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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.

Olive oil is the principal source of fats in the Mediterranean diet and it has been postulated that the components in olive oil can contribute to a lower incidence of coronary heart disease and cancers (prostate, colon, breast, and skin). The positive effects on human health can be attributed to the high level of phenolic compounds present in olive oil, the major ones being oleuropein, hydroxytyrosol and tyrosol. The aim of the present study was to evaluate the effect of oleuropein on enzymes involved in specific pathways of metabolism of proteins, carbohydrates and lipids. In particular, the effects of oleuropein on enzymes, such as trypsin, pepsin, lipase, glycerol dehydrogenase, glycerol-3-phosphate dehydrogenase, and glycerokinase, were investigated. Results demonstrate that oleuropein is able to activate pepsin and shows an inhibitory effect toward all the other enzymes tested, which suggests a new role for this polyphenol. In addition, a new method for lipase activity assay is presented.

The cell egg is in a state of quiescence and only after its fusion with the sperm, a series of pre-programmed metabolic processes will be activated, culminating with embryonic development. The egg/sperm fusion induces a transitory increase of Ca2+ in the cytoplasm, which is responsible for the activation of both precocious and late reactions. The release of Ca2+ occurs by stimulation of the ionic specific channels. In addition to IP3, a new Ca-release inducer was recently evidenced, cyclic ADP ribose (cADPR), in some invertebrates and mammals. Here, we report the first evidence of the cADPR presence in fish. Our data also demonstrate that in the sea bream egg, cADPR is involved in the fertilization process; in fact, its level increases after the entrance of the sperm. By in vitro experiments, it was shown that cADPR induces a release of Ca2+ in the egg homogenate, indicating that in sea bream, the increase of cADPR can induce an intracellular Ca2+ release. Since cADPR is a product of NAD+ metabolism, the activity of several enzymes involved in the NAD+ metabolism was investigated. Sea bream eggs are pelagic and only floating eggs after insemination develop into viable embryos. In the present work, NAD+ metabolism was studied in both types of egg. All the tested enzymes showed similar specific activity in both floating and sinking eggs. In the latter, cADPR was not detectable and the nucleotides content was significantly lower, evidencing a scarce energetic charge in sinking eggs. Mol. Reprod. Dev. 61: 213–217, 2002. © 2002 Wiley-Liss, Inc.

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.

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.

Cytidine deaminase (CDA), is one of the enzymes involved in the pyrimidine salvage pathways, which catalyzes the formation of uridine and deoxyuridine by the hydrolytic deamination of cytidine and deoxycytidine, respectively. Human CDA is a tetrameric enzyme of identical 15 kDa subunits, each containing an essential zinc atom in the active site. The substrate binds to each active site independently and the cooperativity between subunits has not been reported. CDA is able to recognize as substrates some antitumor and antiviral cytidine analogs rendering them pharmacologically inactive. In light of the role played by this enzyme, a deep knowledge of CDA active site and mechanism of catalysis is required. Site-directed mutagenesis, associated with molecular modeling studies, may be an important tool to discover the active site structure of an enzyme and consequently its mechanism of action. In this review are summarized the site-directed mutagenesis experiments performed on human CDA: through these studies it was possible to understand the role exerted by specific amino acid residues in CDA active site and in the contacts between subunits. The obtained results may open a way for designing new cytidine based drugs or more potent CDA inhibitors.

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.

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.

