Identification of rat brain polysomes synthesizing the brain specific enolase (14.3.2 protein), S100 protein and alpha and beta tubulin subunits.

Polysomes prepared from frozen rat brain powder were fractionated by centrifugation in a sucrose gradient. Individual fractions were used to program a reticulocyte lysate in a run-off reaction. The products of cell-free synthesis were assayed for the brain-specific enolase (14.3.2 protein) and S100 protein by immunoprecipitation with specific antisera and for tubulin by two-dimensional electrophoresis in polyacrylamide slab gels. The relative synthesis of these proteins by unfractionated free brain polysomes were 0.1 per cent, 0.05 per cent and 0.7 per cent respectively. After centrifugation in a sucrose gradient polysomes synthesizing S100 protein were separated from those synthesizing the other two markers. There was a threefold enrichment in the specific messenger RNA activity for each of the three proteins studied in their respective peak fractions of polysomes.

Developmental studies with the 14-3-2 antigen and the neuron specific enolase (NSE) associated activities-I.

We have compared the rodent developmental pattern of the 14-3-2 antigen estimated by a microcomplement fixation technique with that of the cerebral enolases. Chromatographic separation of enolase isozymes on microcolumns demonstrates that the embryonic neuron specific enolase is firstly and mostly represented by the ?? isozyme. The most important increase in 14-3-2 antigen and ?? enolase occurs between post-natal days 7th and 15th. By post-natal day 30, adult levels have been reached. An interesting observation is-during embryonic development-the decrease in the specific activity of the cerebral enolase isozyme ??. This could be explained by the replacement-in neuroblasts-of ?? enolase by neuron specific enolase. A comparison between 14-3-2 antigen and neuron specific enolase (??) purified by completely different methods is presented. The 14-3-2 antigen exhibits an enolase specific activity comparable to that of purified enzyme and has the same electrophoretic mobility. Antibodies raised against either antigen have an identical specificity. Pre and post-natal developmental pattern in rodent brains are similar for both proteins. Thus neuron specific 14-3-2 antigen is identical to neuron specific enolase. Thus we have precisely described the ontogenic transition between the three cerebral enolase isozymes at the tissue level. This study is completed by the analysis of these transitions at the neuronal cell level, using homogenous cell lines (Part II of this paper).

Transition between isozymic forms of enolase during in vitro differentiation of neuroblastoma cells-II.

An analysis of enolase expression during differentiation of neuroblastoma clones in homogeneous culture is presented. The enolases expressed in these neuroblast-like cells are identical to those of mouse brain with respect to the examined properties. Our biochemical investigation has premitted us to demonstrate formally that neuroblastoma cells undergo a transition from the embryonic ?? form to the neuronal ?? form and contain both enolases as well as the ?? hybrid form during maturation. These results suggest that the same phenomenon must exist in vivo for neuroblasts. In neuroblastoma cells, an increase in both ?? and ?? neuron specific enolases is related to cell maturation and expression of the ?? form precedes that of the ?? form during differentiation. Modulation of neuronal enolase activities is similar in the various conditions of differentiation studied and appears not to be necessarily related with morphological differentiation, although concomitant with an arrest of cell division. The evolution of specific neuronal enolases in neuroblastoma cells parallels that observed in vivo, in brain from embryonic day 15 to post-natal day 7. Moreover, at least one treatment (dimethylsulfoxide) causes an important decrease in the high specific ?? activity of these cells as occurs in vivo. This enolase can therefore also be considered as a biochemical marker for neuroblastoma maturation. As observed with other markers and other cell types, neuroblastoma cells in culture express an immature biochemical differentiation of the enolase isozymes.

Detection and localization of 14-3-2 protein in primary cultures of embryonic rat brain.

The development of embryonic rat brain in cell cultures was studied by an immunocytochemical method based on the detection of 14-3-2 protein (neuron-specific enolase or NSE), a neuron-specific protein. This protein was already present in undifferentiated neurons (less than 5 days in culture), being dispersed throughout the cytoplasm, though seemingly concentrated in the vicinity of polyribosomal structures. It was not found in nuclei, in mitochondria or in the Golgi apparatus. During neuron differentiation, the location of 14-3-2 protein was related to neurite development insofar as it was detected along the axon and even in what could be taken to be the presynaptic region of numerous interneuron contacts. In contact areas, a thickening of the junction membrane was observed but the presence of 14-3-2 protein was always unilateral demonstrating the absence of a true synapse and reflecting the halt in neurite development observed after 15 days in culture. The presence of 14-3-2 protein in the cell cultures was confirmed by a microcomplement fixation assay. The protein detected in cell cultures had the same immunological properties as that found in the 17-day-old embryo, but was slightly different from that found in adult rat brain. This observation can be confronted with the lack of neuron maturation in the immunocytochemical studies.

Natural messenger ribonucleic acid-directed cell-free protein-synthesizing system of Bacillus subtilis.

A very active in vitro protein-synthesizing system has been developed from Bacillus subtilis. High activity in the extracts is dependent upon precautions taken to reduce proteolytic activity. Endogenous, exogenous natural and synthetic messenger ribonucleic acids (RNAs) are translated by the system. The activity of the B. subtilis system has been compared to that of the Escherichia coli system. With either SPO1 RNA or polyuridylic acid, the activities of the two systems were very similar. Electrophoresis of the products synthesized in vitro by the two systems primed with SPO1 RNA yields similar radioactive profiles. The major bands of radioactivity correspond to proteins of molecular weight between 15,000 and 40,000.

Functional modifications of the translational system in Bacillus subtilis during sporulation.

