Distribution of prothymosin alpha in rat tissues.

A radioimmunoassay, using a rabbit antiserum directed against thymosin alpha 1, was employed to detect the presence of crossreacting peptides in rat tissues. Highest concentrations were present in thymus, but thymosin alpha 1 cross-reacting 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 alpha, a peptide containing approximately equal to 112 amino acid residues. Prothymosin alpha is believed to be the endogenous peptide from which thymosin alpha 1 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 alpha 1 were detected with the extraction procedure employed.     

Parathymosin alpha: a peptide from rat tissues with structural homology to prothymosin alpha.

A peptide, parathymosin alpha, containing approximately equal to 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 alpha 1 and prothymosin alpha. The common sequences do not include residues 2-9, which accounts for the poor reactivity of parathymosin alpha with an antibody directed against this epitope in thymosin alpha 1. Parathymosin alpha appears to modulate the action of prothymosin alpha in protecting sensitive strains of mice against opportunistic infection with Candida albicans.     

Primary structure of rat thymus prothymosin alpha.

The primary structure of prothymosin alpha from rat thymus, containing 113 amino acid residues, is reported as follows: (formula; see text) The sequence of the first 28 amino acids at the NH2 terminus is identical to that of calf thymosin alpha 1. The dicarboxylic amino acids, which account for nearly half of the total residues in prothymosin alpha, 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 alpha-helical regions interrupted by one short extended chain and three short random coils.     

Prothymosin alpha in human blood.

The major cross-reacting peptide in human plasma detected with a radioimmunoassay (RIA) for thymosin alpha 1 was identified as prothymosin alpha, based on its elution properties in gel-filtration chromatography and its amino acid composition after purification by HPLC. A small quantity (less than 10%) of the total cross-reacting material was recovered in fractions corresponding to lower molecular weight thymosin alpha 1-like peptides. The total quantity of cross-reacting material detected in human blood, expressed as thymosin alpha 1 equivalents, was 11-14 pmol/ml (approximately 90% was recovered in the leukocyte fraction, approximately 10% was in the plasma fraction, and 1-2% was in the erythrocyte fraction). The peptide present in leukocytes was also identified as prothymosin alpha. After correction for the 5-times lower molar reactivity of prothymosin alpha in the thymosin alpha 1 RIA employed in these experiments, we estimate that the content of prothymosin alpha in human blood is 55-70 pmol/ml (0.6-0.8 microgram/ml). The relatively small quantities recovered in the erythrocyte and plasma fractions may be attributed to contamination of the former by leukocytes or to leakage from leukocytes into the plasma.     

The human prothymosin alpha gene is polymorphic and induced upon growth stimulation: evidence using a cloned cDNA.

Clones for human prothymosin alpha have been identified in cDNA libraries from staphylococcal enterotoxin A-stimulated normal human lymphocytes and from simian virus 40-transformed fibroblasts. The 1198-base-pair fibroblast clone has been sequenced. The encoded protein is highly acidic (54 residues out of 111) and shares greater than 90% sequence homology with rat prothymosin alpha. The peptide "hormone" thymosin alpha 1 appears at positions 2-29 of the prothymosin alpha amino acid sequence. There is no N-terminal signal peptide. Examination of mouse and human tissues revealed the presence of prothymosin alpha mRNA in kidney, liver, spleen, normal lymphocytes (predominantly T cells), human T-cell leukemia virus-infected T cells, and myeloma cells (B-cell lineage). Prothymosin alpha mRNA is inducible; upon mitogen stimulation it increased greater than 15-fold above the level found in resting lymphocytes. Similarly, serum-deprived NIH 3T3 cells responded to serum restitution with an increase in prothymosin alpha mRNA. Characterization of human genomic DNA by Southern blot analysis disclosed a complicated pattern consistent with genetic polymorphism. These data suggest that prothymosin alpha plays an intracellular role tied to cell proliferation. There is no evidence that it serves as a precursor for secreted thymic peptides. However, given the complexity at the genomic level, multiple functions, including a putative secretory capability, cannot be excluded.     

Purification of thymus mRNA coding for a 16,000-dalton polypeptide containing the thymosin alpha 1 sequence.

A mRNA fraction purified by preparative polyacrylamide disc gel electrophoresis from calf thymus polysomes codes for a polypeptide(s) having a mass of 16,000-17,000 daltons. This polypeptide contains amino acid sequences corresponding to residues 11-18 and 19-25 of thymosin alpha 1. The yield of the octapeptide indicates that the 16,000-dalton peptide is the major product formed in the cell-free synthesis system containing the purified mRNA.     

Translation of mRNA from calf thymus in the wheat germ system: evidence for a precursor of thymosin alpha1.