In children with Cow Milk Protein Allergy (CMPA), when it is not possible to breast feed or to use cow milk, the clinical use of donkey milk is considered since several studies have demonstrated the high similarity of donkey milk compared to human milk. An analysis was performed on donkey milk protein profile by two-dimensional electrophoresis (2-DE) followed by N-terminal sequencing in order to give a panoramic view of the proteins that are present in donkey milk. Furthermore, the interest was focused on the casein fractions and on their phosphorylation degree that may influence the calcium binding ability of caseins. At this purpose experiments on donkey milk casein dephosphorylation have been performed and the dephosphorylated casein fractions have been identified after 2-DE analysis followed by N-terminal sequencing. Among caseins were found mainly αs1- and β-caseins that showed a considerable heterogeneity due to variable degree of phosphorylation and to the presence of genetic variants. Finally, a quantitative determination of some antimicrobial proteins, such as lactoferrin and lactoperoxidase, that could be able to stimulate the development of the neonatal intestine, was performed in donkey milk, with the results being 0.080±0.0035 g/L and 0.11±0.027 mg/L, respectively. From the obtained data is evinced that human and donkey milk contain considerable amounts of lysozyme and lactoferrin but lactoperoxidase is present only in small amounts, confirming the high similarity between donkey and human milk. The present study on donkey milk proteins may be useful to assess the nutritional characteristics of this milk that is used to feed children affected by CMPA, but also may open the possibility of utilizing donkey milk in the general population to benefit subjects with CMPA, such as adults and the elderly.

Yeast alcohol dehydrogenase (ADH) is an enzyme widely studied for biotechnological applications due to its involvement in fermentation industry, and various attempts to improve its catalytic properties and its thermal stability have been carried out. In this paper, the influence of a block copolymer (Poloxamer 407) on ADH enzymatic activity and thermal behaviour has been studied in order to get new insights about the use of poloxamers in formulation of sustained release systems for therapeutic proteins. Poloxamer 407 has the ability to form micelles and gel due to its self-assembling and thermoresponsive properties. The effect of the copolymer towards thermal stress and pH changes, which often reduce enzymes activity it has been investigated by means of enzymatic assays and differential scanning calorimetry. Results showed that at pH 9.1 and 7.3, the Poloxamer in the form of unimeric, micellar and gel state is able to effectively preserve the enzyme from thermoinactivation. In addition by calorimetric data Poloxamer 407 has showed an effect in preserving ADH from aggregation at pH 7.3. In conclusion, Poloxamer 407 seems to be very effective in protecting ADH from stress related events, like alkaline inactivation and aggregation.

Publisher Summary Since antiquity, oil obtained from the fruit of the olive ( Olea europaea ) has been valued for its many medical properties. Peoples as ancient as the Egyptians have recognized the medical benefits and food-preservation properties of olives, olive oil, and olive leaf extracts. Since the 1800s, the bitter component in olives has been used in humans against malaria-induced fevers, and beginning in the last century, researchers have focused considerable attention on the health benefits of the various components of olive oil. In particular, many studies have assigned to polyphenols, the minor components of olive oil, the healthy effects observed. Oleuropein, one of the major polyphenols present in olive oil, possesses antimicrobial, antiviral, and antifungal properties. In addition, further studies reported that it is a powerful antioxidant as well as a hypotensive and hypoglycemic agent. Oleuropein exerts protective effects against heart disease and cancers. Other studies have demonstrated that oleuropein and its two main metabolites (hydroxytyrosol and elenoic acid) are completely non-toxic in several animal species and do not interfere with normal embryonic development. Knowing that oleuropein is a non-toxic compound with healthy properties, this chapter investigates its possible role as metabolism modulator. For this reason, it determines the in vitro effects of oleuropein on enzymes involved in proteic, glucydic, and lipidic metabolism. The results obtained from these studies are summarized.

In the present work the effect of a mutation on tyrosine 33 residue (Y33G) of human cytidine deaminase (CDA) was investigated with regard to protein solubility and specific activity. Osmolytes and CDA ligands were used to increase the yield and the specific activity of the protein. The mutant enzyme was purified and subjected to a kinetic characterization and to stability studies. These investigations reinforced the hypothesis that in human CDA the side chain of Y33 is involved in intersubunit interactions with four glutamate residues (E108) forming a double latch that connects each of the two pairs of monomers of the tetrameric CDA.

