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.

In the presence of micromolar concentrations of Ca2+, both protein kinase C and a cytosolic Ca2+-requiring neutral proteinase of human neutrophils become associated with the neutrophil membrane. Binding to the membrane results in activation of the proteinase, which then catalyzes limited proteolysis of the kinase to produce a form that is fully active in the absence of Ca2' and phospholipid. This irreversibly activated protein kinase is released from the membrane and may thus have access, in the intact cell, to intracellular protein substrates. In the absence of the proteinase, Ca2+ promotes the binding of protein kinase C, but conversion to the Ca2+/phospholipid-independent form does not occur and the kinase remains associated with the membrane fraction.Protein kinase C was originally described in rat brain as a soluble, cAMP-independent proenzyme (1) that was converted to the active kinase by the action of a cytosolic Ca2+-requiring proteinase (2, 3). The native "proenzyme" was later shown to require Ca2' and phospholipid (4,5) and to be further activated by diacylglycerol (6), which markedly increased its affinity for both Ca2' and phospholipid (for reviews, see refs. 7 and 8). Activation of protein kinase C in stimulated platelets has been attributed to the formation of diacylglycerol generated by phospholipase C from inositol phospholipids (7,9). An irreversible activation by limited proteolysis has also been described in platelets treated with phospholipase C (10) or phorbol 12-myristate 13-acetate (11) Isolation of Neutrophils. This was based on the procedure of Boyum (14). Freshly collected, heparinized human blood (100 ml) from healthy donors was treated with 1.6% (wt/vol) dextran (final concentration) and left at 25-28°C for -1 hr. The sedimented erythrocytes were removed and the supernatant solution (40 ml) was collected and layered onto 10 ml of 6% Ficoll 400 solution containing 0.17% (vol/vol) Urovison. The gradient was centrifuged at 800 x g for 20 min and the pellet obtained was resuspended in 10 ml of 0.2% NaCl to lyse the contaminating red cells. After 30 sec, 10 ml of 1.6% NaCl was added; the cells were recovered by centrifugation at 400 x g for 5 min and washed three times with 0.01 M sodium phosphate, pH 7.4/5 mM KCl/0.12 M NaCl/24 mM NaHCO3/5 mM glucose. Prior to use, the cells were maintained in an ice bath in the same medium at a concentration of 15-20 x 106 cells per ml. The cell population obtained consisted of 96% neutrophils, as evaluated by microscopic examination. The remaining 4% consisted of 3.5% eosinophils and 0.4% monocytes.Isolation of Human Platelets. Fresh human blood platelet concentrates were obtained from a blood bank and washed platelets were prepared as described by Baenziger and Majerus (15). The platelets were washed and suspended at a final concentration of 1010 cells per ml in the same buffer employed for the neutrophils.Isolation of the Soluble and Particulate Fractions from Neutrophils and Platelets. These were prepared from lysates obtained by sonicating ...

To test the hypothesis that prothymosin and parathymosin contain amino acid sequences that cause them to be targeted to the cell nucleus, expression vectors were constructed containing a simian virus 40 promoter and cDNAs that would code for chimeric proteins composed of truncated human growth hormone (hGH) linked to the NH2 terminus of prothymosin or parathymosin. The truncated hGH lacked the signal peptide sequence required for its secretion. After transfection of these constructs into HeLa S3 cells, which do not normally synthesize hGH, the use of indirect immunofluorescence staining to follow the localization of the hGH chimeras demonstrated that both prothymosin and parathymosin caused targeting to the cell nucleus. Controls with a construct coding for native hGH only, and one coding for the truncated hGH lacking the signal peptide, revealed secretion into culture medium and staining in the endoplasmic reticulum and Golgi apparatus in the first case, and diffuse staining throughout the cytoplasm in the second. The results provide direct evidence, with proteins synthesized in situ, for the presence of nuclear localization signals in both prothymosin and parathymosin.Interest in proteins of thymic origin was stimulated by the observation that a crude preparation from calf thymus, designated thymosin fraction 5, showed immunomodulatory properties in a number of in vitro test systems (reviewed in ref. 1). Many of these properties were attributed to a single peptide purified from this fraction, named thymosin a, (2), which was proposed to function as a "thymic hormone" influencing lymphocyte maturation (3). Based on evidence suggesting that this thymosin a, might be a proteolytic artifact that arose during the preparation ofthymosin fraction 5 (4, 5), a search was initiated for the native protein or polypeptide precursor. A procedure designed to minimize endogenous proteolysis led to the isolation of two homologous polypeptides, named prothymosin (6) and parathymosin (7), the first of which contained the 28-amino acid sequence of thymosin a, at its NH2 terminus. The proposed role of thymosin a, as a thymic hormone was brought into question when the putative precursor, prothymosin, and the mRNA coding for this polypeptide were shown to be present in all mammalian tissues examined (8,9). In addition, the cDNAs coding for both human (10, 11) and rat (12) prothymosins were found to lack sequences coding for the signal peptides expected in secretory proteins. An extracellular function was also rendered unlikely by the observation that the mRNA for prothymosin was localized exclusively on free polysomes (13).The experimental findings relating to parathymosin have closely mimicked those for prothymosin, with quantitative differences in tissue polypeptide and mRNA content (7,9,12,14).More recent studies have suggested a relationship between prothymosin and cell growth. Increased prothymosin mRNA in response to a variety of stimulators of mammalian cell division has been reported (11), and elevated mRNA was also ...

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