A polypeptide containing -112 amino acid residues, with the thymosin a, sequence at its NH2 terminus, has been isolated from rat thymus by using a radioimmunoassay with an antibody prepared against synthetic thymosin ap. The new polypeptide, named "prothymosin a," was found to be the major substance crossreacting with thymosin a, antiserum in rat thymus extracts; peptides corresponding to thymosin a, or thymosin a,, were not detected. In gel filtration at pH 2.8, prothymosin a emerged as a single symmetrical peak corresponding to an apparent molecular weight of 32,000, approximately 3 times larger than the minimum molecular weight calculated from its amino acid composition. Thymosin a,, a peptide containing 28 amino acid residues, was isolated by Goldstein and coworkers (1) from thymosin fraction 5, a mixture of peptides from calf thymus (2). Thymosin fraction 5 had been reported earlier to restore parameters of immunocompetence in neonatally thymectomized mice (3). Thymosin a1 was found to be active in some of the in vitro tests used for thymosin fraction 5 (4), and it was considered to be one of the factors that modulated steps in the maturation of T cells (5).We have reported (6) our inability to detect thymosin a1 in guanidinium chloride extracts of calf thymus. The suggestion that thymosin a1 might represent a proteolytic fragment of a larger native polypeptide was supported by the finding that preparations of calf thymosin fraction 5 contained at least two other related peptides (7). One of these, designated des-(25-28)-thymosin a1, contained only the first 24 amino acid residues; the other, named thymosin all, contained the sequence of thymosin a1 plus seven additional residues at the COOH terminus.In an effort to isolate the native thymic polypeptide from which these fragments appeared to be derived, we developed a radioimmunoassay based on an antibody prepared against synthetic thymosin a1. With this assay and a procedure designed to eliminate any possibility of proteolytic modification, we isolated a major polypeptide, =112 amino acid residues long, that contains the thymosin a1 sequence at its NH2 terminus. We named this polypeptide prothymosin a because it appears to be the source of the thymosin a1-related peptide fragments found in preparations of thymosin fraction 5.
MATERIALS AND METHODSRat thymuses from male Charles River CD rats, 5 weeks old, were excised immediately after sacrifice of the animals by decapitation, quickly frozen in liquid nitrogen, and stored at -700C. Synthetic thymosin a, (8) was provided by A. Felix of Hoffmann-La Roche. Trypsin (L-1-tosylamido-2-phenylmethyl chloromethyl ketone-treated) and Staphylococcus aureus V8 protease were from Worthington and Miles Laboratories respectively. Fluorescamine was a gift of W. E. Scott of Hoffmann-La Roche. Sephacryl S-200 (superfine) was purchased from Pharmacia. Other reagents and solvents were chromatography-grade commercial preparations; the solvents were redistilled as required.For preparation of the antibody, rabbits were in...