We report the kinetics and molecular properties of CD38 purified from bovine lung microsomal membranes after its solubilization with Triton X-100. The enzyme was found to be a novel member of a multicatalytic NAD+-glycohydrolase (NADase, EC 3.2.2.6). It was able to utilize NAD + in different ways, producing nicotinamide (Nam) and either adenosine diphosphoribose (ADPR, NADase activity) or cyclic ADPR (cADPR, cyclase activity); it also catalyzed the hydrolysis of cADPR to ADPR (cADPR, hydrolase activity). In addition, the enzyme catalyzed the pyridine base exchange reaction with conversion of NAD + into NAD analogues. These data are evidence that CD38 is involved in the regulation of both NAD+ and calcium-mobilizing agents, the concentration resulting in an essential enzyme that plays a key role in cellular energy and signal-transduction systems.

We have previously reported for the first time the purification to homogeneity of the enzyme NMN adenylyltransferase (EC 2.7.7.1) from yeast and its major molecular and catalytic properties. The homogeneous enzyme was found to be a glycoprotein containing 2% carbohydrate and 1 mol of adenine residue and 2 mol of phosphate covalently bound per mole of protein. Such a stoichiometry, apparently consistent with that of ADP-ribose, prompted us to further investigate the possibility that NMN adenylyltransferase could be subjected to poly(ADP-ribosylation) in vitro in a reconstituted system. Poly(ADP-ribose) polymerase was purified to homogeneity from bull testis by means of a rapid procedure involving two batchwise steps on DNA-agarose and Reactive Blue 2 cross-linked agarose and a column affinity chromatography step on 3-aminobenzamide-Sepharose; the optimal conditions for the poly(ADP-ribosylation) of exogenous substrates were determined. When pure NMN adenylyltransferase was incubated in the presence of the homogeneous poly(ADP-ribose) polymerase, a marked inhibition of the polymerase was observed, both in the presence and in the absence of histones, while the activity of NMN adenylyltransferase was not affected. The inhibition could not be prevented by increasing the concentrations of either DNA or NAD. Mg2+ did not affect the activity or the inhibition. The significance of such a phenomenon is at present unknown, but it may be of biological relevance in view of the close topological and metabolic relationship between the two enzymes.

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.

An intersubunit interactions study related to the active site has been performed on the wild-type cytidine deaminase (CDA) and on the mutant enzyme F137W/W113F. F137 is the homologous to the Bacillus subtilis CDA F125 involved in the subunit interactions. In the presence of SDS, wild-type human CDA dissociates into enzymatically inactive monomers without intermediate forms via a non-cooperative transition. Extensive dialysis or dilution of the inactivated monomers restores completely the activity. Circular dichroism measurements show that the secondary/tertiary structure organization of each subunit is unaffected by the SDS concentration, while the mutation Phe/Trp causes weakening in quaternary structure. The presence of the strong human CDA competitive inhibitor 5-fluorozebularine disfavours dissociation of the tetramer into subunits in the wild-type CDA, but not in mutant enzyme F137W/W113F. The absence of tyrosine fluorescence and the much higher quantum yield of the double mutant protein spectrum suggest the occurrence of an energy transfer effect between the protein subunits. This assumption is confirmed by the crystallographic studies on B.subtilis in which it is shown that three different subunits concur with the formation of each of the four active sites and that F125, homologous to the human CDA F137, is located at the interface between two different subunits contributing to the formation of active site.

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.

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. Mol. Reprod. Dev. 66: 291–296, 2003. © 2003 Wiley-Liss, Inc.

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 p I of 4.9 and a K m value for NAD + of 26 μM. High activation of ADPR cyclase activity was observed in the presence of Ag + ions, corresponding to NADase inhibition.

