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- Gustafsson, M, et al.
(author)
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Reverse-phase HPLC of the hydrophobic pulmonary surfactant proteins: detection of a surfactant protein C isoform containing Nepsilon-palmitoyl-lysine
- 1997
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In: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 326326 ( Pt 3), s. 799-806
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Journal article (peer-reviewed)abstract
- A reverse-phase HPLC protocol for analysis of strictly hydrophobic peptides and proteins was developed. Peptide aggregation is minimized by using only 25–40% water in methanol or ethanol as initial solvents and subsequent elution with a gradient of propan-2-ol. Analysis of the pulmonary surfactant-associated proteins B (SP-B) and C (SP-C) with this method reveals several features. (1) SP-B and SP-C retain their secondary structures and separate by about 15 min over a 40 min gradient. SP-B is more hydrophilic than SP-C, which in turn behaves chromatographically like palmitoyl-ethyl ester. (2) SP-C exhibits isoforms additional to the major form characterized previously, which contains two thioester-linked palmitoyl groups. The isoforms now observed contain one or three palmitoyl moieties and constitute together 15–20% of the major form. The tripalmitoylated species contains a palmitoyl group linked to the ϵ-amino group of Lys-11, as concluded from the elution position, MS and amino acid sequence analysis. The tripalmitoylated form increases relative to the dipalmitoylated form on incubation of SP-C in a phospholipid environment. An Nϵ-bound palmitoyl moiety constitutes a third mode of fatty acyl modification of proteins, in addition to the established Nα-bound myristoyl groups and S-bound palmitoyl chains. (3) The dimeric structure of SP-B, lacking covalent modifications, is confirmed by MS detection of the dimer. No SP-B isoforms were detected. (4) Denatured, non-helical SP-C can be distinguished chromatographically from the native α-helical peptide. (5) HPLC of SP-C at 60–75 °C reveals an isoform containing an extra 14 Da moiety compared with the main form. This is concluded to arise from inadvertent methyl esterification of the C-terminal carboxy group. In conclusion, this HPLC method affords a sensitive means of assessing modifications and conformations of SP-B or SP-C in different disease states and before functional studies. It might also prove useful for analysis of other strictly hydrophobic polypeptides.
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| 3. |
- JOHANSSON, J, et al.
(author)
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Secondary structure and biophysical activity of synthetic analogues of the pulmonary surfactant polypeptide SP-C
- 1995
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In: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 307307 ( Pt 2), s. 535-541
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Journal article (peer-reviewed)abstract
- Native pulmonary-surfactant-associated lipopolypeptide SP-C, its chemically depalmitoylated form and several synthetic analogues lacking the palmitoylcysteine residues were analysed for secondary structure in phospholipid micelles and for biophysical activity in 1,2-dipalmitoyl-sn-glycero-3- phosphocholine/phosphatidylglycerol/palmitic acid (68:22:9, by wt.). Compared with the native molecule, with the entire poly-valyl part in a known alpha-helical conformation, depalmitoylated SP-C was found to be still mainly alpha-helical, but with an approx. 20% decrease in the helical content. A synthetic hybrid polypeptide where the entire poly-valyl alpha-helical part of native SP-C had been replaced with the amino acid sequence of a transmembrane helix of bacteriorhodopsin is also predominantly alpha-helical. In contrast, synthetic SP-C analogues lacking only the palmitoyl groups, by replacement of the palmitoylcysteine residues with cysteine, phenylalanine or serine, or lacking the positively charged amino acids by replacement with alanine, are considerably less alpha-helical than both native and depalmitoylated SP-C. The data indicate that the SP-C palmitoyl groups are important for maintenance of the alpha-helical conformation in parts of the polypeptide, and that the poly-valyl alpha-helical conformation is not fully formed in synthetic SP-C polypeptides. Furthermore, the helical structure of both native and depalmitoylated SP-C in dodecylphosphocholine micelles is very resistant to thermal denaturation, exhibiting ordered structure at 90 degrees C. The alpha-helical content grossly parallels the peptide-induced acceleration of the spreading of phospholipids at an air/water interface and the increase of surface pressure. The data suggest that the alpha-helical conformation itself, rather than just the covalent structure, is of prime importance for the biological function of synthetic pulmonary-surfactant peptides.
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