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.

Evidence for the selective release of lysosomal proteinases in fasted rabbits.

The enzyme responsible for the conversion of "neutral" to "alkaline" fructose 1,6-bisphosphatase (EC 3.1.3.11) by removal of a 7000 dalton peptide (converting enzyme, Proteinase I) has been shown to be localized in rat liverlysosomes. Lysosomes also contain a specific proteinase (Proteinase II) that catalyzes the release of a small peptide from the NH2-terminus of the native subunits. In fasted rabbits Proteinase II is released into the cytoplasm, together with Cathepsin A, but Proteinase I remains associated with the lysosomal fraction. Increased osmotic fragility of liver lysosomes in fasted rabbits has also been observed, but this increased fragility does not result in the release of Proteinase I. The appearance of Proteinase II in the cytoplasm may be due either to its selective release from the lysosomes, without release of Proteinase I, or its localization in a different lysosomal fraction. Changes in lysosomal structure induced by fasting may play a dual role in : 1) the mobilization of amino acids for gluconeogenesis and 2) the modulation of activity of gluconeogenic enzymes.

A radioimmunoassay for thymosin alpha 1 that detects the native polypeptide, prothymosin alpha.

A radioimmunoassay, developed for thymosin alpha 1, can also be utilized for the quantitation of the intact native polypeptide, prothymosin alpha, which contains the thymosin alpha 1 sequence at its NH2-terminus (Haritos et al., 1984a). The major epitope was characterized and found to include residues 1-10 at the NH2-terminus of thymosin alpha 1. As little as 5 pmol of prothymosin alpha can be detected in tissue extracts with this radioimmunoassay.

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.B., Blacher, R., Stein, S. and Horecker, B.L. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1050). To evaluate the content of parathymosin in tissues and fluids, we have developed a radioimmunoassay using an antiserum raised in a rabbit against parathymosin isolated from rat liver. Neither thymosin alpha 1 nor prothymosin alpha show significant cross-reactivity with this antiserum. Based on competition experiments with peptide fragments derived from parathymosin and on the lack of cross-reactivity with prothymosin alpha, the major epitope appears to comprise a region in parathymosin including amino acid residues 26-32. Using this radioimmunoassay, which detects as little as 4 pmol of parathymosin, it was confirmed that highest concentrations, ca. 230-240 micrograms/g of fresh tissue, are present in liver and kidney, followed by brain, lung, thymus and spleen. The content of parathymosin in circulating human leukocytes, 2-3 pmol/ml whole blood, was one-fifth of the quantity of prothymosin alpha. With this radioimmunoassay, parathymosin could not be detected in human plasma.

The biochemistry of sugars.

In animal cells the utilization of sugars involves one or more of the three major pathways known to occur in these cells. The glycolytic (Embden-Meyerhof) pathway is quantitatively the most important, and this pathway, coupled to the citric acid cycle, serves as a major source of energy. Glycolysis and the citric acid cycle also provide most of the precursors for the synthesis of proteins, nucleic acids and lipids. The pentose phosphate, or hexose monophosphate oxidation, pathway is a major source of NADPH required for the conversion of carbohydrate to the more reduced lipids and proteins, and also furnishes the ribose and deoxyribose moieties of nucleotides and nucleic acids. The functions of the third pathway, the uronic acid pathway, are less well defined. It is the source of glucuronides for mucopolysaccharides and for detoxification mechanisms, and also in most animals, is the pathway for the synthesis of ascorbic acid. The uronic acid pathway may also function as a point of entry for glucuronides, pentoses, and pentitols.

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. Natalini, and B. L. Horecker (1983) Arch. Biochem. Biophys. 225, 407-413). First-round screening with a synthetic oligodeoxynucleotide probe yielded 55 positive clones, and sequence analysis of 11 of these clones revealed that they all coded for a peptide containing the thymosin beta 10 sequence, except for an additional arginyl residue at position 39. This peptide, designated thymosin beta 10arg, had been identified previously in rabbit tissues and reported as a variant of thymosin beta 10 (S. Ruggieri, S. Erickson-Viitanen, and B.L. Horecker (1983) Arch. Biochem. Biophys. 226, 388-392). Analysis of the 55 positive clones using a specific oligodeoxynucleotide probe constructed to correspond to the mRNA sequence, including the codon for Arg39, confirmed that they all coded for the amino acid sequence including Arg39. Based on these results, the existence of a molecular species lacking Arg39 is considered unlikely, and we conclude that thymosin beta 10 contains 43, rather than 42, amino acid residues, with identity to thymosin beta 4 in 32 of the 43 residues. We propose that the name thymosin beta 10 be used to refer to the peptide containing Arg39 and that the designation thymosin beta 10arg be dropped. In the cDNA sequence the codons for Ala1 and Ser43 of thymosin beta 10 are flanked by initiator and terminator codons, respectively; thus, both the thymosin beta 4 and thymosin beta 10, which coexist in mammalian cells and tissues, are synthesized without the formation of larger polypeptide precursors.