PRODUCTION OF IMMUNOREACTIVE PITUITARY ADENYLATE-CYCLASE ACTIVATING POLYPEPTIDE (PACAP) BY HUMAN NEUROBLASTOMA-CELLS, IMR-32 - DETECTION AND CHARACTERIZATION WITH MONOCLONAL AND POLYCLONAL ANTIBODIES AGAINST DIFFERENT EPITOPES OF PACAP

Sensitive and specific two-side enzyme immunoassays (two-site EIAs) fo r pituitary adenylate cyclase activating polypeptides, PACAP38, and PA CAP27, have been established using six monoclonal antibodies against P ACAP38, and a rabbit antibody against a C-terminal portion of PACAP27. In extracts of rat hypothalamus, these EIAs detected not only PACAP38 and PACAP27 but also an immunoreactive (ir-) PACAP lacking an epitope of a monoclonal antibody, PA-1C, which recognizes the C-terminal port ion of PACAP38. By the use of these EIAs, it was found that one of the human neuroblastoma cell lines, IMR-32, produced ir-PACAP. In reverse -phase (RP-)HPLC, intracellular and extracellular ir-PACAPs were separ ated into two peaks, of which one was eluted at a position close to th at of PACAP38 and the other in rather hydrophobic fractions. Those ir- PACAPs also lacked PA-1C epitope of PACAP38. SDS-PAGE and immunoblot a nalysis of the two peaks of the RP-HPLC indicated that they consisted of several components including those with apparent molecular weights of 6.5 k and 10 k for the first peak ir-PACAP, and 14 k and 20 k for t he second peak ir-PACAP. These results indicate that IMR-32 produces a precursor of PACAP and related peptides generated in various processi ng steps. Although the significance of the modification in the C-termi nus of PACAP38 is unknown, IMR-32 may be a cell line useful for studyi ng the regulation of the biosynthesis of PACAP.