Ascensión Fernández

Specific ADP-ribose pyrophosphatase from Artemia cysts and rat liver: effects of nitroprusside, fluoride and ionic strength

One specific ADP-ribose pyrophosphatase (ADPRibase) has been identified in Artemia cysts, following a protocol that in rat liver allows the identification of three ADPRibases. Artemia ADPRibase resulted similar, but not identical, to rat liver ADPRibase-I with respect to known and novel properties disclosed in this work. In the presence of Mg2+, Artemia ADPRibase was highly specific for ADP-ribose and showed a low, 0.7 microM Km. Preincubation with the nitric oxide donor nitroprusside and dithiothreitol, elicited dose- and time-dependent, severalfold increase of Km and decrease of Vmax. At saturating ADP-ribose concentrations, fluoride was a strong inhibitor (IC50 approximately equal to 10-20 microM), whereas bringing ionic strength to 0.3-1.3 mol/l doubled the activity measured at lower or higher strengths. The novel fluoride and ionic strength effects were studied also with rat liver ADPRibase-I. Differences between the Artemia enzyme and ADPRibase-I concerned molecular weight (31,000 versus 38,500, respectively), Mn2+ ability to substitute for Mg2+ as the activating cation (better for the rat enzyme), and Vmax decrease by nitroprusside (not seen with the rat enzyme). The results are discussed in relation with the role of specific ADPRibases as protective factors limiting free ADP-ribose accumulation and protein glycation, and as targets for cytotoxic agents.

Diadenosine tetraphosphate activates AMP deaminase from rat muscle

Diadenosine tetraphosphate, Ap4A, doubled the activity of AMP deaminase from rat muscle, with an activation constant of 0.005 mM, in the presence of 0.05 mM AMP. The presence of Ap4A appeared to induce Michaelian kinetic behavior. The activation by Ap4A was not dependent on the presence of either MgCl2 or KCl in the reaction mixture. Diguanosine tetraphosphate was inhibitor of the enzyme. Diadenosine and diguanosine triphosphates, adenylosuccinate and xanthosine monophosphate were neither inhibitors nor activators of the reaction.

Glycine-enhanced inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase-I by EDTA: a full account of the reported inhibition by commercial preparations of acidic fibroblast growth factor (FGF-1).

The earlier reported inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase I (EC 3.1.6.9/EC 3.1.4.1; NPP/PDE) by culture‐grade acidic fibroblast growth factor (FGF‐1) correlates with a low‐M r contaminant. 1H‐NMR analyses revealed EDTA in the total‐volume fractions of a gel‐filtration experiment, where all the inhibitory activity of the FGF‐1 preparation was recovered. NPP/PDE inhibition by EDTA (and by unfractionated FGF‐1 or the EDTA‐containing fractions) was time‐dependent, blocked by the substrate p‐nitrophenyl‐dTMP, and strongly enhanced by glycine. The use of glycine buffers in earlier work was critical to the apparent inhibition by FGF‐1. The results point to a conformational change favored by glycine that may be relevant to the biological role of NPP/PDE.

Hydrolysis of the phosphoanhydride linkage of cyclic ADP-ribose by the Mn2+-dependent ADP-ribose/CDP-alcohol pyrophosphatase

Cyclic ADP‐ribose (cADPR) metabolism in mammals is catalyzed by NAD glycohydrolases (NADases) that, besides forming ADP‐ribose, form and hydrolyze the N 1‐glycosidic linkage of cADPR. Thus far, no cADPR phosphohydrolase was known. We tested rat ADP‐ribose/CDP‐alcohol pyrophosphatase (ADPRibase‐Mn) and found that cADPR is an ADPRibase‐Mn ligand and substrate. ADPRibase‐Mn activity on cADPR was 65‐fold less efficient than on ADP‐ribose, the best substrate. This is similar to the ADP‐ribose/cADPR formation ratio by NADases. The product of cADPR phosphohydrolysis by ADPRibase‐Mn was N 1‐(5‐phosphoribosyl)‐AMP, suggesting a novel route for cADPR turnover.

