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...

A radioimmunoassay, using a rabbit antiserum directed against thymosin a,, was employed to detect the presence of crossreacting peptides in rat tissues. Highest concentrations were present in thymus, but thymosin al crossreacting material was also detected in brain, liver, kidney, lung, and spleen, in amounts ranging from 15% to 65% of the quantities found in thymus. In each case, the major immunoreactive peptide, after extraction and purification by a procedure that avoids proteolytic modification, was identified as prothymosin a, a peptide containing %112 amino acid residues. Prothymosin a is believed to be the endogenous peptide from which thymosin a, and other fragments are formed by proteolytic modification during the preparation of thymosin fraction 5. No peptides corresponding in size and chromatographic behavior to thymosin a, were detected with the extraction procedure employed.

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The primary structure of prothymosin a from rat thymus, containing 113 amino acid residues, is reported as follows: AcSer-Asp-Ala-Ala-Val-Asp-Thr-Ser- Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys- Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Gly-Arg-Asp-Ala- 40 Pro-Ala-Asn-Gly-Asn-Ala-Gln-Asn-Glu-Glu- Gly-Glu-Gln-Glu-Ala-Asp- Glu-Glu-Glu-Glu-Gly-Gly-Gly-Glu-Glu (Asx,Gly,Gly, 70 Glx,Glx,Glx,Glx,Glx,Glx,Glx,Glx,Glx,Glx)Asn-Gly- 80Asp-Glu-Asp-Glu -Glu -Ala -Gl u -Ala-Pro -Th r-Gly -90 100Lys-Arg-Val-Ala-Glu-Asp-Asp-Glu-Asp-Asp-Asp- 110Val-Glu-Thr-Lys-Lys-Gln-Lys-Lys-Thr-Asp-GluAsp-AspOH. The sequence of the first 28 amino acids at the NH2 terminus is identical to that of calf thymosin al. The dicarboxylic amino acids, which account for nearly half of the total residues in prothymosin a, are largely clustered in the central portion of the polypeptide chain. The polypeptide contains no aromatic or sulfur-containing amino acids. A computer analysis of the three-dimensional structure based on the primary sequence suggests that the molecule is composed of at least five a-helical regions interrupted by one short extended chain and three short random coils.A mixture of peptides from calf thymus identified as thymosin fraction 5 (1-3) has been reported to restore immune function in thymectomized mice (1) and to yield positive results in other in vivo and in vitro assays for the induction and maintenance of immune function (ref. 4; for reviews of the earlier literature, see refs. 3, 5, and 6). Thymosin fraction 5 also has shown promise in clinical trials with children with immunodeficiency diseases (7) and with cancer patients (8).Thymosin fraction 5 was shown to be a mixture of peptides ranging in molecular weight from 1000 to 15,000 and in isoelectric point from 4.0 to 7.0 (9). The first component of thymosin fraction 5 to be isolated in pure form and characterized was thymosin a,, an acidic peptide containing 28 amino acid residues (9). This peptide was reported to show many of the biological activities of thymosin fraction 5 (refs. 9-12 (pH 5.5). To this buffer, 10-,/g aliquots of carboxypeptidase Y were added at 0, 4, and 8 hr. At timed intervals 5-10% of the total volume was removed, diluted to 100 ,Al with ice-cold buffer of 67 mM sodium citrate (pH 2.0),

A peptide, parathymosin a, containing %105 amino acid residues, has been isolated from rat thymus, and the sequence of the first 30 residues at the NH2 terminus has been determined. In this region, it shows 43% structural identity with thymosin a, and prothymosin a. The common sequences do not include residues 2-9, which accounts for the poor reactivity of parathymosin a with an antibody directed against this epitope in thymosin a1. Parathymosin a appears to modulate the action of prothymosin a in protecting sensitive strains of mice against opportunistic infection with Candida albicans.We have recently reported (1) a procedure for the isolation from rat thymus of three previously identified peptides, thymosin 834 (2), thymosin 6310 (3), and prothymosin a (4, 5). Prothymosin a, identified as a larger polypeptide containing the thymosin a1 sequence (6) at its NH2 terminus, was shown to account for most, if not all, of the immunoreactivity detected with an antibody directed against the NH2-terminal sequence in thymosin a1 (4).In this paper, we report that the isolation procedure also yields a fourth peptide, named parathymosin a because it shows some structural homology to prothymosin a and because it has a similar size and amino acid composition. Parathymosin a can also be isolated from other rat tissues, and in some tissues, such as liver, kidney, and brain, the concentrations of parathymosin a are much higher than the concentrations of prothymosin a. Preliminary results suggest that parathymosin a may act to modulate the immunoenhancing activity exhibited by prothymosin a.EXPERIMENTAL PROCEDURES Materials. Unless otherwise indicated, materials used were as described (1, 4, 5).Methods. The procedures for processing and extracting tissues and separating peptides from the extracts were as described (4, 5). Briefly, fresh rat thymuses were frozen in liquid N2 and pulverized in the frozen state. The powdered frozen tissue was quickly brought to 95°C-100°C in a relatively large volume of boiling 0.1 M sodium phosphate buffer (pH 7.0) and, after cooling, the suspension was homogenized with a Polytron homogenizer (Brinkmann) and the soluble fraction was collected. The clear extracts were desalted on Sep-Pak C-18 cartridges (Waters Associates), and the peptides were eluted with 1 M HCOOH/0.2 M pyridine, separated by chromatography on a column of Sephacryl S-200, and purified by reversed-phase HPLC (1, 4).Amino acid analyses, automated sequencing of peptides, and isoelectric focusing were carried out as described (4). (10 /11) were dried, hydrolyzed with alkali, and analyzed with fluorescamine (4). For subsequent purification by HPLC, the fractions corresponding to peak a, as indicated by the bar, were pooled and combined with similar fractions from three other gel filtration separations.