Deoxycytidylate deaminase activity in non-stimulated and phytohemagglutinin-stimulated human lymphocytes,and in leukemic cells

Deoxycytidylate deaminase isolated from normal human lymphocytes and from mononuclear leucocytes from patients with acute lymphoblastic leukemia, chronic lymphocytic leukemia and acute monocytic leukemia has been characterized in regard to the substrate, dAMP and the allosteric regulators dCTP and dTTP. The enzymes exhibited sigmoidal initial velocity versus dCMP concentration whereas in the presence of the activator, dCTP, Michaelis-Menten kinetics were obtained.At saturating substrate concentrations dTTP acted as an allosteric inhibitor of the enzyme isolated from non-stimulated as well as from stimulated lymphocytes. However, the enzymes isolated from the leukemic cells had lost the allosteric regulation by dTTP.At low substrate concentrations the competitive inhibitor, dAMP, activated all the enzymes. This activation was abolished in the presence of dCTP which indicates that dAMP might be involved in the regulation of dCMP deaminase activity and thus influence the dCTP and dTTP pools under physiological conditions.Abbreviations dCMP deaminase deoxycytidylate deaminase - PHA Phytohemagglutinin - ALL acute lymphoblastic leukemia - CLL chronic lymphocytic leukemia - AMOL acute monocytic leukemia - WBC white blood cells     

Cyclic AMP-Generating Systems in Cell-Free Preparations from Guinea Pig Cerebral Cortex: Loss of Adenosine and Amine Responsiveness Due to Low Levels of Endogenous Adenosine

The accumulations of radioactive cyclic AMP elicited by adenosine, norepinephrine, and histamine in adenine-labeled vesicular entities of a particulate fraction from guinea pig cerebral cortex are greatly reduced as a result of prolonged preincubation. The presence of adenosine deaminase during preincubations largely prevents the loss of adenosine, norepinephrine and histamine responses. Adenosine deaminase was inactivated by deoxycoformycin prior to stimulation of cyclic AMP accumulation by adenosine or amines. If adenosine deaminase is not inactivated, responses to norepinephrine are not significant and histamine responses are reduced by 50%. Adenosine deaminase cannot restore responsiveness of the cyclic AMP-generating systems. It is proposed that, in particulate fractions of guinea pig cerebral cortex, low levels of adenosine cause a slow loss of receptors and/or coupling of receptors to cyclic AMP-generating systems.     

Assay of activity of a soil deaminase

Summary A simple method of assaying activity of a soil deaminase is described. The substrate used in the assay, 1,2-diamino-4-nitrobenzene, is red in colour, intensity decreasing with deamination of the benzene ring. This colour changes was used as the basis for a new assay in which substrate was incubated in a buffered soil slurry for 20 h, after which, the amount of substrate deaminated was determined. The method is simple and precise and, unlike existing soil deaminase assays, does not rely on measurement of the NH ₄ ⁺ product. After demonstration of enzyme kinetics, the assay was used to assess differences in deaminase activities in soils of differing crop-cover and N-status.     

Induction of porphobilinogen oxygenase and porphobilinogen deaminase in rat blood under conditions of erythropoietic stress

Porphobilinogen is the substrate of two enzymes: porphobilinogen deaminase and porphobilinogen-oxygenase. The first one transforms it into the metabolic precursors of heme and the second diverts it from this metabolic pathway by oxidizing porphobilinogen to 5-oxopyrrolinones. Rat blood is devoid of porphobilinogen-oxygenase under normal conditions while it carries porphobilinogen-deaminase activity. When the rats were submitted to hypoxia (p_O2 = 0.42 atm) for 18 days, the activity of porphobilinogen-oxygenase appeared at the tenth day of hypoxia and reached the maximum at the 14–16th day. It decreased to a half after 2 days (half-life of the enzyme) and disappeared after 4 days of return to normal oxygen pressure. Porphobilinogen-deaminase activity increased after the first day of hypoxia, reached a maximum at the 14–16th day and did not decrease to normal values until the 15th day after return to normal oxygen pressure. The activities of both prophobilinogen-oxygenase and porphobilinogen-deaminase were induced by administration of erythropoietin. When rats were made anaemic with phenylhydrazine, porphobilinogen-oxygenase activity also appeared in the blood cells. Although the reticulocyte concentration was higher when compared to that obtained under hypoxia, the activities of the oxygenase obtained under both conditions were comparable. Porphobilinogen-deaminase activity was always closely related to the reticulocyte content. The appearance of porphobilinogen-oxygenase under the described erythropoietic conditions was due to a de novo induction of the enzyme, as shown by its inhibition with actinomycin D and cycloheximide. Porphobilinogen-oxygenase as well as porphobilinogen-deaminase were present in the rat bone marrow under normal conditions. Their activities increased in phenylhydrazine treated rats. The properties and kinetics of porphobilinogen-oxygenase from the rat blood and bone marrow were determined and found to differ in several aspects.     

