Prothymosin alpha gene in humans: organization of its promoter region and localization to chromosome 2.

A genomic clone encoding prothymosin alpha (gene symbol: PTMA), a nuclear-targeted protein associated with cell proliferation, was isolated and the 5'-regulatory region subcloned and sequenced. Because of previously reported discrepancies between several cDNA clones and a genomic clone for prothymosin alpha, we determined the sequence of the first exon and of a 1.7-kb region 5' to the first exon.

Identification of two calpastatin forms in rat skeletal muscle and their susceptibility to digestion by homologous calpains.

Two forms of calpastatin, differing in their specificity for the homologous calpain isozymes I and II, have been separated from rat skeletal muscle extracts and purified to homogeneity. Calpastatin I, the first form to elute in chromatography on DE32, is more effective against calpain I, while calpastatin II is more effective as an inhibitor of calpain II. Based on their molecular mass (approximately 105 kDa) both calpastatin forms belong to the high molecular mass class found in muscles of other animal species (Murachi, T.

Evidence for nuclear targeting of prothymosin and parathymosin synthesized in situ.

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.

Cellular levels of thymosin immunoreactive peptides are linked to proliferative events: evidence for a nuclear site of action.

Thymosin alpha 1 (T alpha 1), the N-terminal 28-amino acid fragment of prothymosin alpha (ProT alpha), and ProT alpha, although originally isolated from whole thymus extracts, are also present in nonthymic cells and tissues. We used an ELISA with an antibody raised against T alpha 1 to investigate the relationship between intracellular levels of thymosin immunoreactive peptide(s) (TIP) and cell proliferation in a rat small intestinal IEC-6 cell line. Increasing TIP levels were observed during cell proliferation, which decreased when proliferation was halted by cellular contact inhibition.

Identification of the proteolytically activated form of protein kinase C in stimulated human neutrophils.

The proteolytically activated form of protein kinase C has been identified in human neutrophils by using a monoclonal antibody that recognizes both the native kinase and the catalytically active proteolytic fragment (protein kinase M). Stimulation with fMet-Leu-Phe results in the conversion of approximately 30% of native protein kinase C to protein kinase M, with little evidence of further degradation. Stimulation with phorbol 12-myristate 13-acetate, on the other hand, causes only a transient formation of protein kinase M, with complete loss of total kinase activity.

Sequencing of peptides and proteins with blocked N-terminal amino acids: N-acetylserine or N-acetylthreonine.

Many proteins cannot be directly sequenced by Edman degradation because they have a blocked N-terminal residue. A method is presented for deblocking such proteins when the N-terminal residue is N-acetylserine (which occurs frequently in eukaryotic proteins) or N-acetylthreonine. The method has been applied successfully to the determination of the N-terminal amino acid sequence of human, bovine, and rat parathymosins.

Isozymes of protein kinase C in human neutrophils and their modification by two endogenous proteinases.

Two major protein kinase C (PKC) isozymes, accounting for approximately 95% of the total activity in human neutrophils, were separated by hydroxyapatite chromatography and were identified as beta-PKC (60% of the total) and alpha-PKC (35% of the total). No gamma-PKC was detected. A minor Ca2+/phospholipid requiring kinase that eluted from hydroxyapatite after alpha-PKC did not react significantly with any of the specific antisera employed for identification.

Activation of neutrophil calpain following its translocation to the plasma membrane induced by phorbol ester or fMet-Leu-Phe.

Stimulation of human neutrophils with phorbol myristate acetate or fMet-Leu-Phe results in translocation to the plasma membrane of approximately 25-40% of the cellular calpain activity. In the membrane-bound form the Ca2+-requirement for proteolytic activity is substantially reduced. An anti-calpain monoclonal antibody that is internalized by stimulated neutrophils is recovered in the same subcellular fraction that contains the membrane-bound calpain, apparently in the form of pinocytotic vesicles.

Prothymosin alpha and parathymosin: mRNA and polypeptide levels in rodent tissues.

Blot hybridization analyses have established the presence of mRNAs for prothymosin alpha (ProT alpha) and for parathymosin (ParaT) in rat and mouse lung, liver, kidney, and brain, confirming the biosynthesis of these peptides in nonlymphoid tissues. In these tissues the levels of mRNAs paralleled the content of the polypeptides, determined with specific radioimmunoassays. The mRNA levels also confirmed the reciprocal relation between the two polypeptides; ProT alpha and its mRNA were found in highest concentrations in spleen and thymus, followed by lung, kidney, and brain, with lowest concentrations in liver.

The sequence of human parathymosin deduced from a cloned human kidney cDNA.

The amino acid sequence of human parathymosin has been deduced from the cDNA sequence of a clone isolated from a human kidney cDNA library. Screening of the cDNA library with a probe containing a partial rat cDNA sequence yielded two clones containing inserts of 1200 and 1100 base pairs respectively, each including the complete open reading frame for human parathymosin. The open reading frame contains 306 nucleotides, including the codon for the initiator methionine.

