Biosynthesis of multiple forms of beta-endorphin in the reptile intermediate pituitary.
Fractionation of the beta-endorphin-sized material from freshly dissected reptile intermediate pituitaries by ion exchange chromatography on sulfopropyl Sephadex (SP) revealed at least three distinct forms of immunoreactive beta-endorphin. These forms eluted at 0.25 M NaCl, 0.28 M NaCl, and 0.32 M NaCl and represent respectively, 6%, 65% and 29% of the total immunoreactivity. Only the 0.28 M NaCl peak and the 0.32 M NaCl peak exhibited naloxone reversible opiate bioactivity when tested in the isolated guinea pig ileum bioassay system; taking into account the molar amount of immunoreactive peptides the 0.32 M NaCl peak was 6 fold more potent than the 0.28 M NaCl peak. Intermediate pituitaries in culture were incubated with either [3H]tyrosine, [3H]arginine, or [35S]methionine for periods up to 24 hours and beta-endorphin-sized peptides were prepared by immunoprecipitation and gel filtration. Fractionation of the labeled beta-endorphin-sized peptides by ion exchange chromatography yielded profiles nearly identical to the immunoassay analyses of freshly dissected tissue. Further analysis of the major labeled forms of reptile beta-endorphin by chromatography on Sephadex G-50 equilibrated in 6 M guanidine HCl indicated that the 0.32 M NaCl peak had an apparent molecular weight of 3500 +/- 100 and the 0.28 M NaCl peak had an apparent molecular weight of 3200 +/- 100. Furthermore, pulse/chase experiments showed that the 0.32 M NaCl peak was the precursor for the 0.28 M NaCl peak. These results coupled with the relative opiate bioactivities of the major argue that the principal post-translational modification of reptile beta-endorphin is COOH-terminal proteolytic cleavage.
D000818:Animals; D001120:Arginine / Q000378:metabolism; D004723:Endorphins / Q000096:biosynthesis* / Q000302:isolation & purification; D007076:Iguanas / Q000502:physiology*; D007700:Kinetics; D008116:Lizards / Q000502:physiology*; D008297:Male; D008715:Methionine / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014316:Tritium; D014443:Tyrosine / Q000378:metabolism; D001615:beta-Endorphin
D004723:Endorphins; D013462:Sulfur Radioisotopes; D014316:Tritium; D014443:Tyrosine; D001615:beta-Endorphin; D001120:Arginine; D008715:Methionine