Guanosine Triphosphate

In the yeast Saccharomyces cerevisiae, CTP synthetase [EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)] is the product of the URA7 gene. CTP synthetase was purified 503-fold to apparent homogeneity from cells bearing the URA7 gene on a multicopy plasmid that directed a 10-fold overproduction of the enzyme. The purification procedure included ammonium sulfate fractionation of the cytosolic fraction followed by chromatography with Sephacryl 300 HR, Q-Sepharose, Affi-Gel Blue, and Superose 6. The N-terminal amino acid sequence of purified CTP synthetase was identified and aligned perfectly with the deduced sequence of the URA7 gene. The minimum subunit molecular mass (68 kDa) of purified CTP synthetase was in good agreement with the size (64.7 kDa) of the URA7 gene product. Antibodies were raised against a maltose-binding protein-CTP synthetase fusion protein which immunoprecipitated CTP synthetase from wild-type cells. Immunoblot analysis was used to identify CTP synthetase in wild-type cells and cells bearing the URA7 gene on a multicopy plasmid. The results of gel filtration chromatography indicated that the size of native CTP synthetase was consistent with a dimeric structure for the enzyme. CTP synthetase oligomerized to a tetramer in the presence of its substrates UTP and ATP. Maximum CTP synthetase activity was dependent on magnesium ions (4 mM) and 2-mercaptoethanol at the pH optimum of 8.0. CTP synthetase exhibited positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP. CTP synthetase was potently inhibited by the product CTP which also increased the positive cooperativity of the enzyme toward UTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Astrocytes are involved in multiple brain functions in physiological conditions, participating in neuronal development, synaptic activity and homeostatic control of the extracellular environment. They also actively participate in the processes triggered by brain injuries, aimed at limiting and repairing brain damages. Purines may play a significant role in the pathophysiology of numerous acute and chronic disorders of the central nervous system (CNS). Astrocytes are the main source of cerebral purines. They release either adenine-based purines, e.g. adenosine and adenosine triphosphate, or guanine-based purines, e.g. guanosine and guanosine triphosphate, in physiological conditions and release even more of these purines in pathological conditions. Astrocytes express several receptor subtypes of P1 and P2 types for adenine-based purines. Receptors for guanine-based purines are being characterised. Specific ecto-enzymes such as nucleotidases, adenosine deaminase and, likely, purine nu...

The gene encoding Bacillus subtilis UMP kinase (pyrH/smbA) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins. The specific activity of the purified enzyme under optimal conditions is 25 units·mg−1 of protein. In the absence of GTP, the activity of B. subtilis enzyme is less than 10% of its maximum activity. Only dGTP and 3′-anthraniloyl-2′-deoxyguanosine-5′-triphosphate (Ant-dGTP) can increase catalysis significantly. Binding of Ant-dGTP to B. subtilis UMP kinase increased the quantum yield of the fluorescent analogue by a factor of more than three. UTP and GTP completely displaced Ant-dGTP, whereas GMP and UMP were ineffective. UTP inhibits UMP kinase of B. subtilis with a lower affinity than that shown towards the Escherichia coli enzyme. Among nucleoside monophosphates, 5-fluoro-UMP (5F-UMP) and 6-aza-UMP were actively phosphorylated by B. subtilis UMP kinase, explaining the cytotoxicity of the corresponding nucleosides towards this bacterium. A structural model of UMP kinase, based on the conservation of the fold of carbamate kinase and N-acetylglutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B. subtilis and E. coli enzymes.

Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER)-resident proteins known to be involved in learning and memory. Dendritic spines in hippocampal neurons play important roles in neuroplasticity and learning and memory. This study tested the hypothesis that Sig-1Rs might regulate denritic spine formation in hippocampal neurons and examined potential mechanisms therein. In rat hippocampal primary neurons, the knockdown of Sig-1Rs by siRNAs causes a deficit in the formation of dendritic spines that is unrelated to ER Ca 2+ signaling or apoptosis, but correlates with the mitochondrial permeability transition and cytochrome c release, followed by caspase-3 activation, Tiam1 cleavage, and a reduction in Rac1·GTP. Sig-1R-knockdown neurons contain higher levels of free radicals when compared to control neurons. The activation of superoxide dismutase or the application of the hydroxyl-free radical scavenger N-acetyl cysteine (NAC) to the Sig-1R-knockdown neurons rescues dendritic spi...

2-Hydroxybenzyl dansyl cadaverine (DNSCH) was designed as a fluorescent chemosensor for Fe3+. DNSCH exhibited significant FL quenching with increasing concentrations of Fe3+. The presence of 15 M equiv of Fe3+ significantly quenched the FL intensity of DNSCH. The binding constant of DNSCH towards Fe3+ was determined and found to be 1.5 ± 0.4 × 10−4 M−1. DNSCH was employed as an intracellular chemosensor for Fe3+.The novel of this work is to synthesize 2-hydroxybenzyl dansyl cadaverine (DNSCH) as a selective fluorescent chemosensor for Fe3+. Of all screened metals, DNSCH exhibited FL quenching upon the addition of Fe3+. DNSCH was found its bio-application as an intracellular chemosensor for Fe3+.► A novel chemosensor based on 2-hydroxybenzyl dansyl cadaverine (DNSCH) is prepared and evaluated for its fluorescence response to heavy metal ions. ► DNSCH exhibits an “off-type” mode with high selectivity in the presence of Fe3+ ion. ► The addition of EDTA recovered the fluorescence of DNSCH·Fe3+ complex offers DNSCH as a reversible chemosensor. ► DNSCH is able to use as an intracellular chemosensor for Fe3+ ion.

In epithelial tissues, cells are linked to their neighbors through specialized cell-cell adhesion proteins. E-cadherin is one of the most important membrane proteins for the establishment of intimate cell-cell contacts, but the molecular mechanism by which it is recruited to contact sites is largely unknown. We report here that the cytoplasmic domain of E-cadherin interacts with C3G, a guanine nucleotide exchange factor for Rap1. In epithelial cell cultures, ligation of the extracellular domain of E-cadherin enhances Rap1 activity, which in turn is necessary for the proper targeting of E-cadherin molecules to maturing cell-cell contacts. Furthermore, our data suggest that Cdc42 functions downstream of Rap1 in this process. We conclude that Rap1 plays a vital role in the establishment of E-cadherin-based cell-cell adhesion.

Extracellular guanosine, guanosine triphosphate (GTP), and 5′-N′-ethylcarboxamidoadenosine (NECA), each significantly enhanced the proportion of nerve growth factor (NGF)-treated rat pheochromocytoma (PC12) cells which had neurites, greater than that in cultures exposed to NGF alone. Guanosine and NECA, but not GTP, increased intracellular cAMP concentrations. An adenylate cyclase inhibitor, SQ22536, completely blocked the cAMP increase induced by both guanosine and 0.1 μM NECA. However, SQ22536 only partially blocked guanosine enhanced neurite outgrowth, although it completely blocked the neuritogenic effect of NECA. Therefore guanosine-enhanced neurite outgrowth through both cAMP-dependent and -independent mechanisms, while the effect of GTP was cAMP-independent.