Characterization of a common precursor to corticotropin and beta-lipotropin: identification of beta-lipotropin peptides and their arrangement relative to corticotropin in the precursor synthesized in a cell-free system.

Abstract

Radioactive proteins synthesized in an mRNA-dependent reticulocyte cell-free system under the direction of mRNA from AtT-20/D-16v mouse cells were isolated by specific immunoprecipitation using antiserum to either a(1-24) corticotropin or f-endorphin [#(61-91) lipotropin]. Each immunoprecipitate was fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and shown to contain only one labeled protein with an apparent molecular weight of 28,500. Tryptic peptide analysis of the Mr 28,500 corticotropin and fl-lipotropin molecules isolated from the gels demonstrated that the two proteins had the same lysine, methionine, and tryptophan peptides. Four tryptic peptides from the cell-free product exhibited the same electrophoretic and chromatographic mobilities as marker tryptic peptides from bovine ,3-melanotropin and porcine (3-endorphin. The identification of these peptides was confirmed by amino acid composition studies with a variety of labeled amino acids. The jP-lipotropin tryptic peptides were also shown to be located carboxy terminal to the corticotropin tryptic peptides.When messenger RNA from cultures of mouse pituitary cells (AtT-20/D-16v cell line) that secrete corticotropin (ACTH) is translated in a reticulocyte cell-free system, a species of ACTH is synthesized with an apparent molecular weight (Mr) of 28,500 (1). The same size ACTH molecule is made when polysomes from cells direct the cell-free synthesis of protein (1). The Mr 28,500 product contains the same methionine, lysine, and phenylalanine peptides as a class of higher molecular weight ACTH glycoproteins (Mr 31,000) observed in the AtT-20 cells (2). The kinetics of labeling of these glycoproteins with radioactive amino acids shows that they are the precursors of the lower molecular weight forms of ACTH found in the cells (Mr 23,000, 13,000, and 4500 ACTH) (3). Hence, the Mr 28,500 cell-Iree product appears to be the primary translation product from which all the forms of ACTH in the AtT-20 cells are derived. Tryptic peptide analysis of the cell-free product has shown that it contains one copy of a(1-39) ACTH, leaving approximately 210 amino acids unaccounted for (1). The finding that ACTH and fl-melanotropin (f,-MSH) [,(41-58) In this study we have used tryptic peptide analysis to show that several ,B-LPH peptides are present in the cell-free product isolated with antisera specific to ACTH and fl-endorphin. These approaches have also yielded information about the location of ,B-LPH relative to a(1-39) ACTH in the cell-free product.

The Nature of the Immunoreactive Lipotropins in Human Plasma and Tissue Extracts

“…What, then, of the "h( "big" ACTH is a glycopeptide, presumably the analogue of the 31,000 mol wt common precursor of ACTH and the LPH in the mouse (37)(38)(39)(40) (44). Thus, the endorphins may be released into the blood in parallel with ACTH and yLPH under physiological situations, such as the response to stress, or under pathological conditions, such as Cushing's disease or the ectopic ACTH syndrome.…”

60 YEARS OF POMC: N-terminal POMC peptides and adrenal growth

“…The year 1977 will be remembered as the landmark year in POMC research as it was the year that concurrently Mains et al (1977) and Roberts and Herbert (1977) elegantly showed the existence of the precursor molecule and conclusively confirmed that ACTH and β-endorphin were derived from the same molecule. These studies also showed that the precursor contained another, previously unknown glycopeptide that was named the '16K fragment' due to its apparent molecular weight when separated by SDS-PAGE.…”

60 YEARS OF POMC: From the prohormone theory to pro-opiomelanocortin and to proprotein convertases (PCSK1 to PCSK9)

“…Biosynthesis of ACTH and related peptides Although the sequences of ACTH and of α-MSH had been known for almost 15 years, their structural relationship during biosynthesis did not attract much attention until two additional facts were published: (i) the existence of high molecular forms of ACTH and (ii) the discovery of CLIP by Scott et al (1972Scott et al ( , 1974 Roberts and Herbert (1977) and Roberts et al (1978), used a cell-free translation of mRNA from AtT-20 cells. Both studies confirmed the hypothesis that ACTH is biosynthesized as a large precursor form of 28,000-31,000 kDa, which is later transformed into 13-and 4.5-kDa forms.…”