Radioimmunoassays specific for the N and C termini of human prothymosin a and the N terminus of human parathymosin a were employed for the measurement of the levels of a-thymosins in human thymus, spleen, and liver during normal growth and intestine and breast in malignant growth. A differential expression of the two a-thymosins was observed in thymus (prothymosin a-rich) and liver (parathymosin a-rich). A decline in the levels of both a-thymosins was found with age, with prothymosin a in thymus showing the sharpest change (15-to 30-fold). The levels of both a-thymosins were higher in malignant tissues as compared with healthy ones. In breast cancer, in particular, the mean increase for prothymosin a and parathymosin a was 17.9-and 11.5-fold, respectively. The major crossreactive material was characterized in all cases as intact prothymosin a and parathymosin a. These results suggest an in vivo relationship of the expression of a-thymosins with the human tissue cell proliferation activity.Human prothymosin a (1) and parathymosin a (2) are 109 and 101 residues long and partly homologous, particularly at their N termini. They are ubiquitous in mammalian tissues (1, 3).Existing literature for the two a-thymosins points toward an extracellular role of immunologic nature and an intracellular role related to cell growth. In the immunologic direction, thymosin al (the N-terminal fragment 1-28 of prothymosin a) and/or prothymosin a have been reported to increase the expression of interleukin 2 (IL-2) and IL-2 high-affinity receptors in normal human lymphocytes (4,5), cooperate with a,B-interferon in boosting natural killer (NK) activity in immunosuppressed and tumor-bearing mice (6), and restore deficient autologous and allogeneic mixed lymphocyte responses in lymphocytes from patients with clinically active multiple sclerosis (7) and systemic lupus erythematosus (8). In cell growth, increased levels of mRNA for prothymosin a have been observed in lymphocytes stimulated with mitogens and serum reconstitution (1, 9), in lymphocytes of leukemic patients (10), and in regenerating liver (9). The activation of the protooncogene MYC was also reported to lead to rapid increase in transcription of the prothymosin a gene (11), and antisense oligomers for prothymosin a were found to inhibit myeloma cell division (12). J.G. and Ch.M.) from breast and (by D.P.) from colon cancer patients. Tissues were stored at -45°C. Extraction was carried out according to Tsitsiloni et al. (13). MATERIALS AND METHODSThe radioimmunoassay for the N terminus of prothymosin a was carried out as described by Yialouris et al. (14). The radioimmunoassays for the C terminus of prothymosin a and the N terminus of parathymosin a were developed by generating rabbit antisera against the synthetic peptides human prothymosin-a-(90-109) and human parathymosin-a-(1-30), both with an N-terminal extension of Cys-Aca (Aca, aminocaproic acid) and coupled to keyhole limpet hemocyanin (KLH). The synthetic peptides human prothymosin-a-(90-109) and human parathy...
Using a radioimmunoassay for the NH2-terminus of prothymosin alpha, the crossreactive material was measured in subcellular fractions of calf thymus and liver. No significant amount of crossreactive material was found in the nucleus. This provides experimental evidence against a recent hypothesis, based on structural evidence, that prothymosin alpha is a nuclear polypeptide.
According to gel‐filtration experiments, α‐ and β‐thymosins appear to form oligomers, which are 4‐5‐fold larger than the corresponding polypeptides. However, on analysis by sedimentation equilibrium ultracentrifugation, prothymosin α and thymosin β4 showed relative molecular masses of 12 800 and 4600, which are close to the values calculated from their amino acid sequences, confirming their existence in solution as discrete monomeric entities.
A new radioimmunoassay has been developed for thymosin beta 4 by generating rabbit polyclonal antibodies against the synthetic N-terminal peptide fragment 1-15 coupled to KLH. The synthetic analogue [Tyr12]-thymosin beta 4 (1-15) was used as tracer. This radioimmunoassay, with a useful range of 10-1000 pmoles, showed cross-reactivity with the second homologous beta-thymosin of man and rat (thymosin beta 10) but not of calf (thymosin beta 9). This radioimmunoassay, together with an improved radioimmunoassay for the N-terminus of parathymosin alpha, was employed for the measurement of the levels of thymosin beta 4 and parathymosin alpha in nuclear and extranuclear extracts of calf thymus. The bulk of these polypeptides was found in the extranuclear material whereas only traces were observed in the nuclear environment, which indicates the extranuclear localisation of alpha- and beta-thymosins.
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