| Abstract: | Human alpha 2-macroglobulin (alpha 2M) is inhibited by covalent reaction with alkylamines. The site of methylamine incorporation has been proposed to be an activated glutamyl residue (Swenson, R. P., and Howard, J. B. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4313-4316). A large, 56-amino acid residue glycopeptide derived from tryptic cleavage of 14C]methylamine-labeled alpha 2M was isolated. Based upon recovery of the specific radioactivity in the peptide, there appears to be only a single site of incorporation per Mr = 185,000 subunit. The complete amino acid sequence was deduced from Edman degradation and carboxypeptidase Y digestion of the tryptic peptide and of several small peptides derived from it. The structure of the radiolabeled amino acid was determined to be gamma-glutamylmethylamide by mass spectral analysis of the phenylthiohydantoin and N-benzoyl-O-methylester derivatives. The putative structure was confirmed by a comparison of the mass spectral and chromatographic properties of the authentic compound and the protein-derived amino acid residue. The 10 amino acid residues following the methylamine-reactive glutamyl residue were identical with the first 10 amino acid residues of the pyroglutaminase-deblocked, Mr = 65,000 fragment generated by heat denaturation of alpha 2M (Howard, J. B., Vermeulen, M., and Swenson, R. P. (1980) J. Biol. Chem. 255, 3820-3823). |
