Radioimmunoassays for the C-terminus of prothymosin alpha and the N-terminus of parathymosin alpha for the measurement of the levels of alpha-thymosins in human cancer.

A radioimmunoassay specific for the C-terminus of human prothymosin alpha was developed using the synthetic peptide [Cys-Aca degrees]-human prothymosin alpha (90-109)-OH coupled to KLH as antigen and the analogue [Tyr-Aca degrees]-human prothymosin alpha (90-109)-OH labelled with 125I as tracer. The radioimmunoassay measured intact prothymosin alpha, in the range of 2-100 pmol and does not cross-react with the partly homologous polypeptide parathymosin alpha. A major epitope was located in the segment 95-107. A radioimmunoassay specific for the N-terminus of human parathymosin alpha, also measuring intact parathymosin alpha in the range of 1-20 pmol and not cross-reacting with prothymosin alpha, was developed using the synthetic peptide [Cys-Aca degrees]-human parathymosin alpha (1-30)-OH as antigen coupled to KLH and the analogue [Tyr-Aca degrees]-human parathymosin alpha (1-10)-OH labelled with 125I as tracer. A major epitope was located in the segment 1-10. These radioimmunoassays, together with a previously established radioimmunoassay for the N-terminus of prothymosin alpha, permitted the identification of the molecular forms of the cross-reactive materials in both normal and neoplastic breast tissue extracts as intact prothymosin alpha and parathymosin alpha. It was also possible to reveal significantly higher levels of both alpha-thymosins in breast cancer tissue compared to the nearby healthy tissue--the mean of 14 samples was over 14-fold higher--suggesting a role of both prothymosin alpha and parathymosin alpha in cell proliferation. The reported radioimmunoassays are expected to facilitate the search for prognostic and/or diagnostic applications of these polypeptides in human cancer.

Esterases in quail (Coturnix coturnix). Physicochemical and developmental aspects.

1. Soluble esterases of digestive system organs of various developmental stages in the quail (Coturnix coturnix) were resolved by polyacrylamide gel electrophoresis into several molecular forms which were characterized as carboxylesterases, acetylesterases, cholinesterases and esterases sensitive to eserine. 2. The pI of the majority of esterasic activity in several quail and chicken tissues was observed in the range of 5.1-5.6, while the apparent molecular weight in liver extracts was 60,000. 3. The expression of the esterase multiple molecular forms was found to be both tissue- and developmental stage-specific, with electrophoretic patterns becoming more complex in number and/or staining intensity upon hatching and thereafter, especially in liver and intestine.

Evidence for the extranuclear localization of thymosins in thymus.

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.

The complete sequences of trout (Salmo gairdneri) thymosin beta 11 and its homologue thymosin beta 12.

Two forms of beta-thymosins, designated thymosin beta 11 and thymosin beta 12, were isolated from trout (Salmo gairdneri) spleen. This suggests that the presence of two beta-thymosins, previously thought to be a property of mammalian tissues only, is a more general phenomenon in vertebrate species. Both trout beta-thymosins were found to be N-terminally blocked by a group identified as acetyl by m.s. Automated protein sequencing of tryptic, thermolytic and Staphylococcus aureus in 41-residue V8 proteinase fragments revealed that one of the two beta-thymosins corresponds to the previously reported 41-residue-long sequence of thymosin beta 11 with two substitutions at positions 5 and 7, i.e. Asn instead of Asp, and Glu instead of Gln, whereas the other beta-thymosin, designated thymosin beta 12, was found to be a 42-residue polypeptide closely similar in sequence to thymosin beta 11, with five substitutions (i.e. at positions 5, 7, 10, 11 and 41, with Asp, Ala, Ser, Asn and Thr instead of Asn, Glu, Ala, Ser and Ser respectively) and one addition at position 42 (Ala). Comparison of the known six sequences of beta-thymosins together with the sequences reported here showed that the sequence similarity of the two beta-thymosins in trout (86%) is greater than that of the two beta-thymosins in mammalian species (74%) and that residues at 28 positions are identical in all beta-thymosins, the longer conserved segments located at positions 16-26 and 31-38.

A thymosin beta 4 ELISA using an antibody against the N terminal fragment thymosin beta 4 [1-14].

A thymosin beta 4 ELISA was developed in which thymosin beta 4, absorbed on microwells, competed with thymosin beta 4 in solution for the binding sites of an anti-thymosin beta 4 antibody. The antibody molecules finally immobilized on the microwells were detected using a goat anti-rabbit immunoglobulin/horseradish peroxidase conjugate in combination with the substrate 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, and measuring the relevant optical density values. Anti-thymosin beta 4 antibodies were raised in rabbits against intact thymosin beta 4 as well as against selected fragments of the peptide, i.e., the N terminal fragments thymosin beta 4[1-14] and thymosin beta 4[1-11]. The antibody against thymosin beta 4[1-14] was used in the thymosin beta 4 ELISA, because it showed minimal cross-reactivity (0.1%) with the highly homologous peptide thymosin beta 9 as well as exhibiting the highest titre. The ELISA procedure developed, apart from showing a minimal cross-reaction with thymosin beta 9, was fast, easy to perform and exhibited good assay characteristics.