When translated in the wheat germ system, mRNA from fresh calf thymus stimulates incorporation of radioactive amino acids into an acid-insoluble product, and 10--20% of the total radioactivity incorporated is precipitated with antisera to active thymosin fractions. In sodium dodecyl sulfate disc gel electrophoresis, radioactivity was recovered mainly in two peptides, corresponding to 16,000 and 11,000 daltons; the latter probably represents incomplete chains. Tryptic digests of each of these peptides yielded fragments corresponding to the sequence of residues 15--19 of thymosin alpha1; these peptides were characterized by cochromatography with digests of synthetic thymosin alpha1 and by Edman degradation. Thus, the 16,000-dalton peptide synthesized in the cell-free system appears to be q precursor of thymosin alpha1 and possibly of other peptides in the fractions isolated from calf thymus. The results support the conclusion that this peptide is synthesized in the thymus gland.     

Thymosin alpha1: isolation and sequence analysis of an immunologically active thymic polypeptide.

The amino acid sequence of a biologically active polypeptide isolated from calf thymus, termed thymosin alpha1, has been determined. Thymosin alpha1 is a heat stable, highly acidic molecule composed of 28 amino acid residues. This peptide is one of several present in thymosin fraction 5 that may participate in the regulation, differentiation and function of thymus-dependent lymphocytes (T cells). A nomenclature for the family of polypeptides present in thymosin fraction 5 is suggested.     

Complete amino acid sequence of bovine thymosin beta 4: a thymic hormone that induces terminal deoxynucleotidyl transferase activity in thymocyte populations.

The amino acid sequence of thymosin beta 4, a polypeptide isolated from calf thymus, was determined. Thymosin beta 4 is composed of 43 amino acid residues and has a molecular weight of 4982 and an isoelectric point of 5.1. The NH2 terminus of the peptide is blocked by an acetyl group. This molecule induces expression of terminal deoxynucleotidyl transferase (DNA nucleotidylexotransferase, EC 2.7.7.31) in transferase-negative murine thymocytes in vivo and in citro. Thus, it appears that thymosin beta 4 acts on lymphoid stem cells and may control the early stages of the maturation process of thymus-dependent lymphocytes. This peptide is one of several present in thymosin fraction 5 that participates in the regulation, differentiation, and function of thymus-dependent thymocytes.     

Thymosin alpha 11: a peptide related to thymosin alpha 1 isolated from calf thymosin fraction 5.

Two peptides related to thymosin alpha 1 have been isolated from preparations of calf thymosin fraction 5. One, lacking four amino acid residues at the COOH terminus, is designated des-(25-28)-thymosin alpha 1. The other, named thymosin alpha 11, contains seven additional amino acid residues at the COOH terminus. The sequence of this peptide is: AcSer-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu- Lys-Glu-Lys- Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Gly-Arg-Glu-Ala-Pro-Ala-AsnOH. Thymosin alpha 11, in doses of less than 300 ng per mouse, protects susceptible inbred murine strains against opportunistic infections with Candida albicans. It is approximately equal to 30 times as potent as thymosin fraction 5 and approximately equal in potency to thymosin alpha 1.     

Molecular cloning of cDNA for human prothymosin alpha.

A cDNA library was constructed from human spleen mRNA and screened for clones containing cDNAs coding for prothymosin alpha. A clone containing a 503-base-pair insert including the entire coding sequence for the translated portion of the mRNA was isolated. The deduced amino acid sequence confirms and completes the partial sequence of human prothymosin alpha determined by protein sequencing methods. The presence of an initiator codon immediately preceding the codon for the NH2-terminal serine residue and of a terminator codon immediately following the codon for Asp-109, the COOH-terminal residue, suggests that prothymosin alpha is synthesized without formation of a larger precursor polypeptide. Analysis of the 5' sequence preceding the initiator methionine codon excluded the presence of a signal peptide in the translated sequence.     

Prothymosin alpha gene expression correlates with proliferation, not differentiation, of HL-60 cells

Accumulating evidence suggests that prothymosin alpha has an as yet undefined intracellular, perhaps intranuclear, function related to cell proliferation. Prothymosin alpha mRNA and/or peptide levels increase when cells are stimulated to proliferate. Because proliferation and differentiation events are often inversely correlated, we examined prothymosin alpha gene expression during proliferation and differentiation of HL-60 myeloid leukemia cells. Steady-state levels of prothymosin alpha mRNA, which are high in exponentially growing HL-60, decrease within hours after induction of HL-60 to differentiate along the neutrophil pathway with dimethylsulfoxide (DMSO) or along the macrophage lineage with either tetradecanoylphorbol acetate (TPA) or bryostatin 1. The decline in prothymosin alpha mRNA in response to these differentiation signals parallels that of c-myc mRNA under the same conditions. We then determined whether the downregulation of prothymosin alpha and c-myc mRNA were due to differentiation or cessation or proliferation. Recombinant human gamma-interferon induces monocytic differentiation of HL-60, but permits continued proliferation, and, under these conditions, expression of prothymosin alpha, as well as of c-myc, mRNA remains elevated. We conclude that prothymosin alpha and c-myc expression are coregulated in differentiating HL-60 and that their expression correlates with the proliferative state of HL-60 cells, rather than with the differentiated state.     

Purification of human platelet-derived growth factor.

