Identification of pituitary adenylate cyclase-activating polypeptide1-38-binding factor in human plasma, as ceruloplasmin

Abstract

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.