Reconstitution of surfactant activity using purified human apoprotein and phospholipids measured in vitro and in vivo.
The major apoprotein of human lung surfactant was isolated from amniotic fluid obtained at term gestation. It was found to be a disulfide-linked oligomer composed of polypeptide chains of 35,000 daltons. The monomeric unit was shown to be a glycoprotein, and treatment with peptide: N-glycosidase F resulted in a decrease in molecular weight to 31,000 daltons. The isolated apoprotein could be recombined in the presence of Ca++ with the phospholipids dipalmitoylphosphatidylcholine and phosphatidylglycerol (3:1) at a weight ratio of 1:100. The surface tension (gamma min) measured on a pulsating bubble formed in 4 mg/ml of phospholipids was reduced from 32.3 +/- 2.0 dyn X cm-1 to 18.0 +/- 0.6 dyn X cm-1 after 15 s when 1% apoprotein was present. Reduction of disulfide bonds and deglycosylation of the apoprotein did not alter its ability to lower gamma min. Fetal rabbits of 27 days gestation had instilled intratracheally at delivery, saline, phospholipids, phospholipids plus apoprotein, or natural human surfactant. The latter 2 resulted in increased lung compliance and striking improvement in homogeneous alveolar expansion when the lungs were expanded to 10 cm H2O pressure, fixed, and viewed histologically. This effect was also shown to be independent of the disulfide-dependent oligomeric structure of the apoprotein or its state of glycosylation. The surfactant produced by recombination of the phospholipids with the isolated apoprotein was, therefore, shown to be biophysically active both in vitro and in vivo. These data suggest that apoprotein can be recombined with phospholipids to produce a biologically active surfactant for use in clinical trials of human surfactant replacement.
D000818:Animals; D001059:Apoproteins / Q000032:analysis / Q000302:isolation & purification* / Q000502:physiology; D002118:Calcium / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D008170:Lung Compliance / Q000187:drug effects; D008970:Molecular Weight; D010743:Phospholipids / Q000032:analysis / Q000302:isolation & purification* / Q000502:physiology; D011663:Pulmonary Surfactants / Q000032:analysis / Q000502:physiology*; D011817:Rabbits; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013500:Surface Tension
D001059:Apoproteins; D010743:Phospholipids; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins; D002118:Calcium