ABSTRACT
Calf thymus thymidylate synthase was purified to homogeneity using a pteroyltetraglutamyllysine Sepharose affinity column. Inhibition of the purified enzyme by folate or methotrexate was enhanced by addition of gamma-Glu residues. Tetrahydrofolate, tetrahydropteroyltriglutamate and tetrahydropteroylheptaglutamate, all having the natural configuration at C-6, all showed similar Km values near 15 microM. Thus, although calf thymus thymidylate synthase showed a higher affinity for pteroylpolyglutamates than pteroylmonoglutamate, this was not reflected in lowered Km values with the corresponding tetrahydropteroylpolyglutamate substrates.
Subject(s)
Folic Acid/analogs & derivatives , Methyltransferases/metabolism , Pteroylpolyglutamic Acids/pharmacology , Thymidylate Synthase/metabolism , Thymus Gland/enzymology , Animals , Binding Sites , Cattle , Chromatography, Affinity , Kinetics , Protein Binding , Pteroylpolyglutamic Acids/metabolism , Structure-Activity RelationshipABSTRACT
The Clarke-Carbon clone bank of hybrid plasmid Escherichia coli DNA has been screened for plasmids able to complement an E. coli strain deficient for the production of beta-cystathionase. Clone 4-14 had the ability to complement a deletion mutation at this locus and expressed higher levels of beta-cystathionase than the wild-type strain. The transfer of the plasmid carried by this clone to a strain that constitutively expresses all the enzymes of the methionine biosynthetic pathway results in 100-fold overproduction of beta-cystathionase as compared to wild-type levels. With use of this strain, an efficient three-step purification scheme is described that gives 90% pure enzyme in 54% yield with a specific activity of 215 IU/mg. This enzyme is characterized as to molecular weight (280 000), number of subunits (six), pyridoxal phosphate binding (5.7 mol of pyridoxal phosphate bound/mol of protein, Km of 0.005 mM), amino acid composition, substrate specificity, and kinetic properties.
Subject(s)
Cloning, Molecular , Escherichia coli/enzymology , Lyases/genetics , Amino Acids/analysis , Cystathionine/genetics , Homocysteine/genetics , Kinetics , Molecular Weight , Plasmids , Pyridoxal Phosphate/pharmacologyABSTRACT
Germinating Cicer arietinum seeds were used to simulate embryonic tissue for evaluating toxicity of purine and pyrimidine analogues. 6-Mercaptopurine stimulated germination, whereas 8-azaadenine, 8-azaguanine, 2,6-diaminopurine, 6-amino, 2-hydroxypurine and 5-fluorouracil inhibited germination. 6-Methyluracil, 5-aminouracil and methylxanthines, viz. caffeine, theophylline, theobromine and uric acid exhibited little inhibitory effects. RNA synthesis ensued immediately after water imbibition and reached maximum at 12 h of germination. 2,6-Diaminopurine, 8-azaadenine, 8-azaguanine and 5-fluorouracil inhibited RNA synthesis. Cyclic-AMP, adenosine, indole-3-acetic acid (IAA) and folic acid partially reversed the inhibitory effects produced by purine pyrimidine analogues. Several hitherto untested pyrimidine analogues inhibited germination as well as RNA synthesis. Inhibition of Escherichia coli dihydrofolate reductase was used for purposes of comparison.