Localization of the gene coding for parathymosin to chromosome 17 in humans.

Parathymosin is a 101-amino acid polypeptide involved in the regulation of cellular immunity. In situ hybridization of rat parathymosin cDNA to human metaphase chromosomes localized the gene for human parathymosin to the q12----q22 region of chromosome 17.

Bovine parathymosin: amino acid sequence and comparison with rat parathymosin.

Parathymosin has been purified from calf liver and its primary sequence established, except for a segment containing approximately 11 amino acid residues in the central part of the polypeptide chain. Bovine parathymosin contains approximately 101 amino acid residues and shows 90% identity with rat parathymosin, with substitution of Glu for Asp at positions 21, 57, and 58, Asp for Glu at positions 60 and 63, and Ala for Val at position 77. Three non-conservative substitutions were Ala for Thr at position 81, Leu for Arg at position 78, and Val for Lys at position 79.

The amino acid sequence of bovine thymus prothymosin alpha.

Prothymosin alpha has been purified from calf thymus and its amino acid sequence determined. It contains 109 amino acid residues and closely resembles human prothymosin alpha, with only two substitutions, glutamic acid for aspartic acid at position 31 and alanine for serine at position 83. This is in contrast to six differences between rat and bovine prothymosins, including four substitutions and two deletions.

Isolation and partial characterization of prothymosin alpha from porcine tissues.

Prothymosin alpha, an immunoactive polypeptide of 12 kDa, has been isolated from porcine thymus, spleen, lung and kidney. It lacks aromatic and sulfur-containing amino acids and has a high content of glutamic and aspartic acids. Tryptic digestion of porcine thymus prothymosin alpha yielded peptides which on separation, amino acid analysis and alignment with the known sequence of prothymosin alpha from rat and man showed that the amino terminal portion of the molecule is conserved and the few differences present are confined to the carboxy terminal.

Prothymosin alpha and parathymosin: amino acid sequences deduced from the cloned rat spleen cDNAs.

A rat spleen cDNA library was screened for clones carrying the cDNAs for prothymosin alpha and parathymosin. Sequence analysis of a clone carrying the entire coding region for prothymosin alpha confirmed and completed the amino acid sequence for this polypeptide and established the number of amino acid residues as 111. Rat prothymosin alpha differs from human prothymosin alpha at six positions, including four substitutions and two insertions.

An endogenous activator of the Ca2+-dependent proteinase of human neutrophils that increases its affinity for Ca2+.

An endogenous activator of the Ca2+-dependent proteinase (calpain) has been identified in human neutrophils. In the presence of the activator, the affinity of calpain for Ca2+ is increased by greater than 100-fold and maximum catalytic activity is observed with Ca2+ concentration below 1 microM. The activator is a heat-stable protein having an apparent molecular mass of approximately equal to 40 kDa.

Effects of a monoclonal anti-calpain antibody on responses of stimulated human neutrophils. Evidence for a role for proteolytically modified protein kinase C.

A monoclonal antibody directed against the Ca2+-requiring proteinase (calpain) of human neutrophils was employed to assess the role of this proteinase in mediating the responses to stimuli such as phorbol 12-myristate 13-acetate or fMet-Leu-Phe. In the presence of either phorbol 12-myristate 13-acetate or fMet-Leu-Phe the antibody is taken up by the neutrophils, and a marked inhibition of intracellular calpain is observed. The decreased calpain activity is accompanied by (a) a significant decrease in the proteolytic conversion of native protein kinase C (Ca2+/phospholipid-dependent enzyme) to the soluble form that does not require Ca2+ or phospholipids for activity; (b) a marked increase in the production of superoxide anion; and (c) a decrease in the exocytosis of granule contents.

A radioimmunoassay for parathymosin.

Parathymosin, a polypeptide that is structurally related to prothymosin alpha, has been shown to block the in vivo immunoenhancing effects of prothymosin alpha (Haritos, A.A., Salvin, S.

Isovalerylcarnitine is a specific activator of calpain of human neutrophils.

Isovalerylcarnitine (IVC) a product of the catabolism of L-leucine, is a potent activator of the Ca2+-dependent proteinase (calpain) of human neutrophils. At concentrations of Ca2+ in the low micromolar range, activation was 12 to 15-fold, and the activity exceeded that observed with millimolar concentrations of Ca2+ in the absence of the activator. Of the acylcarnitine derivatives tested, IVC was most active; D-isovalerylcarnitine was much less effective and palmitylcarnitine was ineffective.

Molecular cloning of the cDNA for rat spleen thymosin beta 10 and the deduced amino acid sequence.

A rat spleen cDNA library was prepared and employed for the molecular cloning of the cDNA for thymosin beta 10, a peptide that previously had been found to accompany the closely related peptide, thymosin beta 4, in several species of mammals (S. Erickson-Viitanen, S. Ruggieri, P.