IMP dehydrogenase from Artemia embryos: Molecular forms, purification and properties

Abstract 1. 1. IMP dehydrogenase (EC 1.2.1.14) has been purified near homogeneity from Artemia embryos. 2. 2. The K m values for IMP and NAD + were 15 and 200 μM, respectively. 3. 3. GMP, XMP, GTP, guanosine 5′-tetraphosphate and diguanosine tetraphosphate (Gp 4 G) were competitive inhibitors of the reaction towards IMP with K i values of 140, 180, 175, 120 and 87 μM, respectively. 4. 4. The enzyme from the 27,000 g supernatant can occur in a number of oligomeric forms (450, 375, 260 or 220 kDa) depending on the ionic strength of the medium. 5. 5. Upon precipitation with ammonium sulphate (0.3–0.4 saturation) the enzyme aggregates forming complexes of more than 1000 kDa.

Characterization of Danio rerio Mn2+-Dependent ADP-Ribose/CDP-Alcohol Diphosphatase, the Structural Prototype of the ADPRibase-Mn-Like Protein Family

The ADPRibase-Mn-like protein family, that belongs to the metallo-dependent phosphatase superfamily, has different functional and structural prototypes. The functional one is the Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase from Rattus norvegicus, which is essentially inactive with Mg2+ and active with low micromolar Mn2+ in the hydrolysis of the phosphoanhydride linkages of ADP-ribose, CDP-alcohols and cyclic ADP-ribose (cADPR) in order of decreasing efficiency. The structural prototype of the family is a Danio rerio protein with a known crystallographic structure but functionally uncharacterized. To estimate the structure-function correlation with the same protein, the activities of zebrafish ADPRibase-Mn were studied. Differences between zebrafish and rat enzymes are highlighted. The former showed a complex activity dependence on Mn2+, significant (≈25%) Mg2+-dependent activity, but was almost inactive on cADPR (150-fold less efficient than the rat counterpart). The low cADPR hydrolase activity agreed with the zebrafish genome lacking genes coding for proteins with significant homology with cADPR-forming enzymes. Substrate-docking to zebrafish wild-type protein, and characterization of the ADPRibase-Mn H97A mutant pointed to a role of His-97 in catalysis by orientation, and to a bidentate water bridging the dinuclear metal center as the potential nucleophile. Finally, three structural elements that delimit the active site entrance in the zebrafish protein were identified as unique to the ADPRibase-Mn-like family within the metallo-dependent phosphatase superfamily.

Molecular bases of catalysis and ADP-ribose preference of human Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase and conversion by mutagenesis to a preferential cyclic ADP-ribose phosphohydrolase.

Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose K m and unchanged k cat of F37A-ADPRibase-Mn, while the K m values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type.

Nucleotide ester-forming alcoholytic activities of nucleotide pyrophosphatases: implications for practical biotransformation, enzyme mechanisms and biological function

Abstract Nucleotide pyrophosphatases (NPP) hydrolyze phosphoanhydride and phosphodiester derivatives of nucleoside 5′-monophosphates (NMP) yielding NMP as a product. In a water–alcohol mixture, the alcohol (R–OH) competes and substitutes for water as the splitting agent, so a mixture of NMP and NMP-O-alkyl ester (NMP-O-R) is formed. NPPs from snake venom, potato tuber and mammalian tissues have been studied in this regard. Snake and potato NPPs were considered as possible practical biocatalysts to synthesize NMP-O-Rs from various nucleotidic substrates and alcohols. Mammalian NPPs, mainly from human blood and rat liver, were studied considering the possibility that the alcoholytic reactions catalyzed by them could be biologically relevant. Valuable information on the active centers and catalytic mechanisms of NPPs was also obtained.

Gasolines as primary solvents in liquid scintillation counting

Gasolines from several commercial sources have been used as primary solvents in liquid scintillation counting of dry and aqueous samples of either 3H‐ or 14C‐labeled compounds. Dry samples can be counted only by the addition of fluors to the gasolines, and compared to a standard liquid scintillator, efficiencies of around 75% were attained. For the counting of aqueous samples, gasolines must also be supplemented with secondary solvents (i.e., 10% naphthalene, 5% Triton X‐100, or 10% methanol). Simply with Triton X‐100, efficiencies similar to those obtained with a dioxane‐based liquid scintillator were observed in the case of some gasolines. Drawbacks to gasoline are the higher toxicity and the variation of efficiency, probably depending on the presence of color markers. On the positive side is the low price of the gasolines, compared with either toluene or dioxane, and the facility of purchasing.

Adenosine deaminase isozymes in Artemia

Three isozymes of adenosine deaminase, termed II, III and IV, have been detected in Artemia embryos. Their pI values, determined by chromatofocusing, were 4.9, 5.0 and 5.2, respectively. Upon development to larvae, a different isozyme (I) is induced, with a pI value of 4.2 as determined by isoelectric focusing.