Human platelets contain a polypeptide growth factor that stimulates the proliferation of connective tissue cells. Purification of this platelet-derived growth factor (PDGF) was accomplished by heat (100 degrees C) treatment of washed platelets and subsequent ion-exchange chromatography, gel filtration in 1 M acetic acid, isoelectric focusing, and preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis. PDGF has an isoelectric point of 9.8 and a molecular weight ranging from 13,000 to 16,000 as judged by gel filtration in 1 M acetic acid or analytical sodium dodecyl sulfate gel electrophoresis under reducing conditions. The specific activity of the purified PDGF is 20 million times greater than that found in unfractionated human serum. Purified PDGF stimulates replicative DNA synthesis and cell proliferation in quiescent density-arrested cultures of BALB/c 3T3 cells at concentrations of 1 ng/ml (0.1 nM).     

Molecular weight and structural nonequivalence of the mature alpha subunits of Torpedo californica acetylcholine receptor.

A discrepancy of about 20% exists between the molecular weight of the alpha subunit of Torpedo californica electroplax acetylcholine receptor as determined by gel electrophoresis of the mature protein (Mr 40,000 +/- 2000) and by nucleotide sequence analysis of cDNA (Mr approximately equal to 50,000). We demonstrate by amino acid sequence analysis that post-translational processing does not occur and that the mature subunit has a Mr of approximately equal to 50,000. The functional acetylcholine receptor contains two copies of this alpha subunit in addition to one each of related beta, gamma, and delta subunits. The binding sites for cholinergic ligands that are located on the alpha subunits have been shown to be nonequivalent. Amino acid sequence analysis of peptides obtained by proteolytic cleavage of the alpha subunit reveals that N-asparagine glycosylation at a single site (residue 141) occurs to a different extent in the two copies of this polypeptide in the mature protein and provides an explanation for nonequivalence of their binding sites.     

Molecular cloning and sequence determination of cDNAs for alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go from rat brain.

We have cloned cDNAs encoding alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go and determined their nucleotide sequences. Purified preparations of Gi and Go alpha subunits (Gi alpha and Go alpha) from rat brain were completely digested with trypsin, and peptides were subjected to amino acid sequence analysis. By screening of a cDNA library from rat C6 glioma cells with a synthetic probe corresponding to a 17 amino acid sequence, a clone encoding the sequence of Go alpha was obtained. Then, the library was rescreened with a Go alpha cDNA probe to isolate several strongly or weakly hybridizing clones. cDNAs encoding the complete sequences of Gi alpha and Gs alpha were thus obtained. From nucleotide sequence analysis, the amino acid sequences of Gs alpha and Gi alpha were deduced; they contain 394 and 355 amino acid residues (including the initiator methionine), respectively. The calculated molecular weights for Gs alpha and Gi alpha were 45,663 and 40,499, respectively. The Go alpha clone encoded a sequence of 310 amino acid residues that lacked the NH2 terminus. The homology of the alpha subunits of Gs, Gi, Go, transducin, and ras-encoded protein is discussed.     

The primary structure of rat parathymosin.

Parathymosin has been isolated from rat thymus and from rat liver. Its primary structure is reported as follows: (Sequence; see text). The blocking group at the NH2 terminus was identified by mass spectrometry as acetyl. Regions homologous to amino acid sequences in prothymosin alpha were found to be located between residues 14-20, 23-25, 33-39, 41-43, and 83-87 of parathymosin.     

Purification and characterization of Atlantic salmon prolactin

Prolactin was isolated from the Atlantic salmon (Salmo salar) pituitary gland by extraction with acid acetone, gel filtration, ion exchange-chromatography, and reversed-phase high-performance liquid chromatography. The yield was 0.6 mg/g wet tissue. The hormone had a molecular weight of 23.5 kDa as determined by SDS-polyacrylamide gel electrophoresis. Isoelectric focusing gave an isoelectric point of 9.2. The N-terminal sequence and the amino acid composition indicated extensive homology between Atlantic and Pacific salmon prolactin. Antiserum against Atlantic salmon prolactin cross-reacted with chum salmon prolactin, but not with human, rat, or sheep prolactin.     

HeLa cell DNA polymerase alpha is tightly associated with tryptophanyl-tRNA synthetase and diadenosine 5',5"'-P1,P4-tetraphosphate binding activities.

The purified high molecular weight form of HeLa cell DNA polymerase alpha (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) was shown to associate tightly with several aminoacyl-tRNA synthetase activities. Fractionation of the high molecular weight enzyme on hexylagarose followed by gel filtration, chromatography on phosphocellulose, or polyacrylamide gel electrophoresis under nondenaturing conditions demonstrated copurification of only tryptophanyl-tRNA synthetase [L-tryptophan:tRNATrp ligase (AMP-forming), EC 6.1.1.2] along with DNA polymerase alpha. The high molecular weight (660,000) and low molecular weight (145,000) forms of DNA polymerase alpha were shown to possess a highly specific, noncovalent, diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding activity. The dissociation constants were determined to be 16 and 22 microM, respectively, by utilization of a charcoal adsorption procedure. No high-affinity binding of ATP could be detected. These findings suggest a link between the amino acid activation process and DNA replication in mammalian cells.