Identification of pituitary adenylate cyclase-activating polypeptide1-38-binding factor in human plasma, as ceruloplasmin
| Autor: | Jan Fahrenkrug, Anders H. Johnsen, Jeppe W. Tams |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
chemistry.chemical_classification
endocrine system biology Chemistry Immunoprecipitation Neuropeptide Adenylate kinase Biological activity Cell Biology Biochemistry Cyclase Molecular biology Amino acid biology.protein Ceruloplasmin Molecular Biology Peptide sequence hormones hormone substitutes and hormone antagonists |
| Zdroj: | Biochemical Journal. 341:271-276 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj3410271 |
| Popis: | 125I-Pituitary adenylate cyclase-activating polypeptide (PACAP) 1-38 is able to bind a factor in human plasma, which can be displaced by unlabelled PACAP 1-38 and PACAP 28-38 but not by the other biologically active form, PACAP 1-27. Likewise, 125I-PACAP 28-38 binds this plasma factor, whereas 125I-PACAP 1-27 does not. Apparent Kd values were measured to be 12.0±1.3 and 3.4±1.5 nM for PACAP 1-38 and PACAP 28-38, respectively, using a competition assay with 125I-PACAP 28-38. Purification of the PACAP 1-38-binding factor from human blood was made by ethanol precipitation of serum followed by Ni2+-chelating and anion-exchange chromatography. A 120-kDa band on SDS/PAGE, as well as some proteolytic products, was blotted on to PVDF membrane and their N-terminal amino acid sequences determined. In combination with a mass-spectrometric fingerprinting of a tryptic digest of the 120-kDa band, this PACAP 1-38-binding factor was identified as ceruloplasmin. Purified commercial ceruloplasmin shows identical mobility on SDS/PAGE to the PACAP 1-38-binding factor and the same binding characteristics to PACAP 1-38, 1-27 and 28-38, using the same amount of ceruloplasmin as was expected to be found in the human plasma. Furthermore, the ability of plasma to bind 125I-PACAP 1-38 or 28-38 disappeared when ceruloplasmin was immunoprecipitated from plasma with rabbit anti-human ceruloplasmin Ig. |
| Databáze: | OpenAIRE |
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