Search

The AtoS-AtoC two-component signal transduction system positively regulates the expression of the atoDAEB operon in Escherichia coli. Upon acetoacetate induction, AtoS sensor kinase autophosphorylates and subsequently phosphorylates, thereby activating, the response regulator AtoC. In a previous work we have shown that AtoC is phosphorylated at both aspartate 55 and histidine73. In this study, based on known three-dimensional structures of other two component regulatory systems, we modeled the 3D-structure of the receiver domain of AtoC in complex with the putative dimerization/autophosphorylation domain of the AtoS sensor kinase. The produced structural model indicated that aspartate 55, but not histidine 73, of AtoC is in close proximity to the conserved, putative phosphate-donor, histidine (H398) of AtoS suggesting that aspartate 55 may be directly involved in the AtoS-AtoC phosphate transfer. Subsequent biochemical studies with purified recombinant proteins showed that AtoC mutants with alterations of aspartate 55, but not histidine 73, were unable to participate in the AtoS-AtoC phosphate transfer in support of the modeling prediction. In addition, these AtoC mutants displayed reduced DNA-dependent ATPase activity, although their ability to bind their target DNA sequences in a sequence-specific manner was found to be unaltered.

A protein kinase which phosphorylates in vitro the biosynthetic ornithine decarboxylase (ODC) was partially purified from Escherichia coli. In vivo phosphorylation of ODC occurs after incubation of E. coli with [32P]orthophosphate. When the recombinant ODC was incubated with calf intestine alkaline phosphatase it was inactivated and this inactive ODC could be reversibly activated allosterically only by guanyl or uracyl phosphate analogues at a concentration of 10(-4) or 10(-3) M. The pH optimum of the [8-3H]GTP binding was determined and it was shown that the GTP binding is proportional to the amount of ODC. The [8-3H]GTP binds specifically to ODC as was proved by the addition of cold GTP or ATP. High concentration of GTP can dissociate the ODC-antizyme complex and either reactivate or liberate the ODC. Therefore, a working hypothesis is suggested describing the regulation of ODC by phosphorylation-dephosphorylation reaction or by antizyme and nucleotide analogues interaction.

Agents such as dimethylsulfoxide, N,N'-dimethylformamide and bisacetyldiaminopentane that induce erythroid differentiation of Friend leukemia cells, cause a rapid increase in ornithine decarboxylase (EC 4.1.1.17) activity in intact cells during the 'latent' period preceding the accumulation of hemoglobin-containing cells. Blockage of erythroid differentiation with 5-bromo-2'-deoxyuridine did not prevent these alterations in enzyme activity. Addition of each chemical inducer in the extracts of these cells stimulate the basal levels of ornithine decarboxylase activity. These data indicate that the chemical inducers of differentiation modify the normal pattern of ornithine decarboxylase activity in this system.

The purification of the antizymes to ornithine decarboxylase of Escherichia coli to homogeneity is detailed. An acidic component, pI 3.8, and two basic histone-like proteins, pI above 9.5, are described. The two latter proteins constitute approximately 90% of the total antizyme activity.

A protein kinase and an acidic phosphoprotein phosphatase were purified from Tetrahymena pyriformis which phosphorylate and dephosphorylate the purified ornithine decarboxylase (ODC) of this microorganism. The protein kinase and the phosphoprotein phosphatase are copurified with ODC and can be separated in three distinct peaks only by a hydrophobic column of phenyl-Sepharose. The purified kinase is not dependent on cAMP, requires Mg2+ for its catalytic activity and has a molecule mass of 45 kDa. Incubation of [32P]ODC with the purified phosphoprotein phosphatase results in a complete loss of 32P and its catalytic activity. Phosphorylation of the inactive phosphatase-treated ODC by endogenous kinase or rat liver casein kinase-2 results in 100 or 40% reactivation of the initial untreated ODC activity, respectively.

The addition of fresh serum-containing growth medium to L1210 mouse leukemic cells in culture resulted in a 5-fold increase in ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity. The presence of microtubule disrupting agents (colchicine, vinblastine) or cations (5-10 mM K+, Na+ or Mg2+) abolishes this increase of ornithine decarboxylase activity. (Chen, K.Y., Heller, J.S. and Canellakis, E.S. (1976) Biochem. Biophys. Res. Commun. 70, 212-219). Based on these observations we proposed that fluctuation in cellular cation concentrations may act as a link between the membrane structure and ornithine decarboxylase. To test this proposal, we studied the effects of selective membrane perturbing agents such as ionophores and local anesthetics, on the serum-stimulated increase of ornithine decarboxylase activity in L1210 cells. Among the six ionophores tested, valinomycin was the most potent one, with I50 value (concentration that gives 50% inhibition of ornithine decarboxylase activity) of 6.10(-9) M. Dibucaine and tetracaine were also effective inhibitors at 10(-4)-10(-5) M. The I50 values of valinomycin on the protein synthesis and RNA synthesis, however, were greater than 1.10(-6) M. These results substantiate the notion that ornithine decarboxylase activity can be regulated at plasma membrane level and such regulation is related to the perturbation of cellular cation pools.

In Tetrahymena pyriformis, ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activities are present in the cytosolic and nuclear fractions and reach maximal values in the middle and late log phases of growth, respectively. The two activities have been purified to homogeneity by ammonium sulfate fractionation (20-45%), anion-exchange chromatography (DEAE-Bio-Gel A), gel-filtration (Sephadex G-150 and Sephadex G-100 superfine) and hydrophobic chromatography (Phenyl-Sepharose). Both the crude and the purified enzyme preparations are inactivated irreversibly by alpha-difluoromethylornithine, a suicide inhibitor of mammalian ornithine decarboxylase. The enzyme preparations from the nucleus and cytosol each showed a single band on polyacrylamide gel electrophoresis under native and denaturing conditions and on acrylamide gel electrofocusing. Both activities show the same pH optima (8.6) isoelectric point (5.3), molecular weight (64 000) and Kmorn (4.7 microM). The Km for L-lysine is 0.5 mM. The two activities also cross-react with acidic antizyme extracted from E. coli mutant MA 255. Based on the physicochemical properties, one can safely conclude that cytosolic and nuclear activities reside on the same protein molecule.

In Tetrahymena pyriformis the cytosolic ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity is considerably inhibited by the presence of polyamines in the growth medium, while the nuclear ornithine decarboxylase is only slightly affected. Experimental evidence suggests that the presence of putrescine and/or spermidine elicits the appearance of non-competitive inhibitors of ornithine decarboxylase. One of the inhibitors has a molecular weight of 25,000 and properties of antizyme. In addition, two other low molecular weight inhibitors are extracted, one which is a phosphoserine oligopeptide, and the other which is phosphotyrosine. All inhibit non-competitively the homologous and heterologous (Escherichia coli and rat liver) ornithine decarboxylases. Similarly, non-competitive inhibition was obtained when the commercially available phosphoamino acids were tested against the already mentioned ornithine decarboxylases.