Modulation of human cytidine deaminase by specific aminoacids involved in the intersubunit interactions.

An investigation was made of the role exerted by some residues supposed to be involved in the intersubunit interaction and also in the catalytic site of homotetrameric human cytidine deaminase (T-CDA). Attention was focused on Y33, Y60, R68, and F137 residues that are a part of a conserved region in most T-CDAs. Hence, a series of site-directed mutagenesis experiments was set up obtaining seven mutants: Y60G, Y33G, Y33F Y33S, F137A, R68G, and R68Q. Each active purified mutant protein was characterized kinetically, with a series of substrates and inhibitors, and the effect of temperature on enzyme activity and stability was also investigated. Circular dichroism (CD) experiments at different temperatures and in presence of small amounts of sodium dodecyl sulphate (SDS) were performed in all the soluble mutant CDAs. The results obtained by site-directed mutagenesis studies were compared to the crystallographic data of B. subtilis CDA and E. coli CDA and to molecular modeling studies previously performed on human CDA. The mutation of Y60 to glycine produced an enzyme with a more compact quaternary structure with respect to the wild-type; this mutation did not have a dramatic effect on cytidine deamination, but it slightly affected the binding with the substrate. None of the mutant CDAs in Y33 showed enzymatic activity; they existed only as monomers, indicating that this residue, located at the intersubunit interface, may be responsible for the correct folding of human CDA. The insertion of an alanine instead of phenylalanine at position 137 led to a soluble but completely inactive enzyme unable to form a tetramer, suggesting that F137 residue may be important for the assembling of the tetramer and also for the arrangement of the CDA active site. Finally, R68G and R68Q mutations revealed that the presence of the amino group seems to be important for the catalytic process but not for substrate binding, as already shown in B. subtilis CDA. The quaternary structure of R68Q was not affected by the mutation, as shown by the SDS-induced dissociation experiments and CD studies, whereas R68G dissociated very easily in presence of small amounts of SDS. These experiments indicated that in the human CDA, the side chain of arginine 68 involved in the catalytic process in one subunit active site might come from another subunit. The data obtained from these studies confirmed the presence of a complicated set of intersubunit interactions in the active site of human CDA, as shown in other T-CDAs.

Studies on thermal stability of human cytidine deaminase.

The thermal stability of human cytidine deaminase (CDA), an enzyme involved in pyrimidine metabolism was investigated. With this in view, the residues R68 and Y60, supposed to be involved in the intersubunit interactions and in the catalytic site of CDA, were mutated to glutamine and glycine, respectively. Thermal stability experiments were performed on the purified mutants by means of circular dichroism and enzymatic assays. The results obtained should be useful for designing more efficient cytidine based drugs for chemotherapy.

NAD+ metabolism in lymphocytes of patients affected by B-cell chronic lymphocytic leukaemia.

In the present work we have quantified some enzymatic activities, related to NAD+ metabolism, in lymphocytes of healthy donors and of patients affected by B-cell chronic lymphocytic leukemia (B-CLL): NADase activity for NAD+ degradation and NMN adenylyltransferase (NMNAT) activity for NAD+ biosynthesis. Most of the samples under investigation (12 B-CLL patients and 12 healthy donors) presented the enzymatic activities assayed. No significant differences in terms of specific activity have been found between ill and healthy subjects. Nevertheless by expressing the activity as mU/10(9) cells (nmol min(-1) 10(9) cells) we observed significantly higher values of both enzymatic activities, involved in the NAD+ metabolism, in healthy donors with respect to the B-CLL patients.

Isoenzymatic forms of human cytidine deaminase.

Cytidine deaminase (CDA) purified from human placenta revealed the presence of five isoenzymatic forms that differ only in their isoelectric point. Since human cytidine deaminase exists in two variants (CDA 1 and CDA 2) with a non-conservative amino acid substitution at codon 27, in this work we demonstrate that these two variants may combine together in vitro, giving five CDA isoforms as observed in vivo from human placenta. For this purpose, each of the two forms of CDA was purified close to homogeneity and dissociated into monomers in the presence of a small amount of sodium dodecyl sulfate as a dissociating agent. The monomers were mixed together and subjected to anion-exchange chromatography and to chromatofocusing analysis in order to visualize the formation of the five isoforms. Furthermore, for both CDA 1 and CDA 2 some substrates and inhibitors of CDA were assayed, with the aim of demonstrating different kinetic behavior between the two natural variants.

Apoptosis in sea bream Sparus aurata eggs.

In the present study, the sea-bream Sparus aurata, a pelagic egg spawner, was used as experimental model, in order to establish the occurrence of apoptosis in vertebrates with external reproduction. The same female ovulates floating and nonfloating eggs, but only the former, after fertilization, proceed to embryo development. The eggs were divided into floating and nonfloating and both were analyzed for the presence of several apoptosis markers. The results here reported provide evidence that the nonfloating cells present severe shrinkage and highly express both FAS receptor and FAS ligand on their surface. Furthermore, DNA fragmentation and mitochondria swelling were found, suggesting that the nonfloating eggs were cells programmed to die.

NAD+ catabolism in pheochromocytoma rat cells.

The catabolic pathway of nicotinamide adenin dinucleotide (NAD) in cultured pheochromocytoma rat cells (PC12) was investigated. The first evidence obtained in these studies was that, despite inducing cell differentiation, NGF treatment did not modify NAD catabolism. Following incubation of PC12 homogenate with NAD, ADP-ribose, AMP, IMP, and HYP was produced. The catabolic fate of AMP and ADPR so obtained was followed by monitoring to a final production of inosine and hypoxanthine through several enzymatic steps. When intact PC12 cells were incubated with NAD in the culture medium AMP, IMP and HYP were found but, no ADPR and cADPR were present in the growth medium. "Nucleotides analyses" carried out on the homogenate obtained from these cells, confirmed the absence of cADPR and an increase of intracellular ADPR. These results led us to believe that in PC12 cells the ADP ribosyl cyclase activity is absent and that NADase is an ecto-enzyme able to transfer the ADPR, produced from NAD catabolism, inside the cells.

Cytidine deaminase from two extremophilic bacteria: cloning, expression and comparison of their structural stability.

We cloned, purified and characterized two extremophilic cytidine deaminases: CDA(Bcald) and CDA(Bpsy), isolated from Bacillus caldolyticus (growth at 72 degrees C) and Bacillus psychrophilus (growth at 10 degrees C), respectively. We compared their thermostability also with the mesophilic counterpart, CDA(Bsubt), isolated from Bacillus subtilis (growth at 37 degrees C). The DNA fragments encoding CDA(Bcald) and CDA(Bpsy) were sequenced and the deduced amino acid sequences showed 70% identity. High sequence similarity was also found with the mesophilic CDA(Bsubt). Both enzymes were found to be homotetramers of approximately 58 kDa. CDA(Bcald) was found to be highly thermostable, as expected, up to 65 degrees C, whereas CDA(Bpsy) showed higher specific activity at lower temperatures and was considerably less thermostable than CDA(Bcald). After partial denaturation at 72 degrees C for 30 min, followed by renaturation on ice, CDA(Bcald) recovered 100% of its enzymatic activity, whereas CDA(Bpsy) as well as CDA(Bsubt) were irreversibly inactivated. Circular dichroism (CD) spectra of CDA(Bcald) and CDA(Bpsy) at temperatures ranging from 10 to 95 degrees C showed a markedly different thermostability of their secondary structures: at 10 and 25 degrees C the CD spectra were indistinguishable, suggesting a similar overall structure, but as temperature increases up to 50-70 degrees C, the alpha-helices of CDA(Bpsy) unfolded almost completely, whereas its beta-structure and the aromatic amino acids core remained pretty stable. No significant differences were seen in the secondary structures of CDA(Bcald) with increase in temperature.

NAD(P)(+)-glycohydrolase from human spleen: a multicatalytic enzyme.

NAD(P)(+)-glycohydrolase (NADase, EC 3.2.2.6) was partially purified from microsomal membranes of human spleen after solubilization with Triton X-100. In addition to NAD+ and NADP+, the enzyme catalyzed the hydrolysis of several NAD+ analogues and the pyridine base exchange reaction with conversion of NAD+ into 3-acetylpyridine adenine dinucleotide. The enzyme also catalyzed the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and the hydrolysis of cADPR to adenosine diphosphoribose (ADPR). Therefore, this enzyme is a new member of multicatalytic NADases recently identified from mammals, involved in the regulation of intracellular cADPR concentration. Human spleen NADase showed a subunit molecular mass of 45 kDa, a pI of 4.9 and a Km value for NAD+ of 26 microM. High activation of ADPR cyclase activity was observed in the presence of Ag+ ions, corresponding to NADase inhibition.

Possible role of two phenylalanine residues in the active site of human cytidine deaminase.

Site-directed mutagenesis on human cytidine deaminase (CDA) was employed to mutate specifically two highly conserved phenylalanine residues, F36 and F137, to tryptophan; at the same time, the unique tryptophan residue present in the sequence at position 113 was mutated to phenylalanine. These double mutations were performed in order to have for each protein a single tryptophan signal for fluorescence studies relative to position 36 or 137. The mutant enzymes thus obtained, W113F, F36W/W113F and F137W/W113F, showed by circular dicroism and thermal stability an overall structure not greatly affected by the mutations. The titration of Trp residues by N-bromosuccinimide (NBS) suggested that residue W113 of the wild-type CDA and W36 of mutant F36W/W113F are buried in the tertiary structure of the enzyme, whereas the residue W137 of mutant F137W/W113F is located near the surface of the molecule. Kinetic experiments and equilibrium experiments with FZEB showed that the residue W113 seems not to be part of the active site of the enzyme whereas the Phe/Trp substitution in F36W/W113F and F137W/W113F mutant enzymes had a negative effect on substrate binding and catalysis, suggesting that F137 and F36 of the wild-type CDA are involved in a stabilizing interaction between ligand and enzyme.

Identification of four amino acid residues essential for catalysis in human cytidine deaminase by site-directed mutagenesis and chemical modifications.

By site-directed mutagenesis on human cytidine deaminase (CDA), five mutant proteins were obtained: C65A, C99A, C102A, E67D and E67Q. The three cysteine mutants were completely inactive, whereas E67D and E67Q showed a specific activity about 200- and 200000-fold lower, respectively, than the wild-type CDA. Zinc analysis revealed that only E67D, E67Q and C65A contained 1 mol Zn2+/mol subunit as in the wild-type CDA. Kinetic measurements with the specific carboxylic group reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline performed on wild-type CDA suggest that Glu67 is essential for the catalytic process. Furthermore, when both native and denatured CDA was titrated with 5,5'-dithiobis(2-nitrobenzoic acid) six sulfhydryl groups were detected, whereas in the denatured and reduced enzyme nine such groups were found, according to the sequence data. When p-hydroxymercuriphenyl sulfonate was used, nine sulfhydryl groups were detectable and the release of 1 mol of zinc per mole of CDA subunit was revealed by the metal indicator dye 4-(2-pyridylazo)resorcinol. It seems plausible that the limiting step for the maintenance of zinc in the active site is the formation of coordination between Cys99 and Cys102, whereas Cys65 could lead the zinc to the correct position and orientation within the active site.

Human placenta cytidine deaminase: a zinc metalloprotein.

Cytidine deaminase, a tetrameric enzyme purified from human placenta, was shown to contain a single atom of tightly bound zinc per subunit by Inductively Coupled Plasma Optical Emission Spectrometry analysis. The metal appears to be involved in catalysis, as suggested by the inhibition exerted by 1,10-phenanthroline and dipicolinic acid. This hypothesis is further supported by the finding that the presence of substrate protects the enzymatic activity from dipicolinic acid inhibition. Furthermore the total cysteine residues per subunit were investigated by sulphydryl groups titrating agents.

Human placenta cytidine deaminase: proton-linked enzyme activity and substrate binding.

In this work we focused our attention on some catalytic site requirements for human placenta cytidine deaminase (CDA). The effect of pH on substrate binding and catalysis was studied between pH 3.0 and pH 11.0. The results could be discussed postulating the presence of two classes of ionizable groups in the active site of CDA. The kinetic parameters pH-dependence has been discussed considering the presence of four zinc atoms per each enzyme tetramer. Furthermore fluorescence studies on the enzyme and on enzyme-inhibitor complexes, examined by using polar and non polar quenchers, allowed to define the substrate(inhibitor)-dependent accessibility of the tryptophan-containing pocket on each CDA monomer.

NMN adenylyltransferase from bull testis: purification and properties.

The purification procedure of NMN adenylyltransferase from bull testis presented here consists of a heat step and an acidic precipitation followed by four chromatographic steps, including dye ligand, adsorption and hydrophobic chromatography. The final enzyme preparation subjected to non-denaturing and denaturating PAGE with silver nitrate staining exhibited a single band. At this step the enzyme appeared to be homogeneous. The M(r) value of the native enzyme calculated by gel filtration was about 133,000. The protein appeared to possess a quaternary structure with four subunits of apparent M(r) 33,000 without disulphide interchain bonds. Isoelectric experiments gave a pI of 6.2, and pH studies showed the possible presence of an acidic group in the active site having a pKa of 4.9. Analysis of the amino acid composition showed the presence of more acidic residues than basic ones, according to the pI value calculated by Mono P FPLC. The Ea calculated by Arrhenius plot gave an apparent value of 55.7 kJ/mol. The Km values for NMN, ATP, NAD+ and PPi were 0.11, 0.023, 0.37 and 0.16 nM respectively. The polyclonal antiserum produced against the NMN adenylyltransferase reacted with the purified enzyme at different dilutions and recognized the enzyme in the homogenate as well.

Assay methods for nicotinamide mononucleotide adenylyltransferase of wide applicability.

NMN adenylyltransferase (NMNAT) reversibly catalyzes the synthesis of NAD+ or NaAD+ from ATP and NMN or NaMN. In this work, we describe a continuous coupled spectrophotometric assay that can be rapidly and routinely used in place of the previous cumbersome two-step assay. The reaction rates measured with the coupled assay display a linear dependence with respect to enzyme concentration over the range investigated. The method yields accurate and reliable estimates of the enzyme activity in the direction of NAD+ synthesis. Furthermore, we developed an HPLC-based method suitable for the assay activity both in the forward and reverse directions of the enzymatic reaction. The method appears particularly useful for measuring the NMNAT activity when the product is not NAD+ (e.g., in studies using alternative substrates), and offers the possibility of monitoring simultaneously both the NMNAT-catalyzed reaction and interfering side reactions. This is achieved through the HPLC identification and quantitation of metabolites and derivatives produced in the reaction mixture during the assay. The two methods described here should cover most needs for the assay of NMNAT activity.

The antitumor drug, 1,3-bis(2-chloroethyl)-1-nitroso-urea, inactivates human nicotinamide mononucleotide adenylyltransferase.

Nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) from human placenta is rapidly inactivated by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). A similar inactivation is observed with other C- and N-nitroso compounds. The inactivation by BCNU is dependent on incubation time, temperature and BCNU concentration. Protective reagents for -SH groups, dithiothreitol and beta-mercaptoethanol, and the substrate NMN are very effective in protecting NMN adenylyltransferase from BCNU inactivation and in preserving its catalytic properties, while ATP is less efficient. Incubation of BCNU-inactivated and dialysed NMN adenylyltransferase with dithiothreitol results in a partial recovery of the enzymatic activity.

