Processing of proenkephalin-A in bovine chromaffin cells - Identification of natural derived fragments by N-terminal sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry

A large variety of proenkephalin-A-derived peptides (PEAPs) are present in bovine adrenal medulla secretory granules that are cosecreted with catechol amines upon stimulation of chromaffin cells. In the present paper, after re verse phase high performance liquid chromatography of intragranular soluble material, PEAPs were immunodetected with antisera raised against specific proenkephalin-A (PEA) sequences (PEA63-70 and PEA224-237) and analyzed by m atrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, Thirty PEAPs were characterized in addition to enkephalins an d whole PEA, indicating that preferential proteolytic attacks occurred at b oth N- and C-terminal regions. A similar approach was used to characterize PEA-derived fragments exocytotically released into the extracellular space that showed five additional minor PEAPs. Among all these naturally generate d peptides, enkelytin, the antibacterial bisphosphorylated C-terminal pepti de (PEA209-237), was predominantly generated, as shown by MALDI-TOF mass sp ectrometry analysis, which constituted an efficient method for its identifi cation. Finally, the data on PEA intragranular and extracellular processing in adrenal medulla are discussed in regard to the known enzymatic processi ng mechanisms. We note the high conservation of the cleavage points in evol utionarily diverse organisms, highlighting an important biological function for the released PEAPs.