Binding Thermodynamics of the Transition State Analogue Coformycin and of the Ground State Analogue 1-Deazaadenosine to Bovine Adenosine Deaminase

Binding of the transition state analogue coformycin and the ground state analogue 1-deaazadenosine to bovine adenosine deaminase have been thermody-namically characterized. The heat capacity changes for coformycin and 1-deazaadenosine binding are - 4.7 × 0.8 kJ/mole-K and -1.2 × 0.1 kJ/mole-K, respectively. Since the predominant source of heat capacity change in enzyme interactions are changes in the extent of exposure of nonpolar amino acid side chains to the aqueous environment and the hydrophobic effect is the predominant factor in native structure stabilization, we propose that the binding of either class of ligand is associated with a stabilizing enzyme conformational change with coformycin producing the far greater effect Analysis of the T dependence of the second order rate constant for formation of the enzyme/coformycin complex further reveals that the conformational change is not rate limiting. We propose that the enzyme may facilitate catalysis via the formation of a stabilizing conformation at the reaction transition state.     

Psychrotolerant bacteria isolated from the leaf apoplast of cold-adapted wild plants improve the cold resistance of bean (Phaseolus vulgaris L.) under low temperature

We have isolated psychrotolerant bacteria from the leaf apoplast of cold-adapted wild plants and aimed to investigate their effect on the cold resistance of bean (Phaseolus vulgaris L.). Based on the findings of 16S rRNA gene sequence analysis, 20 isolates belonging to 5 bacteria species (Pseudomonas fragi, P. chloropaphis, P. fluorescens, P. proteolytica and Brevibacterium frigoritolerans) were identified in the leaf apoplastic fluid of Draba nemorosa, Galanthus gracilis, Colchicum speciousum, Scilla siberica, Erodium cicutarium, respectively. We have determined that 6 of the 20 isolates have exhibited ACC (1-aminocyclopropane-1-carboxylate) deaminase activity and secreted different extracellular proteins under cold condition (+4 °C) compared to normal growth condition (28 °C). The six isolates were then inoculated independently of each other to the leaves of 10-day-old bean seedlings growing under normal conditions (25/22 °C, 16/8 h photoperiod), and the inoculated and uninoculated (control) seedlings were transferred to cold (9/5 °C, 16/8 h photoperiod) for 3 days. The bacterial inoculations have decreased freezing injury, ice nucleating activity and lipid peroxidation content in parallel with the decrease of reactive oxygen species level such as O₂^.- and H₂O₂ in the inoculated seedlings compared to the control. In addition, the inoculations of the isolates have stimulated the activity of apoplastic antioxidant enzymes including superoxide dismutase, catalase, peroxidase, and glutathione reductase. The results show that the inoculations improve the cold resistance of bean seedlings and the psychrotolerant bacterial isolates can be evaluated within the group of plant growth promoting bacteria (PGPB) which can increase tolerance of cold-sensitive crops.     

2′,3′-Isopropylidene Group,A Molecular Scaffold to Study the Activity of Adenosine and Adenylate Deaminase on Adenosine Analogues Modified in the Ribose Moiety

2 ′,3 ′-Isopropylidene group can be used as a molecular scaffold for the introduction of modifications at 5 ′ and 1 ′ positions of adenosine and these modified nucleosides are used to evaluate the biocatalytic activity of adenosine and adenylate deaminase.     

Commercial-Scale Synthesis of Protected 2′-Deoxycytidine and Cytidine Nucleosides

Abstract

Transformation of 2′-deoxyuridine and uridine analogs to protected 2′-deoxycytidine and cytidine analogs has been investigated by two different methods. First, traditional triazolation protocol and second p-nitrophenoxylation method. Our studies conclude that the triazolation method is better and suitable for commercial scale--up.     

Rhizobacterial mediation of plant hormone status

Plant growth-promoting rhizobacteria are commonly found in the rhizosphere (adjacent to the root surface) and may promote plant growth via several diverse mechanisms, including the production or degradation of the major groups of plant hormones that regulate plant growth and development. Although rhizobacterial production of plant hormones seems relatively widespread (as judged from physico-chemical measurements of hormones in bacterial culture media), evidence continues to accumulate, particularly from seedlings grown under gnotobiotic conditions, that rhizobacteria can modify plant hormone status. Since many rhizobacteria can impact on more than one hormone group, bacterial mutants in hormone production/degradation and plant mutants in hormone sensitivity have been useful to establish the importance of particular signalling pathways. Although plant roots exude many potential substrates for rhizobacterial growth, including plant hormones or their precursors, limited progress has been made in determining whether root hormone efflux can select for particular rhizobacterial traits. Rhizobacterial mediation of plant hormone status not only has local effects on root elongation and architecture, thus mediating water and nutrient capture, but can also affect plant root-to-shoot hormonal signalling that regulates leaf growth and gas exchange. Renewed emphasis on providing sufficient food for a growing world population, while minimising environmental impacts of agriculture because of overuse of fertilisers and irrigation water, will stimulate the commercialisation of rhizobacterial inoculants (including those that alter plant hormone status) to sustain crop growth and yield. Combining rhizobacterial traits (or species) that impact on plant hormone status thereby modifying root architecture (to capture existing soil resources) with traits that make additional resources available (e.g. nitrogen fixation, phosphate solubilisation) may enhance the sustainability of agriculture.