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, t he N-acetylation of ACTH(1-13)-NH2 to yield alpha-MSH occurs as a cose cretory processing event, whereas the N-acetylation of beta-endorphin occurs as a posttranslational processing event. To understand how thes e two N-acetylation reactions are segregated, B. marinus intermediate pituitary cells were analyzed by immunogold labeling electron microsco py, and by using an ultracentrifugation procedure. The immunogold labe ling studies indicated that ACTH(1-13)-NH2-related immunoreactivity wa s colocalized with N-acetylated beta-endorphin-related immunoreactivit y in secretory granules. Furthermore, ACTH(1-13)-NH2-related immunorea ctivity was not detected in either the ER or the Golgi. N-Acetylated b eta-endorphin-related immunoreactivity, however, was detected in the G olgi. Ultracentrifugation analysis revealed that in an ER/microsomal f raction, beta-LPH-sized and nonacetylated beta-endorphin-sized immunor eactive material were present in a molar ratio of 1:2. No N-acetylated forms of beta-endorphin were detected in the ER/microsomal fraction. In a Golgi/secretory granule fraction, the molar ratio of beta-LPH to beta-endorphin was 1:9 with 58% of the beta-endorphin being N-acetylat ed. Collectively, these data support the following hypotheses. The pro teolytic cleavage of ACTH(1-39) to yield ACTH(1-13)-NH2 is a late proc essing event occurring in secretory granules. The cleavage of beta-LPH to yield nonacetylated beta-endorphin is an early processing event th at may occur in the ER or the Golgi. Because N-acetylated beta-endorph in and nonacetylated ACTH(1-13)-NH2 are colocalized in secretory granu les, it appears, therefore, that the N-acetylation of beta-endorphin i s completed prior to loading into secretory granules. Thus, there is a spatial and temporal separation of the posttranslational processing e vents associated with the beta-LPH portion and ACTH portion of the POM C biosynthetic pathway in amphibian intermediate pituitary cells.