Evidence for a common precursor for alpha MSH and beta-endorphin in the intermediate lobe of the pituitary of the reptile Anolis carolinensis.

In order to investigate the biosynthesis of alpha MSH and beta-endorphin in a non-mammalian vertebrate, individual lizard intermediate pituitaries were incubated in complete medium containing a radioactive amino acid, using either a steady label or a pulse/chase protocol. Following incubation, acid extracts of the tissue were immunoprecipitated with either an NH2-terminal ACTH antiserum or a beta-endorphin antiserum and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After a 24 hr steady label in medium containing [3H]tyrosine, multiple molecular weight forms of beta-endorphin-related and NH2-terminal ACTH-related radioactivity were detected. The major peak of beta-endorphin-related radioactivity co-migrated with synthetic beta-endorphin(1-31); minor peaks of beta LPH-sized material and precursor-sized material were also detected. The major peak of NH2-terminal ACTH-related material co-migrated with synthetic alpha MSH; in addition, smaller amounts of material designated ACTH biosynthetic intermediate 1, ACTH biosynthetic intermediate 2, and precursor-sized material were detected. Sequential immunoprecipitation experiments revealed that the precursor-sized material had antigenic determinants for both alpha MSH and beta-endorphin. Pulse/chase experiments established that this material is the common precursor for alpha MSH and beta-endorphin. Based on gel filtration chromatography in 6 M guanidine HCl, the molecular weights of these various peptides are: common precursor, 23,300 daltons; ACTH biosynthetic intermediate 1, 12,200 daltons; ACTH biosynthetic intermediate 2, 4,200 daltons; alpha MSH, 1,500 daltons; beta LPH, 8000 daltons; beta-endorphin, 3,400 daltons. None of the peptides precipitated with either antiserum incorporated [3H]glucosamine; thus glycosylation does not appear to be involved in this biosynthetic pathway in the lizard. The results of the kinetic experiments and molecular weight determinations indicate that the major biosynthetic pathway involves the following events: common precursor is first cleaved to yield ACTH biosynthetic intermediate 1 plus beta LPH; subsequently, beta LPH is cleaved to produce beta-endorphin; ACTH biosynthetic intermediate 1 is cleaved to produce ACTH biosynthetic intermediate 2 which is subsequently cleaved to produce alpha MSH. The pulse/chase experiments indicate minor pathways exist for cleaving beta-endorphin directly from the common precursor or via a high molecular weight form intermediate in size between the common precursor and beta LPH.