Capillary electrophoresis in biochemical and clinical laboratoriesp selected attractive examples.

As demonstrated by selected examples from our laboratories, CE is a unique methodology for purity control of synthetic as well as natural tissue-isolated biopolymers, a prerequisite before reliable biotestings should be performed. A combination of rapid matrix-assisted laser desorption ionization mass and CE electrophoretic mobility determinations facilitates primary sequence determinations of enzymatic peptide digest mixtures often making costly Edman degradations unnecessary. The enormous separation efficiency and large variety of different possible separation modes in CE, allow detection of single components in complex mixtures which is demonstrated by the apolipoprotein A-I determination in human blood serum in this communication.

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.

Expression of alpha-thymosins in human tissues in normal and abnormal growth.

Radioimmunoassays specific for the N and C termini of human prothymosin alpha and the N terminus of human parathymosin alpha were employed for the measurement of the levels of alpha-thymosins in human thymus, spleen, and liver during normal growth and intestine and breast in malignant growth. A differential expression of the two alpha-thymosins was observed in thymus (prothymosin alpha-rich) and liver (parathymosin alpha-rich). A decline in the levels of both alpha-thymosins was found with age, with prothymosin alpha in thymus showing the sharpest change (15- to 30-fold). The levels of both alpha-thymosins were higher in malignant tissues as compared with healthy ones. In breast cancer, in particular, the mean increase for prothymosin alpha and parathymosin alpha was 17.9- and 11.5-fold, respectively. The major crossreactive material was characterized in all cases as intact prothymosin alpha and parathymosin alpha. These results suggest an in vivo relationship of the expression of alpha-thymosins with the human tissue cell proliferation activity.

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.

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.

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.

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.