Chapter 8 Glycosylation Steps Involved in Processing of Pro-Corticotropin-Endorphin in Mouse Pituitary Tumor Cells

Abstract

Publisher Summary This chapter discusses the glycosylation steps involved in processing of pro-corticotropin–endorphin in mouse pituitary tumor cells. β -endorphin, adrenocorticotropic hormone (ACTH) and α - and β -melanocyte-stimulating hormones (MSH) are all derived from the same precursor protein (pro-ACTH–endorphin) in the pituitary. A portion of the amino-acid sequence of the precursor in mouse pituitary cells is revealed using recombinant deoxyribonucleic acid (DNA) technology. The chapter discusses approaches used to study proteolytic processing and glycosylation of the precursor in mouse pituitary tumor cells. To study the processing of oligosaccharides, AtT-20 cells were incubated with different radioactive sugars. Pro-ACTH–endorphin, ACTH intermediates, and N -terminal fragments were purified from cell extracts and culture medium by immunoprecipitation and sodium dodecyl sulfate (SDS) gel electrophoresis. The purified components were digested with pronase and the pronase glycopeptides were analyzed by gel filtration to determine their size, susceptibility to various glycosidases ( α -mannosidase, glucosidases, and endoglucosidases) to determine if terminal mannose or glucose residues are present, and sugar composition. The first proteolytic cleavage occurs within 15–25 minutes of the labeling of pro-ACTH–endorphin with radioactive amino acids.