POMC-related products in the intermediate pituitary of the amphibian, Bufo marinus: Differential subcellular processing in the Golgi and secretory granules

In the intermediate pituitary of the anuran amphibian, Bufo marinus, the N-acetylation of ACTH(1–13)-NH 2 to yield α-MSH occurss as a cosecretory processing event, whereas the N-acetylation of β-endorphin occurs as a posttranslational processing event. To understand how these two N-acetylation reactions are segregated, B. marinus intermediate pituitary cells were analyzed by immunogold labeling electron microscopy, and by using an ultracentrifugation procedure. The immunogold labeling studies indicated that ACTH(1–13)-NH 2-related immunoreactivity was colocalized with N-acetylated β-endorphin-related immunoreactivity in secretory granules. Furthermore, ACTH(1–13)-NH 2-related immunoreactivity was not detected in either the ER or the Golgi. N-Acetylated β-endorphin-related immunoreactivity, however, was detected in the Golgi. Ultracentrifugation analysis revealed that in an ER/microsomal fraction, β-LPH-sized and nonacetylated β-endorphin-sized immunoreactive material were present in a molar ratio of 1:2. No N-acetylated forms of β-endorphin were detected in the ER/microsomal fraction. In a Golgi/secretory granule fraction, the molar ratio of β-LPH to β-endorphin was 1:9 with 58% of the β-endorphin being N-acetylated. Collectively, these data support the following hypotheses. The proteolytic cleavage of ACTH(1–39) to yield ACTH(1–13)-NH 2 is a late processing event occurring in secretory granules. The cleavage of β-LPH to yield nonacetylated β-endorphin is an early processing event that may occur in the ER or the Golgi. Because N-acetylated β-endorphin and nonacetylated ACTH(1–13)-NH 2 are colocalized in secretory granules, it appears, therefore, that the N-acetylation of β-endorphin is completed prior to loading into secretory granules. Thus, there is a spatial and temporal separation of the posttranslational processing events associated with the β-LPH portion and ACTH portion of the POMC biosynthetic pathway in amphibian intermediate pituitary cells.