Biochemistry zyxwvut 1982, 21, 5341-5346 5341 Tai, T., Yamashita, K., zyxwvutsrq & Kobata, A. (1977) Biochem. Bio- phys. Res. Commun. 78, 434-441. Tarentino, A. L., Trimble, R. B., & Maley, F. (1978) Methods Enzymol. 5, 574-580. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leim- gruber, W., & Weigele, M. (1972) Science (Washington, Vliegenthart, J. F. G., Van Halbeek, H., & Dorland, L. (1981) Warren, L. (1959) J. Biol. Chem. 234, 1971-1974. D.C.) 178, 871-872. Pure Appl. Chem. 53, 45-77. Weber, K., & Osborn, M. (1969) J. Biol. Chem. 244, White, H. B., 111, & Hughes, A. R. (1981) Poult. Sci. 60, Wuthrich, K. (1976) NMR in Biological Research: Peptides Yamashina, I. (1972) in Glycoproteins (Gottschalk, A., Ed.) Yamashita, K., Tachibara, Y., & Kobata, A. (1978) J. Biol. 4406-44 12. 1 454- 1 4 5 7. and Proteins, pp 43-50, Elsevier, New York. Part B, pp 1187-1200, Elsevier, New York. Chem. 253, 3862-3869. Characterization of Multiple Forms of Porcine Anterior Pituitary Proopiomelanocortin Amino-Terminal Glycopeptide? Guy Boileau, Normand Larivitre, Kuo-Liang Hsi, Nabil G. Seidah, and Michel Chritien* ABSTRACT: Chromatography on a molecular sieve column of a preparation of porcine proopiomelanocortin N-terminal glycopeptide purified from anterior pituitary resulted in the isolation of three forms of the peptide with respective apparent M, 21 000, 17 500, and 13 500 on polyacrylamide/sodium dodecyl sulfate gel. Determination of the amino acid corn- position of each peptide revealed that the form with a mo- lecular weight of 17 500 corresponds to the 80 amino acid residue porcine N-terminal glycopeptide (PNT 1-80) previ- ously characterized [LariviEre, N., Seidah, N. G., & Chritien, M. (1981) Znt. J. Pept. Protein Res. 18,487-4911. The forms with molecufar weight of 21 000 and 13 500 correspond re- spectively to longer and shorter forms of the N-terminal Adrenocorticotropic hormone (ACTH), 6-lipotropic hor- mone (P-LPH), zyxwvutsrqpo a- and /3-melanotropic hormones (a- and P-MSH), and @-endorphin (P-END) are synthesized in the pituitary gland from a common glycoprotein precursor (Roberts & Herbert, 1977; Mains et al., 1977; Crine et al., 1978) called proopiomelanocortin (POMC) (Chritien et al., 1979). These hormones are located on the central and C- terminal portions of the POMC structure leaving a large N-terminal fragment. Using recombinant DNA technology, Nakanishi et al. (1979) determined the nucleotide sequence of the mRNA coding for pre-POMC in bovine pars in- termedia. This study revealed an amino acid sequence in the N-terminal portion of POMC homologous to the sequence of a- and P-MSH in ACTH and P-LPH, respectively. This new sequence was called y-MSH (Nakanishi et al., 1979). More recently, investigations on the genomic DNA structure of human (ChaFg et al., 1980), bovine (Nakanishi et al., 1980), and rat (Drouin & Goodman 1980) POMC were published confirming the finding of Nakanishi et al. (1979). Pulse and pulse-chase experiments in rat pars intermedia (Crine et al., 1979, 1980a,b; Gossard et al., 1980) have shown that during maturation of POMC, the N-terminal portion of the molecule is released as two glycosylated peptides with apparent mo- lecular weights (M,) on polyacrylamide/sodium dodecyl sulfate (NaDodSOd gel of 17 000 and 19 000. From the Clinical Research Institute of Montreal, Montreal, Canada H2W 1R7. Received February 19, 1982. This work was supported by a program grant from the Medical Research Council of Canada and by the National Institutes of Health (NS-16315-02). G.B. is a MRC fellow. 0006-2960/82/042 1-534 1$01.25/0 glycopeptide. The high molecular weight form contains 107 amino acid residues. Sequencing of the fragments obtained after cleavage of the molecule with cyanogen bromide and Myxobacter Lys-C protease indicated that an extension of 27 amino acid residues is linked to PNT 1-80 through a -Lys-Arg- sequence. The sequence of the extension is reported. The low molecular weight form corresponds to the first 61 residues of PNT 1-80. Pronase digestion of the peptide and dansylation of the digest revealed the presence of a residue of phenylalanine amide at position 61. A general model for the maturation of the N-terminal glycopeptide of proopiomelanocortin in porcine anterior pituitary is presented: Recently, we purified the N-terminal fragment of POMC from the anterior lobe of porcine pituitary glands and from whole human pituitary glands and reported their complete amino acid sequence (Larivitre et al., 1981; Seidah & Chritien, 1981). The peptide from porcine anterior lobe was found to be 80 amino acids long while its human homologue was 76 amino acids long. However, during the purification procedure, a certain heterogeneity of the preparations could be detected by gel filtration and two-dimensional polyacryl- amide gel electrophoresis. In this paper we present the chemical characterization of two additional forms of the N-terminal fragment of porcine POMC differing in their length from the peptide of 80 amino acids. One has 107 amino acid residues and represents probably the N-terminal portion of POMC up to the putative -Lys-Arg- sequence preceding the ACTH sequence (Nakanishi et al., 1979, 1980; Chang et al., 1980; Drouin & Goodman 1980); the other has 61 amino acids, it contains the y-MSH sequence, and its C-terminal phenylalanine is amidated. This latter form is possibly a maturation product of the 80 amino acid peptide. Materials and Methods Isolation zyxwv of Porcine N- Terminal Peptides and Two-Di- mensional Polyacrylamide Gel Electrophoresis. The methods for the isolation and purification of the peptides by high- performance liquid chromatography (HPLC) have been pre- viously reported (Larivitre et al., 1981). One subsequent step of gel filtration on a column (125 zyx X 1.5 cm) packed with Sephadex G-75 superfine (Pharmacia Fine Chemicals) eluted 0 1982 American Chemical Society