The posttranslational processing of prodynorphin in the rat anterior pituitary.
The posttranslational processing of prodynorphin (Pro-Dyn) is not well understood. The rat anterior pituitary is an interesting tissue which merits examination to address this issue since it is known that Dyn immunoreactivity is stored as high mol wt (HMW) intermediates and not as free products such as dynorphin-A-(1-17) (Dyn-A17) or dynorphin-B-(1-13) (Dyn-B). The aim of our study is to characterize the Pro-Dyn products in the rat anterior pituitary quantitatively as well as qualitatively by keeping a close account of each of the possible domains that are known to compose the protein structure. This was achieved by a convergence of tools: designing RIA with antibodies to each of these domains, including antibodies to Dyn-A17, Dyn-B, alpha-neo-endorphin, bridge peptide, and Pro-Dyn carboxyl-terminal peptide (C-peptide), and using these antisera with gel filtration chromatography, reverse phase HPLC, immunoaffinity, and immunoprecipitation techniques. Our data indicate the presence of at least six distinct molecules which are classified as HMW intermediates (greater than 3.5K). By gel filtration chromatography they have apparent mol wt of 16,000, 10,000, 8,000, 6,000, 4,000, and 3,500, respectively. Each of these structures is characterized by multiple immunoreactivities to account for the observed mass. Based on the relative content of each structure we present a scheme for the posttranslational processing pathway of Pro-Dyn in the rat anterior pituitary. We also analyze other tissues, spinal cord and hypothalamus, for their content of Pro-Dyn HMW intermediates. Our results indicate that these tissues store Pro-Dyn HMW molecules of similar sizes and immunoreactive properties, suggesting that Pro-Dyn may be processed in a similar manner, at least in the initial phases, across tissues.
Day
R
R
University of Michigan, Mental Health Research Institute, Ann Arbor 48109.
Akil
H
H
D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004745:Enkephalins / Q000378:metabolism*; D007031:Hypothalamus / Q000032:analysis; D007158:Immunologic Techniques; D008297:Male; D008970:Molecular Weight; D010903:Pituitary Gland, Anterior / Q000032:analysis / Q000378:metabolism*; D011233:Precipitin Tests; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D013116:Spinal Cord / Q000032:analysis; D014020:Tissue Extracts / Q000032:analysis
D004745:Enkephalins; D011498:Protein Precursors; D014020:Tissue Extracts; C033660:preproenkephalin