Małgorzata Balińska

At least four regulatory genes control sulphur metabolite repression in Aspergillus nidulans

Mutations in four genes: sconA (formerly suA25meth, mapA25), sconB (formerly mapBl), sconC and sconD, the last two identified in this work, relieve a group of sulphur amino acid biosynthetic enzymes from methionine-mediated sulphur metabolite repression. Exogenous methionine has no effect on sulphate assimilation in the mutant strains, whereas in the wild type it causes almost complete elimination of sulphate incorporation. In both mutant and wild-type strains methionine is efficiently taken up and metabolized to S-adenosylmethionine, homocysteine and other compounds. scon mutants also show elevated levels of folate-metabolizing enzymes which results from the large pool of homocysteine found in these strains. The folate enzymes apear to be inducible by homocysteine and repressible by methionine (or Sadenosylmethionine).

Induction of betaine: Homocysteine methyltransferase in some murine cells cultured in vitro

Betaine when present in the culture medium could induce the activity of betaine: homocysteine methyltransferase (EC 2.1.1.5) in mouse L-cells, and leukemic L1210 cells, as well as in mouse embryo fibroblasts grown in vitro. We found this process to be time- and concentration-dependent. A persisting contact of the cells with betaine was indispensible for expressing and maintaining the enzyme activity. The treatment of cells with cycloheximide or actinomycin D abolished the process of induction. Methionine as well as homocysteine, when present either in the culture medium or in the reaction mixture, strongly depressed the activity of this enzyme. The L-cells with the induced betaine:homocysteine methyltransferase survived but did not multiply in the methionine-deficient medium, therefore, they did not become prototrophs with respect to methionine.

Synthesis and Biological Activity of 5-Fluoro-2-thiocytosine Nucleosides

Abstract Two pathways are described for the synthesis of the 2′-deoxynucleosides of 2-thiocytosine and 5-fluoro-2-thiocytosine: (a) by nucleoside condensation, (b) by amination of the corresponding nucleosides of 2,4-dithiouracil. Biological activities vs two cell systems are described. The nucleosides are moderate to weak substrates of deoxycytidine kinase and, partly as a result of this, reasonable good inhibitors of the enzyme

Betaine-homocysteine methyltransferase in the fungus Aspergillusnidulans

Abstract A betaine:homocysteine methyltransferase activity was demonstrated in the cell-free extracts from the fungus Aspergillus nidulans . Among methionine-requiring mutants which do not grow on homocysteine one class responds to betaine indicating that this compound can serve as a methyl donor in methionine synthesis in vivo . Mutants of the second class which grow only on methionine were shown to have betaine: homocysteine — and methyltetrahydrofolate: homocysteine methyltransferases simultaneously impaired.

Biosynthesis of methionine in mouse cells cultured in vitro.

Abstract In the mouse cell-lines cultured in vitro, viz. L-cells and mouse embryo fibroblasts, the methylation of homocysteine to methionine is carried out by vitamin B12-dependent 5-methyltetrahydrofolate:L-homocysteine methyltransferase only. In these cells grown in the standard Eagle medium, the activity of another methyltransferase, which utilizes betaine as the methyl donor, was not detected. The high activity of the vitamin B12-dependent methionine synthetase is typical for mouse cells from the logarithmic phase of growth. In L-cells 60%, and in the mouse fibroblasts 30% of the enzyme exist in the holo-form; the ratio between the holo- and apoenzyme activity remains stable in cells from logarithmic and stationary cultures. The level of the activity of methionine synthetase strongly depends on the presence of vitamin B12, folate and methionine in the culture medium and is greater after prolonged contact of the cells with these agents.

Regulation of folate-dependent enzyme levels in Aspergillus nidulans: studies with regulatory mutants.

The synthesis of folate-dependent enzymes in Aspergillus nidulans appears to be regulated by intracellular pools of homocysteine and methionine. The results are consistent with the view that homocysteine acts as an inducer and methionine as a corepressor, but the molecular mechanism of the regulation is still unknown. Methionine-requiring mutants, metH2 and metD10, apparently allelic, show deregulation of folate-dependent enzymes. Most characteristic of the mutants is a repressed level of folylpolyglutamate synthetase. New mutations suppressing the metH2 lesion which render folate enzymes insensitive to methionine-mediated repression have been isolated. These mutations are likely to identify new regulatory genes in folate metabolism.

