Distribution of phosphoneuroprotein 14 (PNP 14) in vertebrates: its levels as determined by enzyme immunoassay.
We have established an enzyme immunoassay for phosphoneuroprotein 14 (PNP 14) which is mainly localized in the cytoplasmic matrix in presynaptic axon terminals and which is phosphorylated in vivo, as well as in vitro. Fab' prepared from rabbit IgG antibodies against bovine PNP 14 was conjugated with maleimide-horseradish peroxidase. The enzyme-conjugated Fab' was used as a second antibody in a sandwich enzyme immunoassay. This assay was able accurately to quantify 0.5-100 ng of rat PNP 14, as well as bovine PNP 14, and it was used for the determination of concentrations of PNP 14 in various rat tissues, neuroblastoma cells, and brains of other vertebrates. The concentrations of PNP 14 in the rat cerebrum, cerebellum, and testis were 1.1, 1.0, and 0.28 micrograms/mg of protein, respectively, and those in other tissues examined were less than 0.1 microgram/mg of protein. PNP 14 was also found in cultured cells, such as rat pheochromocytoma PC12 cells, NG108-15 cells, which are a hybrid between a mouse neuroblastoma and a rat glioma, mouse neuroblastoma Neuro-2a cells, and human neuroblastoma IMR32 cells. Furthermore, PNP 14-specific immunoreactivity was evaluated in the brains of various vertebrates, such as fish, frog, snake and chicken by immunoblot and enzyme immunoassay. The results revealed the immunoreactivity in the brains of all vertebrates examined and the levels were determined to be 0.6-2.1 micrograms bovine PNP 14 equivalents per mg of protein, suggesting that PNP 14 might be an essential component of the central nervous systems of vertebrates.
Nakajo
S
S
Laboratory of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan. nakajo@pharm.showa-u.ac.jp
Tsukada
K
K
Kameyama
H
H
Furuyama
Y
Y
Nakaya
K
K
D000818:Animals; D001923:Brain Chemistry / Q000502:physiology; D001932:Brain Neoplasms / Q000378:metabolism; D002417:Cattle; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006735:Horseradish Peroxidase; D006801:Humans; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification; D008297:Male; D008301:Maleimides / Q000737:chemistry; D051379:Mice; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D051843:Synucleins; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D051846:beta-Synuclein
D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008301:Maleimides; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D051843:Synucleins; D051846:beta-Synuclein; D006735:Horseradish Peroxidase