a lipid-protein complex that promotes alveolar stability by lowering the surface tension at the air-fluid interface in the peripheral air spaces. A group of hydrophobic surfactant-associated proteins has been shown to be essential for rapid surface film formation by surfactant phospholipids. We have purified a hydrophobic surfactant protein of :5 kDa that we term SP5 from bronchopulmonary lavage fluid from a patient with alveolar proteinosis and shown that it promotes rapid surface film formation by simple mixtures of phospholipids. We have derived the full amino acid sequence of human SP5 from the nucleotide sequence of cDNAs identified with oligonucleotide probes based on the NH2-terminal sequence of SP5. SP5 isolated from surfactant is a fragment of a much larger precursor protein (21 kDa). The precursor contains an extremely hydrophobic region of 34 amino acids that comprises most of the mature SP5. This hydrophobicity explains the unusual solubility characteristics of SP5 and the fact that it is lipid-associated when isolated from lung.Pulmonary surfactant is a phospholipid-protein complex that lowers surface tension at the air-liquid interface in the alveolus (1). The lipid composition of surfactant has been studied in detail (2), and the major lipid components by weight are dipalmitoylphosphatidylcholine, monoenoic phosphatidylcholine, and phosphatidylglycerol. Four surfactant protein species have been identified in canine and human bronchoalveolar lavage. The most abundant species is a glycoprotein with a collagen-like 4). This protein has been shown to enhance uptake of surfactant lipids into alveolar type II cells (5) and inhibit secretion of surface-active material from these cells (6). The three other surfactant proteins, SP5 (5 kDa), SP8 (8 kDa), and SP18 (18 kDa), are very hydrophobic and have proved difficult to purify to homogeneity. The NH2-terminal amino acid sequences of the canine hydrophobic proteins have been determined, and it was shown that SP5 and SP8 share a common NH2 terminus (7). The complete amino acid sequence of SP18 deduced from canine and human cDNA sequences does not contain any region corresponding to the NH2 terminus of SP5 or SP8 (7,8).Various groups have studied the ability of the surfactant proteins to enhance surface activity of phospholipids in vitro using a surface balance. It has been shown that the small molecular weight hydrophobic proteins isolated from bovine surfactant enhance surface film formation by phospholipids (9-12). We have shown that mixtures of SP5, -8, and -18 or that SP18 alone, isolated from canine surfactant, stimulated phospholipid surface film formation (7). Unfortunately, detailed comparison of data between groups is difficult due to different methods of isolation and characterization of surfactant proteins.In this article we report the effect of purified human SP5 on the surface activity of simple phospholipid mixtures and the amino acid sequence of the precursor of SP5 derived from the sequences of near full-leng...

Pulmonary surfactant is a mixture of phospholipids and proteins which stabilizes lung alveoli and prevents respiratory failure. The surfactant-associated protein of Mr = 28,000-36,000 (SP-A) influences the structure, function (film formation), and metabolism of surfactant. We have characterized glucocorticoid regulation of SP-A and SP-A mRNA in explants of fetal human lung. The time course of response to dexamethasone was biphasic, with early stimulation and later inhibition of SP-A accumulation. Maximal induction of SP-A occurred with 3-10 nM dexamethasone and =300 nM cortisol for 72 hr, and stimulation diminished at higher concentrations. SP-A mRNA accumulation was maximally stimulated at 24 -48 hr of exposure to dexamethasone (10 nM) and was generally inhibited by 4-6 days. Stimulation was also observed with cortisone and corticosterone but not with sex steroids, suggesting a receptor-mediated process. When explants were exposed to cortisol for only 24 hr, SP-A content was transiently increased above the level in continuously treated tissue and subsequently was similar to control. The content of SP-A and its mRNA was also increased by dibromo-cAMP, terbutaline, and forskolin, and effects were approximately additive with those of dexamethasone. However, elevated in tracellular cAMP did not alter the biphasic time course or dose-response patterns of dexamethasone. We propose that glucocorticoids have both stimulatory and inhibitory effects on SP-A gene expression. This biphasic regulation is not consistent with generalized toxic effects, product-feedback inhibition, or receptor down-regulation, and it appears to be specific for SP-A among the various surfactant components.Pulmonary surfactant, which is composed of both proteins and lipids, lowers surface tension in alveoli and is necessary for normal lung function. The surfactant-associated proteins are thought to have important roles in modifying surfactant phospholipid function and metabolism (1-7). The largest of these proteins, surfactant protein A (SP-A: Mr = 28,000-