Identification and characterization of N-glycosylated and phosphorylated variants of proenkephalin A-derived peptides in bovine adrenal medulla, spinal cord and ileum.
Recent studies have shown that during its biosynthesis in bovine adrenal medulla, the opioid precursor proenkephalin A, may be both N-glycosylated and phosphorylated. To investigate whether these chemical modifications were common to proenkephalin A processing in other tissues, we have sought to characterize enkephalin-containing peptides from bovine adrenal medulla, spinal cord and ileum. The peptides were identified using antiserum L189, specific for the C-terminus of Met-enkephalin Arg6Gly7Leu8 (MERGL), and L152, specific for the C-terminus of Met-enkephalin Arg6Phe7 (MERF). Glycosylated MERGL-immunoreactive peptides of 23, 20, 16 and 13 kDa were identified in adrenal medulla using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and concanavalin A-Sepharose affinity chromatography. Sephadex G50 gel filtration fractionated the glycosylated peptides into two immunoreactive peaks. Similar peaks of concanavalin A-binding MERGL immunoreactivity were detected in extracts of spinal cord and ileum, although there were differences in relative proportions of the two peaks. Antiserum L152 identified phosphorylated N-terminally extended variants of MERF when boiling water extracts of adrenal medulla, spinal cord and ileum were separated by anion exchange chromatography. In adrenal medulla these peptides were more than 99% phosphorylated, whereas in both ileum and spinal cord there was a relatively higher proportion of the unphosphorylated peptide. The results indicate that N-glycosylation and phosphorylation of proenkephalin A occurs in adrenal medulla, spinal cord and ileum, although there are tissue-specific differences in the relative proportions of the modified and unmodified peptides.
Watkinson
A
A
MRC Secretory Control Group, University of Liverpool, U.K.
Dockray
G J
GJ
D000313:Adrenal Medulla / Q000032:analysis* / Q000187:drug effects; D000581:Amidohydrolases; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003208:Concanavalin A / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004745:Enkephalins / Q000032:analysis* / Q000235:genetics; D014644:Genetic Variation; D006031:Glycosylation; D007082:Ileum / Q000032:analysis* / Q000187:drug effects; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010766:Phosphorylation; D011498:Protein Precursors / Q000032:analysis* / Q000235:genetics; D013116:Spinal Cord / Q000032:analysis* / Q000187:drug effects
D004745:Enkephalins; D011498:Protein Precursors; C029992:proenkephalin; D003208:Concanavalin A; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase