RESUMO
Nucleoside diphosphate kinase (NDK; EC 2.7.4.6) is an enzyme that catalyzes the third phosphorylation of nucleoside diphosphates, leading to nucleoside triphosphates for DNA replication. Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al. (2012), J. Bioenerg. Biomembr. 44, 325-331]. In order to investigate the differences in selectivity, LvNDK was crystallized as binary complexes with both acceptor (dADP and dCDP) and donor (ADP) phosphate-group nucleoside diphosphate substrates and their structures were determined. The three structures with purine or pyrimidine nucleotide ligands are all hexameric. Also, the binding of deoxy or ribonucleotides is similar, as in the former a water molecule replaces the hydrogen bond made by Lys11 to the 2'-hydroxyl group of the ribose moiety. This allows Lys11 to maintain a catalytically favourable conformation independently of the kind of sugar found in the nucleotide. Because of this, shrimp NDK may phosphorylate nucleotide analogues to inhibit the viral infections that attack this organism.
Assuntos
Crustáceos/enzimologia , Núcleosídeo-Difosfato Quinase/metabolismo , Nucleosídeos de Purina/metabolismo , Nucleosídeos de Pirimidina/metabolismo , Animais , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Núcleosídeo-Difosfato Quinase/química , Conformação ProteicaRESUMO
Here we described an nucleoside triphosphate diphosphohydrolase (NTPDase) activity in living trophozoites of Trichomonas gallinae. The enzyme hydrolyzes a variety of purine and pyrimidine nucleoside di- and triphosphates in an optimum pH range of 6.0-8.0. This enzyme activity was activated by high concentrations of divalent cations, such as calcium and magnesium. Contaminant activities were ruled out because the enzyme was not inhibited by classical inhibitors of ATPases (ouabain, 5.0 mM sodium azide, oligomycin) and alkaline phosphatases (levamisole). A significant inhibition of ATP hydrolysis (38%) was observed in the presence of 20 mM sodium azide. Sodium orthovanadate inhibited ATP and ADP hydrolysis (24% and 78%), respectively. The apparent K(M) (Michaelis constant) values were 667.62+/-13 microM for ATP and 125+/-5.3 microM for ADP. V(max) (maximum velocity) values were 0.44+/-0.007 nmol Pi min(-1) per 10(6) trichomonads and 0.91+/-0.12 nmol Pi min(-1) per 10(6) trichomonads for ATP and ADP, respectively. Moreover, we showed a marked decrease in ATP, ADP and AMP hydrolysis when the parasites were grown in the presence of penicillin and streptomycin. The existence of an NTPDase activity in T. gallinae may be involved in pathogenicity, protecting the parasite from the cytolytic effects of the extracellular nucleotides.
Assuntos
Inibidores Enzimáticos/farmacologia , Penicilinas/farmacologia , Pirofosfatases/metabolismo , Estreptomicina/farmacologia , Trichomonas/efeitos dos fármacos , Trichomonas/enzimologia , Animais , Cálcio/farmacologia , Ativadores de Enzimas/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Magnésio/farmacologia , Nucleosídeos de Purina/metabolismo , Nucleosídeos de Pirimidina/metabolismo , Azida Sódica/farmacologia , Especificidade por Substrato , Trofozoítos/efeitos dos fármacos , Trofozoítos/enzimologiaRESUMO
This study was conducted to determine the mode(s) of uptake of selected pyrimidine bases and nucleosides by axenically grown Entamoeba histolytica. Two-minute uptake studies with 3H-labeled compounds showed the following: 1) uridine and cytidine are taken up, in part, by a carrier mediated system; 2) uracil, cytosine, thymine and thymidine enter amebae via passive diffusion; both cytidine and uridine are taken up by passive diffusion when present at exogenous concentration greater than 500 muM; 3) specific carrier sites are used for transport of uridine-cytidine and uridine-adenosine; 4) cytidine and uridine uptake is markedly inhibited by iodoacetate and N-ethylmalemide; 5) hypoxanthine (10mM) stimulates uptake of uridine; 6) ribose fails to inhibit uptake of cytidine and uridine; 7) cytidine uptake exceeds that of uridine (i.e., apparent Vmax for: a) cytidine = 10.0 nmoles/2 min/10(6) amebae; b) uridine = 3.0 nmoles/2 min/10(6) amebae); 8) Kt for: cytidine = 0.30 mM; uridine = 0.21 mM; 9) Q 10's at 35.5 C vs 25.5 C for: a) cytidine = 3.9; b) uridine = 2.6.