V. Busiello

Selenalysine and protein synthesis.

Selenalysine is a lysine analog having the gamma-methylene group substituted by a selenium atom. It has been demonstrated that selenalysine is activated and transferred to tRNAlys by either Escherichia coli or rat liver aminoacyl-tRNA synthetases, and inhibits lysine incorporation into polypeptides in protein-synthesizing systems from E. coli, rat liver or rabbit reticulocytes. All tests were performed in comparison with thialysine, a lysine analog having the gamma-methylene group substituted by a sulfur atom. In all the reactions studied, both thialysine and selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine shows a slightly lower activity as lysine inhibitor.

Action of thiazolidine-2-carboxylic acid, a proline analog, on protein synthesizing systems.

Thiazolidine-2-carboxylic acid, or beta-thiaproline, is a proline analog in which the beta methylene group of proline is substituted by a sulfur atom. It has been deomonstrated that beta-thiaproline is activated and transferred to tRNAPro by Escherichia coli and rat liver aminoacyl-tRNA synthetases, and inhibits proline incorporation into polypeptides in protein synthesizing systems from E. coli, rat liver or rabbit reticulocytes. In mammalian systems beta-thiaproline inhibits also leucine incorporation; in rabbit reticulocyte lysate it inhibits ribosome run-off. Both these effects may be explained by the fact that beta-thiaproline once incorporated into the growing polypeptide chain impairs its further elongation, as shown by experiments made with puromycin. All tests were performed in comparison with thiazolidine-4-carboxylic acid, or gamma-thiaproline, another proline analog having the gamma methylene group substituted by a sulfur atom; it was shown that in all the reactions studied both compounds act as competitive inhibitors of proline. Some differences in the effects of the two analogs have been evidenced: in almost all the reactions and mainly in the whole protein synthesizing systems, beta-thiaproline shows an higher inhibitory activity.

Selenaproline and protein synthesis

Abstract Selenaproline is a proline analog having the C-4 methylene group substituted by a selenium atom. It has been demonstrated that selenaproline is activated and transferred to tRNA Pro by either Escherichia coli or rat liver aminoacyl-tRNA synthetases in competition with proline, and that it inhibits polypeptide synthesizing systems from E. coli , rat liver or rabbit reticulocytes. Analyzing the polysomal pattern of reticulocyte lysate it has been shown that selenaproline inhibits the ribosome run-off. Studying the effect of the addition of puromycin to the polypeptide synthesizing system, data have been obtained indicating that selenaproline may be incorporated in the growing polypeptide chain but impairs further chain elongation. All tests were performed in comparison with thiaproline, a proline analog having the C-4 methylene group substituted by a sulfur atom, which is known to act as a competitive inhibitor of proline. In all the reactions studied both thiaproline and selenaproline show similar inhibitory effects. The main difference between thiaproline and selenaproline is that the inhibitory effects of the latter are evident at lower concentrations and are not completely reverted by proline.

Thialysine utilization by E. coli and its effects on cell growth.

SummaryThialysine can be utilized for growth by a wild type K12 strain of E. coli. It is incorporated into proteins in substitution and in competition with lysine; up to 17% of protein lysine can be substituted by thialysine. Nevertheless the presence of thialysine in the culture medium gives rise to an inhibition of cell growth rate. This effect has been correlated to the inhibition of protein synthesis rate by thialysine and to the extent of protein lysine substitution by the analog. On the other hand this substitution does not affect cell viability.

Thiaisoleucine and protein synthesis.

Thiaisoleucine is an isoleucine analogue having the gamma-methylene group of the valerianic carbon chain substituted by a sulphur atom. It has been demonstrated that thiaisoleucine is activated and transferred to tRNAIle by rat liver aminoacyl-tRNA synthetase and inhibits isoleucine incorporation into polypeptides in protein synthesizing systems from rat liver or rabbit reticulocytes, whereas it does not affect either leucine incorporation or ribosome run-off or polypeptide chain elongation rate. All tests were performed in comparison with O-methyl-threonine, an isoleucine analogue with the gamma-methylene group substituted by an oxygen atom. In all the reactions studied, both thiaisoleucine and O-methyl-threonine act as competitive inhibitors of isoleucine. With respect to O-methyl-threonine, thiaisoleucine shows higher activity as an isoleucine inhibitor.