Nuclear matrix-associated NMN adenylyltransferase activity in human placenta.

This paper presents data about the presence of the NMN adenylyltransferase at the nuclear matrix level of human placenta nuclei. It was found that 40-45% of the activity (depending on the extraction procedure) referred to the total nuclear NMN adenylyltransferase was tightly associated with this subnuclear compartment. The matrices purified by two different procedures exhibited DNA, RNA and protein contents comparable with those described in literature. Extensive digestion of human placenta nuclei with DNase I was not able to solubilize the NMN adenylyltransferase activity. Therefore, the data we present are consistent with the conclusion that a part of the total nuclear NMN adenylyltransferase is associated with the nuclear matrix.

NAD biosynthesis in human placenta: purification and characterization of homogeneous NMN adenylyltransferase.

Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from human placenta. The purification procedure consists of several chromatographic steps, including dye-ligand, adsorption, and hydrophobic interaction chromatography. The final enzyme preparation is homogeneous as judged by a single silver stainable band on both nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 132,000, as determined by gel filtration on a Superose 12 HR 10/30 fast protein liquid chromatography column. The protein possesses a quaternary structure and is composed of four apparently identical M(r) 33,000 subunits. Isoelectrofocusing experiments give multiple pI values ranging from pH 4.7 to 6.6. Optimum pH study shows a plateau extending from pH 6.0 to pH 9.0. Km values for NMN, ATP, NAD+, and PPi are 38, 23, 67, and 125 microM, respectively. Kinetic analysis reveals a behavior consistent with an ordered sequential Bi-Bi mechanism. Among several metabolites tested only ADP-ribose and beta-NMNH were found to significantly inhibit the enzyme activity.

Purification of human cytidine deaminase: molecular and enzymatic characterization and inhibition by synthetic pyrimidine analogs.

Cytidine deaminase has been purified to homogeneity from human placenta by a rapid and efficient procedure consisting of affinity chromatography followed by hydrophobic interaction chromatography. The final enzyme preparation showed a specific activity of 64.1 units/mg, corresponding to about 46,000-fold purification with respect to the crude extract. The enzyme is a 52-kDa oligomeric protein composed of four apparently identical subunits. The acidic isoelectric point is 4.5. The enzyme's stability is strictly dependent on the presence of reducing agents. Amino acid analysis reveals the presence of five thiol groups per monomer which cannot be titrated by Ellman's reagent in the native enzyme. However, the presence of sulfhydryl groups involved in the catalytic activity was evidenced by the inhibition exerted by p-chloromercuribenzoate and heavy metal ions. In addition, the protection effected by the substrate against the p-chloromercuribenzoate inhibition and the competitive inhibition exerted by 5-(chloromercuri)cytidine suggest the presence of a thiol group(s) in the catalytic site of the enzyme. pH studies have shown that the rapid decline of activity occurring at pH 4.5 might result from the protonation of the pyrimidine ring at the N-3 position. The enzyme catalyzes the deamination of cytidine, deoxycytidine, and several analogs, including antineoplastic agents, thus abolishing their pharmacological activity. Therefore, several pyrimidine nucleoside analogs have been tested as potential inhibitors of the enzyme. The competitive inhibition exerted by cytidine analogs having the ribose moiety replaced by aliphatic chains is interesting.

Presence of NAD pyrophosphorylase in skeletal muscle in dystrophic mice.

NAD pyrophosphorylase (ATP:NMN adenylyltransferase) activity has been measured in the skeletal muscle of dystrophic mice. The amount of this enzyme in the dystrophic mice, as determined by three different methods, was about one half of that in the controls. In addition, the concentration of ATP was too low to be detected in crude extracts of dystrophic mouse skeletal muscle, which were prepared using Tris buffer alone or Tris buffer containing either 3 M KCl, or 1 mM PMSF.