Extracts of sporulating cells were found to be defective in vitro translation of phage SP01 ribonucleic acid (RNA) and vegetative Bacillus subtilis RNA. The activity of washed ribosomes from sporulating cells was very similar to that of washed ribosomes from vegetative cells in translating polyuridylic acid, SP01 RNA, and vegetative RNA. The S-150 fraction from either vegetative or sporulating cells grown in Difco sporulation medium contained an apparent inhibitor of protein synthesis. The crude initiation factor fraction from ribosomes of sporulating cells was defective in promoting the initiation factor-dependent translation of SP01 RNA. The crude initiation factor preparations from sporulating cells were as active as the corresponding preparations from vegetative cells in promoting the initiation factor-dependent translation of either phage Qbeta or phage T4 RNA by washed Escherichia coli ribosomes. The crude initiation factors from sporulating cells were perhaps more active than those from vegetative cells in promoting the initiation factor-dependent synthesis of phage T4 lysozyme by E. coli ribosomes. The crude initiation factor preparations from either vegetative or stationary-phase cells of an asporogenous mutant showed similar ability to promote the in vitro translation of SP01 RNA.

Changes in the expression of the alpha alpha form of enolase during neuroblastoma differentiation.

The relative amounts of the different enolase isozymes present in neuroblastoma cells change during differentiation. When differentiation is induced by low serum in the presence of DMSO (dimethyl sulfoxide), there is a 50% decrease in the concentration of enolase activity associated with the form alpha alpha, and an increase in the activity associated with the gamma-containing isozymes (alpha gamma plus gamma gamma); in the absence of DMSO, there is no decrease in alpha alpha or in total enolase activity. In order to study the mechanism of the changes in alpha alpha, cells differentiated with low serum with and without DMSO were compared. Measurements of the concentration of the alpha antigen by microcomplement fixation and by immunotitration demonstrate that the decreased enolase activity in DMSO cells is due to a decreased concentration of the alpha antigen. Measurements of the relative rate of synthesis of the antigen show that the decreased concentration of the alpha antigen is due to a decreased rate of synthesis. Enolase in differentiated cells is sufficiently stable (t1/2 greater than 100 h) that a comparison of the relative rates of degradation has not been possible. The decreased synthesis of the alpha subunit of enolase that occurs under these conditions appears to be a useful model system for studying the de-expression of the alpha gene that occurs in vivo during neuronal differentiation.

Developmental expression of alpha- and gamma-enolase subunits and mRNA sequences in the mouse brain.

Nonneuronal alpha alpha- and neuron-specific alpha gamma- and gamma gamma-enolase activities were measured in the mouse brain during development. The corresponding mRNA sequences were quantified directly by hybridization with cDNA probes. The variations in alpha- and gamma-monomer levels inferred from the enzymatic activities were very similar to those of their respective mRNAs. We conclude that monomer levels are primarily controlled by the amounts of their mRNAs during mouse brain development.

Isolation of murine neuron-specific and non-neuronal enolase cDNA clones.

cDNA clones corresponding to subunits of neuron-specific (gamma gamma and alpha gamma) and non-neuronal (alpha alpha) enolase isozymes were characterized from two mouse brain cDNA libraries. Our hybridization data revealed a partial homology of the coding sequences of mouse alpha, mouse gamma and rat gamma mRNAs. The noncoding sequences, however, appear to be specific for each mouse mRNA. Although coding for two polypeptides of the same molecular weight, the mRNA for the gamma subunit (2600 bases) is larger than that for the alpha subunit (1900 bases). The noncoding sequences for neuron-specific gamma mRNA (about 1300 bases) are therefore longer than those of the non-nervous tissue specific alpha mRNA (about 600 bases).

Developmental changes in translatable mRNAs for the cerebral enolase isozymes alphaalpha and gammagamma.

Using the rabbit reticulocyte cell-free translation system we have estimated during ontogenesis the proportions of in vitro translatable alpha and gamma brain enolase mRNAs, which are two minor mRNA species. No polypeptide precursor to these enzyme subunits appears to be synthesized during translation in vitro. During brain development, the changes in translatable alpha and gamma mRNA content seem to parallel those of the corresponding antigens. The proportion of each of the enolase mRNAs is highest in adult mouse brain. Mechanisms controlling alpha and gamma antigen expression are discussed. In order to prepare the specific cDNA probes, purification of alpha and gamma mRNAs was undertaken.

Murine muscle-specific enolase: cDNA cloning, sequence, and developmental expression.

In vertebrates, the glycolytic enzyme enolase (EC 4.2.1.11) is present as homodimers and heterodimers formed from three distinct subunits of identical molecular weight, alpha, beta, and gamma. We report the cloning and sequencing of a cDNA encoding the beta subunit of murine muscle-specific enolase. The corresponding amino acid sequence shows greater than 80% homology with the beta subunit from chicken obtained by protein sequencing and with alpha and gamma subunits from rat and mouse deduced from cloned cDNAs. In contrast, there is no homology between the 3' untranslated regions of mouse alpha, beta, and gamma enolase mRNAs, which also differ greatly in length. The short 3' untranslated region of beta enolase mRNA accounts for its distinct length, 1600 bases. It is known that a progressive transition from alpha alpha to beta beta enolase occurs in developing skeletal muscle. We show that this transition mainly results from a differential regulation of alpha and beta mRNA levels. Analysis of myogenic cell lines shows that beta enolase gene is expressed at the myoblast stage. Moreover, transfection of premyogenic C3H10T1/2 cells with MyoD1 cDNA shows that the initial expression of beta transcripts occurs during the very first steps of the myogenic pathway, suggesting that it could be a marker event of myogenic lineage determination.