Date of Award
1995
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
Surfactant-associated protein C (SP-C) contributes to the surface active properties of pulmonary surfactant. Processed from a 21 kDa precursor, the mature form of SP-C is 34-35 amino acids in length. Both the precursor and the mature protein are modified by palmitoylation. Due to its small size and extreme hydrophobicity, SP-C has been difficult to isolate, detect and study. Using molecular exclusion chromatography, SP-C was isolated from calf lung surfactant extract, characterized by partial sequencing, amino acid analysis, tricine polyacrylamide gel electrophoresis, and absence of immune-reactivity with antibodies raised against surfactant-associated protein B (SP-B). A method was developed to label, detect and quantify SP-C based on the reaction of iodoacetamide with sulfhydryl groups released upon the treatment of the protein with mild alkali to cleave the thioester bond through which the palmitates are bound to the protein. The surface active properties of lipid mixtures of dipalmitoylphosphatidylcholine (DPPC): phosphatidylglycerol (PG) with SP-C were studied using the pulsating bubble surfactometer. The presence of SP-C enhanced lipid adsorption onto the air-liquid interface. Upon compression, the mixture exhibited low surface tension, but the surface tension did not approach zero values except at moderately acidic pH values (2.5-3.5) or after prolonged pulsation. This partially clarified the inconsistency in the literature regarding the role of SP-C in lowering the surface tension as a pH effect. The function of palmitoylation was studied using both the pulsating bubble and the captive bubble surfactometers. Palmitoylation was found to contribute to enhancing lipid adsorption but to be absolutely essential for the stability of DPPC:PG:SP-C monolayers and for lipid re-spreading to form surface active layers after depletion of surfactant from the subphase. SP-C palmitoylation was carried out in vitro in the presence of palmitoyl CoA as the lipid donor. No other factors were needed for the re-palmitoylation of chemically depalmitoylated SP-C, indicating that the process is likely to be non-enzymatic. In addition to donating palmitate for covalent incorporation, palmitoyl CoA associated with SP-C in a highly specific but non-covalent manner. Palmitic acid, CoA, and surfactant lipids did not compete with palmitoyl CoA in its interaction with SP-C. This specificity suggests the possibility that the palmitoylation of SP-C may be a self-catalyzed process.
Recommended Citation
Qanbar, Riad, "On The Palmitoylation Of Surfactant-associated Protein C" (1995). Digitized Theses. 2507.
https://ir.lib.uwo.ca/digitizedtheses/2507