Stathmin is a ubiquitous cytoplasmic protein, phosphorylated in response to agents regulating the proliferation, the differentiation and the specialized functions of cells, in a way possibly integrating the actions of diverse concomitant regulatory signals. Its expression is also regulated in relation with cell proliferation and differentiation and reaches a peak at the neonatal stage. To assess the possible role of stathmin at earlier stages of development, we examined its expression and regulation in embryonal carcinoma (EC) and derived cell lines as well as in the early mouse embryo. Interestingly, stathmin is highly abundant in the undifferentiated, multipotential cells of the F9, 1003 and 1009 EC cell lines. Its high expression markedly decreased, both at the protein and mRNA levels, when F9 cells were induced to differentiate into endodermal-like cells with retinoic acid and dibutyryl-cAMP. Stathmin was also much less abundant in differentiated cell lines such as the trophectodermal line TDM-1, as well as in several F9- and 1003-derived cell lines committed to differentiate towards the mesodermal and neuroectodermal lineages but still proliferating. Therefore, the observed decrease of stathmin expression is not related to the reduced proliferation rate but rather to the differentiation of the multipotential EC cells. The immunocytochemical pattern of stathmin expression during early mouse development indicated that stathmin is also highly abundant in the multipotential cells of the inner cell mass of the blastula, whereas it is much lower in the differentiated trophectodermal cells. These results confirm the physiological relevance of the observations with EC cells, and suggest that stathmin, in addition to its high expression at later stages of development and in the adult nervous system, may be considered as a new marker of the multipotential cells of the early mouse embryo.

F9 embryonal carcinoma (EC) cells were transformed using a recombinant plasmid carrying simian virus 40 (SV40) early genes linked to the promoter-enhancer region of the early transcription unit 1A (E1A) of adenovirus type 5. One clone of transformed cells, F9-K4B2, contained several integrated copies of the plasmid and expressed SV40 early genes (T antigens) only when it was induced to differentiate in vitro by the addition of retinoic acid and dibutyryl-cAMP. From the cell population positive for T antigen, it is possible to isolate permanent cell lines with high efficiency. Most of these cells exhibit stable traits that are characteristic of parietal endoderm. We also obtained another cell type which did not express the specific markers of either undifferentiated EC cells or endoderm cells; this type might represent a different stage of commitment in the differentiation of F9 cells. All clones are tumorigenic when injected into irradiated syngeneic mice, and they maintain their phenotype after successive passages in vivo as well as in vitro. The introduction of SV40 early genes into other EC cell lines and early mouse embryos appears to be a promising approach for the immortalization of early embryonic cells.

The extensively purified multienzyme complexes from sheep and rabbit livers containing seven aminoacyl-tRNA synthetases specific for Ile, Leu, Met, Gln, Glu, Lys, and Arg displayed characteristic one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic patterns composed of 11 and 10 major polypeptide components, respectively. Their polypeptide compositions revealed by two-dimensional electrophoresis, including isoelectric focusing in 9 M urea, were not significantly more complex. The isoelectric point of each component from the two complexes fell within the pH range of 6.2 to 7.1, with the notable exception of the common polypeptide of Mr = 43,000 which was distinctly basic. The apparent molecular weight of each component from both complexes was determined by SDS-polyacrylamide gel electrophoresis. Four polypeptides, corresponding to molecular weights of 139,000, 129,000, 43,000, and 38,000 were common to both complexes. The other components from the two complexes displayed similar yet clearly distinct molecular weights. The molar ratios of the polypeptides, estimated by densitometry scanning of stained SDS-polyacrylamide gels, indicated that several components from each complex may be present as more than one copy. Following SDS-polyacrylamide gel electrophoresis, the methionyl-tRNA synthetase component from each complex was identified by the protein blotting procedure, using specific antibodies and 125I-labeled protein A. The unique labeled bands from the complexes of sheep and rabbit precisely matched the major polypeptides of Mr = 103,000 and 108,000, respectively. Mild trypsin treatment of the two native complexes generated fully active forms of methionyl-tRNA synthetase, with molecular weights of 68,000 and 69,500, respectively. The kinetics of proteolysis showed that modification proceeded sequentially through discrete intermediates.

