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) for pituitary adenylate cyclase activating polypeptides, PACAP38, and PACAP27, have been established using six monoclonal antibodies against PACAP38, 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 portion 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 separated into two peaks, of which one was eluted at a position close to that of PACAP38 and the other in rather hydrophobic fractions. Those ir-PACAPs also lacked PA-1C epitope of PACAP38. SDS-PAGE and immunoblot analysis 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 the second peak ir-PACAP. These results indicate that IMR-32 produces a precursor of PACAP and related peptides generated in various processing steps. Although the significance of the modification in the C-terminus of PACAP38 is unknown, IMR-32 may be a cell line useful for studying the regulation of the biosynthesis of PACAP.
Suzuki
N
N
Discovery Research Laboratories, Takeda Chemical Industries, Ltd., Ibaraki.
Harada
M
M
Kitada
C
C
Ohkubo
S
S
Matsumoto
H
H
Watanabe
T
T
Coy
D H
DH
Tsuda
M
M
Arimura
A
A
Fujino
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D006801:Humans; D007031:Hypothalamus / Q000737:chemistry / Q000378:metabolism*; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D009447:Neuroblastoma / Q000378:metabolism*; D009479:Neuropeptides / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051381:Rats; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
C495401:ADCYAP1 protein, human; C495403:Adcyap1 protein, rat; D000911:Antibodies, Monoclonal; D000939:Epitopes; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide