Abstract
Surfactant-associated protein of Mr = 35,000, SAP-35, is the major glycoprotein present in mammalian pulmonary surfactants. In this study, canine SAP-35 and several of its COOH-terminal peptides were purified and characterized by amino acid composition and NH2-terminal sequencing analysis. These proteins were then studied in terms of their specific lipid-binding characteristics and surface activity when combined with a synthetic phospholipid mixture, SM, chosen as an approximation of lung surfactant phospholipids. Purified, delipidated SAP-35 bound SM strongly. In contrast, SAP-21 (a non-collagenous fragment generated by collagenase digestion) bound phospholipid weakly; SAP-18 (an acidic COOH-terminal fragment comprising residues Gly-118 to Phe-231) did not bind phospholipid, demonstrating the importance of hydrophobic amino acid residues Gly-81 to Val-117 and the NH2-terminal collagenous domain in interaction of the SAP-35 with phospholipids. In surface activity experiments, purified SAP-35 enhanced the adsorption of SM phospholipids in terms of both rate and overall surface tension lowering. However, the adsorption facility of the SM-SAP-35 mixture did not approach that of either whole surfactant or the surfactant extract preparations, calf lung surfactant extract or surfactant-TA, used in exogenous surfactant replacement therapy for the neonatal respiratory distress syndrome. In addition, the dynamic surface activity of the SM-SAP-35 mixture was well below that of natural surfactant or surfactant extracts. This was also true of mixtures of SM phospholipids combined with the SAP-18 and SAP-21 fragments of SAP-35.
Highlights
From the $Departments of Pediatrics, University of Cincinnati, Cincinnati, Ohio 45267-0541 and $University of Rochester, Rochester, New York 14627and Abbott Laboratories, North Chicago,IIEinois 60064
SAP-21-We have recently reported an initial characterization of the major non-collagenous domain of SAP-35 [27]
King and MacBeth [51] have reported weak biophysical effects of purified SAP-35 on lipids by monitoring SAP-35 association with dipalmitoyl phosphatidylcholine vesicles.Hawgood et al [28] have shown that SAP-35 reduces surface tension in adsorption experiments when added to lung surfactant lipid extracts, but interpretations are complicated because such lipid extractscontainother distinct apoprotein components [11, 22, 23]
Summary
Surfactants for Binding or Surface Activity Studies-A number of synthetic phospholipids and other surfactants were used in this study in addition to the surfactant-associated protein SAP-35 and itsvarious molecular segments described below. For delipidation and purification of surfactant-associated protein, the pelleted material from each dog lung was resuspended in 20 ml of ether/ethanol (3:l) and incubated a t -20 "C overnight. SAP-35 binding was assessed using two-antibody ELISA and SDS-PAGE analysis of the resultant lipid pellet and aqueous fractions. Interfacial Biophysical Methods-Biophysical activity of lipid-protein mixtures was evaluated by measurements of adsorption facility in the absence of diffusion resistance [12,13,14] and dynamic surface tension-lowering ability on an oscillating bubble apparatus [46]. Phospholipid concentration was defined by phosphatedeterminations using the method of Chen et al [48]
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