Isolation and characterization of lipid-associated and neurosecretory polypeptides

Abstract: Lipid-interacting proteins play important roles in all living organisms. This thesis focuses on isolation and characterization of an enzyme in the triacylglycerol biosynthesis (phosphatidic acid phosphatase, PAP), hydrophobic polypeptides in bile, and polypeptides in cerebrospinal fluid. These fields constitute methodological challenges and mean development of suitable tools in between lipid and protein biochemistry. Two methods used to measure PAP activity were compared. It was established that the discrepancy between the methods was mainly due to different acyl chains of the substrate. In purification of PAP from the yeast Saccharomyces cerevisiae, an apparent molecular weight of 35 and/or 40 kDa (determined by SDS-PAGE) and approximately 67 kDa (determined by size exclusion chromatography) was obtained. The discrepancy in molecular size indicates that PAP forms a multimeric structure. The results suggest a smaller subunit size than previously deduced. A method to determine protein content in extremely lipid-rich samples (90-98% lipids), where the protein content is low (1-5%), was developed. By derivatization of hydrolysed samples to their phenylisothiocarbamyl forms, and subsequent separation by reversed-phase HPLC, the amino acid content was determined. In addition, phosphatidylethanolamine and phosphatidylserine could be detected together with the amino acid constituents. A method to isolate polypeptides from lipid-rich material was evaluated. Separation by cromatography of chloroform/methanol extracts of lipid-rich tissues as spinal cord, lung and bile on Lipidex-5000 in the solvent system methanol/ethylene chloride, the polypeptides were well separated from the majority of the lipids. Several hydrophobic polypeptides in human gallbladder bile were identified, as ATP-synthase lipid-binding protein 9, a fragment of monocyte differentiation antigen CD14, and a fragment of mac25/insulin-like growth factor-binding protein 7, all not previously identified in bile. Several novel proteolytic fragments of neuroendocrine proteins were identified in human cerebrospinal fluid. Proteolytically processed fragments of chromogranin A, chromogranin B, neuroendocrine protein 7B2, proenkephalin, neuroendocrine specific protin VGF, osteopontin, testican and IGF-II E-peptide were characterized, several of them not previously reported.