Prothymosin alpha is not a nuclear polypeptide.

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.

Evidence for the monomeric nature of thymosins.

According to gel-filtration experiments, alpha- and beta-thymosins appear to form oligomers, which are 4-5-fold larger than the corresponding polypeptides. However, on analysis by sedimentation equilibrium ultracentrifugation, prothymosin alpha and thymosin beta 4 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.

Physicochemical characterization and tissue distribution of esterases in two salamandridae species (Mertensiella luschani and Salamandra salamandra).

1. Tissue- and species-specificity of the electrophoretic patterns of the multiple molecular forms of esterases were observed in the urodele amphibians Mertensiella luschani luschani, M.l. helverseni and Salamandra salamandra. All esterases--distributed into two electrophoretic mobility areas in gonads, muscles and brain and into four areas in liver, stomach and intestine--were characterized as carboxylesterases. 2. M. l. luschani and S. salamandra liver esterases were electrofocused into nine and eleven major bands with pIs ranging from 4.60 to 5.65 and from 4.40 to 6.20, respectively. 3. Two size groups of esterases were observed in liver extracts of the above three subspecies by Sephadex G-200 gel filtration. The mean values of their apparent molecular weights were 70,000 and 230,000 respectively.

A radioimmunoassay for parathymosin alpha using antibodies to synthetic N-terminal peptide 1-30.

Antibodies against the N-terminus of rat parathymosin alpha have been raised in rabbits by conjugating parathymosin alpha (1-30) to hemocyanin. A radioimmunoassay for parathymosin alpha was established by utilizing antibodies against the above polypeptide and parathymosin alpha(1-12)[Tyr] as tracer. The useful range was 5-450 pmol for parathymosin alpha. An epitope was located in the amino acid sequence 1-12. The antiserum failed to crossreact with the same molar concentrations of the partly homologous thymosin alpha 1 or prothymosin alpha. With this radioimmunoassay, parathymosin alpha was isolated from calf thymus after separation from prothymosin alpha by reversed phase HPLC. Endogenous proteases did not appear to generate N-terminal fragments of parathymosin alpha in rat liver extracts in a similar fashion to that observed for prothymosin alpha. Parathymosin alpha has a ubiquitous distribution in the human tissues examined, with levels ranging from 93 (brain) to 1043 (liver) ng of parathymosin alpha(1-30) equivalents/g (wet weight).

The identification of prothymosin alpha-like material in vertebrate lymphoid organs by a radioimmunoassay for the N-terminal decapeptide.

A radioimmunoassay (RIA) is described for the detection and quantitation of prothymosin alpha (ProT alpha), and its N-terminal fragments containing as a minimum the first ten amino acid residues. This range of peptides includes thymosins alpha 1 (T alpha 1) and alpha 11 (T alpha 11). Antibodies against T alpha 1 and the tracer T alpha 1(1-10)Tyr11(125I), an analogue of the major epitope, were utilized in this RIA. 50% displacement of the ligand was observed with 1.3 pmol of T alpha 1 and 6.4 pmol of ProT alpha. The partially homologous parathymosin alpha (ParaT alpha) showed less than 2% crossreactivity with ProT A. Sephacryl S-200 gel filtration separation of the peptides of calf thymus, chicken spleen and trout spleen extracts prepared by a method eliminating proteolysis, combined with the above RIA, showed the presence of a major immunoreactive peak. Its elution volume corresponded to that of rat ProT alpha (apparent mol. weight 36,000) for both calf (37,000) and chicken (35,000) tissues. In trout it corresponded to a significantly higher molecular weight (62,000). No detectable levels of shorter fragments, including T alpha 1, were observed in any of the above species. The levels of ProT alpha-like peptides in calf thymus, chicken spleen and trout spleen were found to be 246, 8.6 and 7.7 micrograms respectively, of rat ProT alpha equivalents per gram of fresh tissue. The significance of the presence of ProT alpha-like polypeptides in vertebrate species as distant as fish and mammals, the absence of short T alpha 1-like fragments, and the relative conservation of the N-terminus as suggested by the RIA is discussed.

Physicochemical characterization and tissue distribution of multiple molecular forms of fish (Trachurus trachurus) esterases.

