Calcium dependent association of surfactant protein A with pulmonary surfactant: application to simple surfactant protein A purification.
Surfactant protein A (SP-A) is an abundant lipoprotein component of pulmonary surfactant that plays multiple roles in surfactant homeostasis within the lung. A simple and rapid purification procedure for SP-A is described. Purified surfactant is washed by centrifugation with Ca2+ containing buffer to remove residual soluble proteins. Following the Ca2+ buffer wash, the surfactant pellet is washed in buffer containing EGTA and Mg2+ which releases the bound SP-A in almost pure form. Subsequent chromatography of the SP-A on Sephacryl S-500 yields homogeneous preparations of the protein. The SP-A purified using this procedure requires no exposure to either detergents or organic solvents to remove lipid. SP-A prepared by this new method inhibits lipid secretion from alveolar type II cells as effectively as SP-A prepared by other methods. In addition, the SP-A depleted surfactant produced in the first step of this procedure is capable of binding exogenous SP-A in a time dependent, saturable and Ca2+ dependent manner.
Suwabe
A
A
Lord and Taylor Laboratory for Lung Biochemistry, Department of Medicine, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, USA.
Mason
R J
RJ
Voelker
D R
DR
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002122:Calcium Chloride / Q000494:pharmacology; D002850:Chromatography, Gel; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D008168:Lung / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding; D011510:Proteolipids / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011650:Pulmonary Alveoli / Q000378:metabolism; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D004533:Egtazic Acid; D002122:Calcium Chloride; D002118:Calcium