β-Selenaproline as competitive inhibitor of proline activation

Abstract β-Selenaproline, a proline analog having the β-methylene group substituted by a selenium atom, has been tested in ATP-PPi exchange reaction catalyzed by either Escherichia coli or rat liver aminoacyl-tRNA synthetases. It has been shown that with both enzymatic systems β-selenaproline does not give rise to ATP-PPi exchange, but specifically inhibits proline activation. The inhibition is of fully competitive type and the K i values, lower than the K m values for proline, show that β-selenaproline binds to the synthetases with high affinity. The inability to form the complex with AMP, taking into account also the behavior of γ-selenaproline and other proline analogs, has been ascribed to the presence of the selenium atom in the β-position.

DNA polymerase activities in friend cells during the differentiation process

DNA synthesis and DNA polymerase activities were followed in FL cell cultures (clone 5.86) at different stages of differentiation. A temporary block of growth and DNA synthesis was observed after the addition of the inducers (DMSO or HMBA). This delay in the growth and in the DNA synthesis initiation is not observed in cultures of DMSO-resistant variants after treatment with DMSO. In both uninduced and induced cultures, during growth, the DNA gamma-polymerase activity is constant and the alpha-polymerase activity is strictly dependent on the DNA synthesis rate. On the other hand, a different behaviour between induced and uninduced cultures is observed for the DNA polymerase beta: its activity is constant in uninduced cultures, whereas it changes in induced cultures at various stages of differentiation, dropping to lower values at early times and rising to values similar to those observed in uninduced cultures at later times. This behaviour is not observed in cultures of a DMSO-resistant variant clone: in this case the beta-polymerase activity is constant in both the absence and the presence of DMSO or HMBA.

DNA hypomethylation and differentiation in Friend leukemia cell variants.

The occurrence, upon differentiation, of a transient DNA hypomethylation has been observed in Friend erythroleukemia cells. Treatment with hexamethylenebisacetamide (HMBA) induces within 24 h a 20% hypomethylation of newly synthesized DNA, that is followed by re-methylation before completion of the differentiative process, as measured by the appearance of benzidine-positive cells. We examined a series of mutant clones which continue to grow in the presence of an inducer. Methylcytosine content of DNA was measured by HPLC, after cell labeling with [3H]uridine. We found that one of these continuously growing clones, which was still capable of hemoglobin synthesis, showed the same degree of hypomethylation as the parental one. The re-methylation process did not occur, however, unless erythroid differentiation was reverted by the removal of the inducer. In another clone which had lost the capacity to synthesize hemoglobin, no DNA hypomethylation was detectable. These experiments show that DNA hypomethylation is an early event strictly related to cell differentiation but not to cell growth arrest.

Thialysine utilization by a lysine-requiring Escherichia coli mutant.

SummaryThialysine cannot completely substitute lysine as growth factor for a lysine-requiring E. coli mutant. However it can be utilized for growth in the presence of limiting amounts of lysine, in substitution of, and in competition with this latter. The effects of thialysine on growth rate, protein synthesis rate and cell viability, and its incorporation into proteins were studied in function of lysine and thialysine concentration in the culture media. Up to 60% of protein lysine substitution by thialysine is observed, without appreciable effects on cell viability.

In vivo incorporation of selenalysine in Escherichia coli proteins and its effects on cell growth

The presence of selenalysine in the culture medium at concentration ranging from 0.05 to 0.3 mM inhibits Escherichia coli growth rate and cell viability. The inhibition of cell growth rate can be imputed to the inhibition of protein synthesis and can be only partially reverted by lysine. Selenalysine is incorporated into cellular proteins in substitution of and in competition with lysine, reaching the value of about 1% as molar fraction with respect to the total amino acids, and substituting up to 14% of protein lysine. The effect of selenalysine on cell viability can be correlated to the extent of its incorporation into proteins, and can be completely reverted by lysine. However, substitution up to 5% of protein lysine by selenalysine does not affect the viability, thus indicating that some degree of substitution can be well tolerated by the cell.