Both the tRNA aminoacylation and amino-acid-dependent ATP-PPi exchange activities of monomeric trypsin-modified methionyl-tRNA synthetase from sheep liver are lost upon incubation with oxidized initiator tRNAMet. The inactivation, which reflects the formation of a Schiff's base between the 5'-terminal adenosine of tRNA and a lysine within the catalytic site of the enzyme, is accompanied by the covalent attachment of one tRNA molecule per enzyme molecule. The affinity labeling method is applied to the sheep liver complex of Mr 10(6) carrying seven aminoacyl-tRNA synthetase activities, from which the monomeric trypsin-modified methionyl-tRNA synthetase (Mr 68 000) was derived. Upon incubation with oxidized initiator tRNAMet, the methionyl-tRNA synthetase activity of the complex is lost. Of the eleven polypeptide chains composing the high-molecular-weight complex, only one polypeptide chain with Mr 103 000 reacts with the modified tRNAMet. The blocking by periodate-treated tRNA of the methionyl-tRNA synthetase activity in the complex has no effect on the other aminoacyl-tRNA synthetase activities. This strongly argues in favor of the independent parallel functioning of the seven aminoacyl-tRNA synthetases associated in a high-molecular-weight complex.

Using a three-step procedure designed to minimize the risks of proteolysis, high molecular weight complexes containing the same seven aminoacyl-tRNA synthetases specific for isoleucine, leucine, methionine, lysine, arginine, glutamic acid, and glutamine were purified from sheep liver and spleen, as well as from rabbit reticulocytes and liver.The polypeptide composition of these complexes, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is characteristic of the animal species from which they are derived.The complexes from sheep liver and spleen display indistinguishable polypeptide patterns composed of 11 major components.Of the 10 common components which characterize the complexes of rabbit reticulocytes and liver, 4 are also shared by the complexes from sheep, while 6 have distinctly different electrophoretic mobilities.Furthermore, in the case of the complex from rabbit reticulocytes, it is shown that the enzyme and polypeptide composition of the complex is independent of the purification method employed.The isolation of high molecular weight complexes of identical aminoacyl-tRNA synthetase and polypeptide compositions from two cell types as radically different as rabbit reticulocytes and hepatocytes suggests that these multienzyme complexes do not arise as artifacts of preparation and supports the view that they reflect a structural organization existing within the cell.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).

Methionyl-tRNA synthetase from sheep lactating mammary gland is found predominantly in the form of high-molecular-weight complexes. Controlled proteolysis of these aggregates generates a low-molecular-weight species of the enzyme with full maintenance of activity as assessed by the rate of aminoacylation of tRNA. The product of proteolysis, which has been purified to homogeneity with a yield of 23%, is a monomeric enzyme of molecular weight 78 000. It has a specific activity of 405 units/mg at 25 degrees C. These findings clearly demonstrate that the aggregated state of methionyl-tRNA synthetase is not a prerequisite for full expression of catalytic activity. Furthermore, the results emphasize the need to provide effective protection against proteolytic damage in studies dealing with the characterization of high-molecular-weight complexes of aminoacyl-tRNA synthetases.

The size distribution of methionyl-tRNA synthetase in extracts from sheep liver is compared to that of lysyl-tRNA, isoleucyl-tRNA, leucyl-tRNA and seryl-tRNA synthetases by gel filtration on Biogel A-5m. Extraction conditions are described which lead to isolation of methionyl-tRNA synthetase exclusively in the form of complexes of molecular weight close to 10(6). Limited trypsin treatment of these aggregates releases a fully active low-molecular-weight form of methionyl-tRNA synthetase which was purified to a specific activity of 674 units/mg at 25 degrees C with a yield of 40%. The homogeneous enzyme appears to be undistinguishable from the corresponding enzyme derived from sheep lactating mammary gland, as judged by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by titration with antibodies raised against the enzyme purified from liver.