Prothymosin alpha: isolation and properties of the major immunoreactive form of thymosin alpha 1 in rat thymus.

Abstract

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...

Retrovirus-mediated transfer of prothymosin gene inhibits tumor growth and prolongs survival in murine bladder cancer

“…1 Prothymosin α (ProT), the putative precursor of thymosin α1 that contains 113 amino acid residues with the thymosin αl sequence at its N-terminus, was originally isolated from rat thymus. 2 Many data show the behavior of ProT, like thymosin α1, as a biological response modifier. Furthermore, ProT has been reported to be more effective than thymosin α1 in the protection of mice infected with Candida albicans, suggesting that the biological activity of ProT is not solely determined by its N-terminal region that includes the thymosin α1 fragment.…”

Overexpression of hepatic prothymosin alpha, a novel marker for human hepatocellular carcinoma

“…The a-prothymosin is an acidic nuclear protein containing 111 amino acids, which was first isolated from rat thymus (Haritos et al, 1994) and was thought to be associated with regulation of cellular immunity (Zatz and Goldstein, 1995). However, an accumulating body of evidence suggests that a-prothymosin is associated with cell proliferation, although the precise mechanism remains to be elucidated.…”

Prothymosin α in Vivo Contains Phosphorylated Glutamic Acid Residues

“…The protein is neither a precursor for processed polypeptides nor specifically associated with the thymus nor a member of a family with ␤ or ␥ homologues (1)(2)(3). Instead, it is probably the most acidic naturally occurring polypeptide in the eukaryotic world, with 54 carboxyl groups in 109 amino acids, resulting in an isoelectric point at or below pH 3.5 (1,2,4). The mRNA for prothymosin ␣ is distributed ubiquitously among mammalian nucleated cells and tissues (1).…”

“…Furthermore, our laboratory has shown that nucleoplasmin, also, has glutamyl phosphate. 2 With so many features in common, it is tempting to speculate that prothymosin ␣, like nucleoplasmin, interacts with chromatin. Further evidence for a role involving chromatin comes from studies of the half-life of prothymosin ␣'s glutamyl phosphates in vivo (36).…”

“…The only other known example is that of frog nucleoplasmin. 2 Homologies between prothymosin ␣, whose specific function is unknown, and nucleoplasmin, a protein involved in remodeling chromatin, have been noted. Both proteins are abundant, acidic, and located exclusively in the nucleus (germinal vesicle in Xenopus) (Refs.…”