Distribution of phosphoneuroprotein 14 (PNP 14) in vertebrates: its levels as determined by enzyme immunoassay

Abstract

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 μg of protein, respectively, and those in other tissues examined were less than 0.1 μg/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 μg bovine PNP 14 equivalents per mg of protein, suggesting that PNP 14 might be an essential component of the central nervous systems of vertebrates.