Comparison of brain and pituitary immunoreactive prolactin by peptide mapping and lectin affinity chromatography.
Immunoreactive PRL (IR-PRL) has been identified in many areas of the rat brain. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses we have recently shown that the primary IR-PRL protein in the rat brain has an apparent mol wt (Mr) of 24,000, which was identical to that of pituitary PRL. In these studies, brain and pituitary 24,000 Mr PRL were compared by peptide mapping and lectin chromatography. PRL-enriched fractions were prepared from the pituitary, hypothalamus, hippocampus, and pons-medulla and labeled with 125I. This material was further purified by immunoprecipitation, and immunopurified 24,000 Mr PRL was isolated by sodium dodecyl sulfate-gel electrophoresis. Cleavage of 125I-labeled 24,000 Mr IR-PRL prepared from the pituitary and the three brain regions with chymotrypsin resulted in identical peptide maps with two primary labeled peptide fragments (5,500 and 6,000 Mr), and approximately five less intense fragments. Similarly, trypsin cleavage of brain and pituitary 24,000 Mr IR-PRL resulted in the production of two major fragments (6,200, and 5,200 Mr), and three less intense fragments. Cleavage of the 24,000 Mr IR-PRL with Staphylococcus V8 protease resulted in identical fragment patterns with two primary peptide fragments (14,000 and 6,200 Mr). When the pituitary and brain 24,000 Mr PRL were applied to Concanavalin-A-Sepharose columns no 24,000 Mr PRL was absorbed. The similarity of the peptide fragments obtained from the cleavage of the 24,000 Mr IR-PRL from the brain and pituitary clearly indicate that the IR-PRL found in the brain has an amino acid sequence that shares a high degree of structural homology with pituitary PRL.
DeVito
W J
WJ
Division of Endocrinology, University of Massachusetts Medical School, Worcester 01655.
D000818:Animals; D001923:Brain Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D007016:Hypophysectomy; D008297:Male; D008970:Molecular Weight; D009928:Organ Specificity; D010449:Peptide Mapping; D010902:Pituitary Gland / Q000032:analysis*; D011388:Prolactin / Q000032:analysis* / Q000302:isolation & purification; D051381:Rats
D003208:Concanavalin A; D011388:Prolactin