1. Tissue-specific electrophoretic patterns of multiple molecular forms of esterases were observed in fish Trachurus trachurus. They were composed of three (in intestine) to nine (in liver) bands characterized mainly as carboxylesterases and acetylesterases. 2. Two major esterase activity bands of pI 4.60 and 4.77 were accompanied by minor ones of higher pH in tissue extracts. 3. Under optimum assay conditions, liver was the richest source of esterase activity followed by intestine, stomach, brain, red muscles, heart, white muscles and gills. 4. Esterases of 70,000 and 420,000 mol. wt were resolved by gel-filtration in liver, intestine and brain. Low size esterases prevailed in liver while the opposite was the case in brain.

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.

On the molecular size of thymosins.

The immunoregulatory polypeptide prothymosin alpha and its biologically active N-terminal fragment thymosin alpha 1m, with relative molecular masses of 12,500 and 3108 respectively, were found to behave as oligomers (trimers to hexamers) in gel-filtration measurements. This phenomenon of an apparent association of polypeptides has been reported for other thymosins--parathymosin alpha, thymosin beta 4 and thymosin beta 10. In contrast, sedimentation equilibrium ultracentrifugation shows that thymosin alpha 1 is a monomer with a relative molecular mass of 3000 +/- 200. Measurement of the diffusion coefficient as 221 micron2/s suggests that the molecule is approximately spherical. The implications for the molecular species of prothymosin alpha, parathymosin alpha, and beta-thymosins are discussed.

Human prothymosin alpha: amino acid sequence and immunologic properties.

Prothymosin alpha has been purified from human thymus and its amino acid sequence determined, except for a 15 amino acid segment including 10 glutamyl residues near the middle of the molecule. Like prothymosin alpha from rat thymus [A. A. Haritos, R. Blacher, S. Stein, J. Caldarella, and B. L. Horecker (1985) Proc. Natl. Acad. Sci. USA 82, 343-346], human prothymosin contains the thymosin alpha 1 sequence at its NH2-terminus. It contains a total of 109-110 residues compared to 111-112 for rat prothymosin alpha, with deletions corresponding to positions Gln39 and Lys108 of the rat polypeptide. Human prothymosin alpha also differs from rat prothymosin alpha at positions corresponding to residues 87, 92, and 102 of the latter, with substitutions of alanine for proline, alanine for valine, and aspartic acid for glutamic acid, respectively. Human prothymosin is significantly less active than rat prothymosin in protecting mice against infection with Candida albicans and in stimulating release in vivo of migration inhibitory factor. Thus, the differences in amino acid sequences, present mainly the COOH-terminal half of the polypeptides, may determine species specificity in biological properties.

Purification and physical properties of hexokinase from human erythrocytes.

A bulk purification is described for hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) from human erythrocytes. Following a 110,000-fold purification from 40 litres of blood, 5 mg protein with a specific activity of 22 units/mg were obtained. On application of various separation techniques, the enzyme activity co-migrated with the main protein component. The physical properties, such as the relative molecular mass of 108,000 and sedimentation coefficient of 5.5 S, are similar to those of the enzyme from human heart. In particular, there is a correspondence in the conformational response to glucose 6-phosphate as shown by an association of the enzyme promoted by this metabolite.

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.

Multiple molecular forms of soluble esterases in the digestive system of the developing chicken.

Soluble esterases in tissues of endodermic origin and of various developmental ages of Gallus gallus were analysed by polyacrylamide slab gel electrophoresis and characterized as carboxylesterases, acetylesterases and cholinesterases. Esterase bands were observed from day 9 in the liver and from day 6 in stomach, intestine and yolk sac. The electrophoretic profiles became more complex after hatching with concomitant increase in the staining intensity. On isoelectric focusing of liver extracts only a major form with pI 5.4 was observed. An eserine sensitive band designated EL-1 was found to be tissue (liver) and age (upon hatching) specific. EL-1-like isozymes were also observed in other species of the Galliformes order.

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.

Isolation and glucose-6-phosphate-mediated dimerization of hexokinase from human heart.

Soluble hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified from human heart. 1 kg of tissue provided 25 mg hexokinase with a specific activity of 58 units/mg, representing a 1700-fold purification and 47% yield. The purification involved six steps, including affinity chromatography with glucosamine attached to Sepharose. The material was homogeneous according to electrophoresis, gel-filtration and sedimentation in the ultracentrifuge, but gave two main components on electrophoresis in denaturing conditions. From determination of the sedimentation and diffusion coefficients, the relative molecular mass was calculated to be 105 000. The enzyme is monomeric, but glucose 6-phosphate promotes an association to dimers. This effect is reversible and is independent of the concentrations of glucose or inorganic phosphate. The results support the postulate that soluble and mitochondrion-bound hexokinases are identical.