Synthesis and antitumour properties of 2-thio-5-chloro-nucleosides

Abstract Four methods are described for the synthesis of 2-thio-5-chlorouracil (1). β- and α-5-Chloro-2-thio-2′-deoxyuridines (12 and 13) were obtained by Lewis acid catalysed condensation of TMS derivative of 1 with 2-deoxy-3,5-di-O-p-toluyl-α-D-ribosyl chloride and deblocking of toluylated derivatives with methanolic ammonia. Selective enzymatic phosphorylation of 12 led to its 5′-monophosphate, the latter being a moderate inhibitor of thymidylate synthase, while 12 showed moderate cytotoxicity in vitro against mouse leukemic cells L15178Y.

Quinazoline Antifolate Thymidylate Synthase Inhibitors: Replacement of Glutamic Acid by Aminophosphonic Acids

The synthesis of six analogues of the potent thymidylate synthase (TS) inhibitor N -[4-[ N -[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinoyl)-methyl]- N -prop-2-ynylamino]benzoyl]- L -glutamic acid 2 is described in which the glutamic acid residue has been replaced by DL -aminophosphonic acids. New antifolates were tested as inhibitors of TS isolated from mouse L1210 leukemic cells as well as inhibitors of growth mouse leukemic L5178Y cells. In general these modifications result in compounds that are considerably less potent than 2 as TS inhibitors with K i s 0.17-1.10 w M. Very poor solubility in water limited their proper assay of growth cells inhibition.

Regulation of Folate Metabolizing Enzymes In Fungus Aspergillus nidulans

Summary The regulation of methionine synthase. serine hydroxymethyltransferase. methylenetetrahydrofolate oxidorelluctase and dihydrofolate reductase in Aspergillus nidulans have been found to be controlled by endogenous pools methionine and homocysteine. Mutants impaired in sulfur metaholite repression. which have highly elevated pools of these amino acids. also show elevated levels of folate metabolizing enzymes mentioned above. This effect was found to he the result of the accumulation of endogenous homocysteine. which induces folate enzymes. High concentration (up to 5 mM) or methionine in growth medium leads to repression of these enzymes. It appears. therefore. that the levels of folate metaholizing enzymes arc determined by the ratio of cellular levels or methionine and homocysteine.

Target and non-target metabolic effects of aminofolates and of a 5-methylquinazoline antifolate in mouse cells

Abstract Target and non-target response of mouse L-cells to aminofolates, aminopterin and amethopterin, as well as to a 5-methylquinazoline antifolate CB 3703 were compared. When added into the culture medium for 48 hr, each of those antifolates strongly inhibited the cell proliferation; the respective 10.5 values were of the order of 10−8M for aminopterin and of the order of 10−7 M for amethopterin and the quinazoline antifolate. However, even at concentrations which did not limit cell growth, each of the antifolates accumulated intracellularly far above the level of dihydrofolate reductase, and this accumulation was concentration dependent. Simultaneously, the activity of the target enzyme, dihydrofolate reductase, decreased considerably with a concomitant decrease in the activity of lactate dehydrogenase and in lactate content, whilst the content of pyruvate increased in the cells. Consequently, the ratio of lactate to pyruvate decreased. At growth-limiting concentrations, the antifolates did not only reduce the activity of dihydrofolate reductase nearly to zero, but also decreased to a smaller extent the activities of other folate-related enzymes such as methionine synthetase and serine hydroxymethyltransferase, and changed those of thymidylate synthetase and formyltetrahydrofolate synthetase to a greater or lesser degree and in a differentiated manner. Thus, the quinazoline antifolate substantially diminished the activity of thymidylate synthetase, whose activity was apparently enhanced by both aminofolates as compared with the respective controls. On the other hand, the activity of formyltetrahydrofolate synthetase was elevated by the quinazoline antifolate, being greatly decreased by aminopterin and only slightly lowered by amethopterin. Each of the antifolates also caused a significant decrease in the activity of glucose-6-phosphate dehydrogenase and a sharp decrease in that of lactate dehydrogenase. A simultaneous decrease in lactate content and an increase in pyruvate content led to a significant decrease in the ratio of lactate to pyruvate, indicating an increase in intracellular NAD+ content and glycolysis impairment in the L-cells. In conclusion, we suggest considering the ratio of lactate to pyruvate as a kind of simple measure of the non-target effects of antifolates in mammalian cells.