Publisher Summary The enzyme nicotinamide–mononucleotide adenylyltransferase plays a key role in pyridine nucleotide turnover, being involved in both de novo and salvage synthesis of nicotinamide adenine dinucleotide (NAD). Sustained degradation and resynthesis of NAD is a well-recognized phenomenon both in eukaryotic and in prokaryotic microorganisms. Sulfolobus solfataricus is a thermophilic archaeon living under extreme conditions of temperature and pH. Comparison of the molecular and catalytic properties of nicotinamide–mononucleotide adenylyltransferase from mesophilic and thermophilic microorganisms can provide significant information for the elucidation of the significance of NAD turnover in an evolutionary perspective. In addition, the identification of the molecular determinants of the enzyme thermostability and thermophilicity appears to have possible biotechnological interest. Owing to the high reaction temperature, a continuous spectrophotometric assay cannot be used. Therefore, the activity of the enzyme from Sulfolobus solfataricus is measured with two different discontinuous assays. The effect of temperature on the enzyme activity shows Arrhenius activation energy of 95 kJ/mol, indicating a remarkable thermophilicity, even when compared with that of other enzymes from the same microorganism.

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.

School of Veterinary Medical Sciences University of Camerino, via Gentile III da Varano, 62032 Camerino, MC, Italy School of Biosciences and Biotechnologies, via Gentile III da Varano, 62032 Camerino, MC, Italy Laboratory of Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, 20892 Bethesda, MD, United States School of Pharmacy, University of Camerino, via Gentile III da Varano, 62032 Camerino, MC, Italy

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 Mr 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 μ m , respectively. Kinetic analysis reveals a behavior consistent with an ordered sequential Bi-Bi mechanism. Among several metabolites tested only ADP-ribose and β-NMNH were found to significantly inhibit the enzyme activity.

The nuclear location of NMN adenylyltransferase (NMNAT), the last enzyme in the main biosynthetic pathway of NAD+, prompted us to investigate about a possible involvement of this enzyme in the regulation of the cellular activity by hypothesizing its eventual relationship with poly(ADP-ribose) polymerase (ADPRP), another chromatin bound enzyme. This hypothesis was based on the discovery of an inhibitory effect exerted by NMNAT on ADPRP in vitro in reconstituted systems, composed by enzymes purified both from different sources (heterologous system) (1) and from identical sources (homologous system) (2). In order to verify such a phenomenon in man and to better elucidate its functional significance we carried out the purification of NMNAT from human placenta, where the system involved in NAD+ comsumption has been fully characterized (3).

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.

Despite the increasing interest on both the physiological and pathological role of ADP-ribosylation reactions, little appears to be known about the mechanism by which the intracellular level of NAD+ is regulated with respect to its participation in the redox metabolism on the one hand and to its utilization in poly(ADP-ribosy)lation on the other. Conditions enhancing poly(ADPribosyl)ation can significantly lower NAD+ concentration thereby influencing NAD+ dependent metabolic processes in the whole cell, while the cellular redox status can be signaled to chromatin thereby influencing the level of ADPribosylation of nuclear proteins (1).

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.

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.

In mammalian cells the main biosynthetic pathway leading to the formation of NAD involves the enzyme NMN adenylyltransferase (NMNAT), which catalyzes the following reaction

The molecular and kinetic properties of cytidine deaminase from E. coli and chicken liver show several interesting differences and similarities: 1. 1. Both enzymes possess an oligomeric structure, and linear kinetics. 2. 2. The chicken liver enzyme is strictly dependent on the presence of reducing agents and presents a microheterogeneity in the pure preparation. 3. 3. Both enzymes display identical specificity and share a rapid-equilibrium random Uni-Bi mechanism of catalysis. 4. 4. The chicken liver enzyme is inhibited competitiely by dTTP, CMP and dCMP.

Modification of rabbit liver fructose-1,6-bisphosphatase (EC 3.1.3.11) with pyridoxal phosphate in the presence of the substrate fructose 1,6-biphosphate has been shown to decrease its sensitivity to inhibition by AMP. Two adjacent lysyl residues, located at positions 137 and 138 in the primary sequence, have now been identified as the site of modification, each being modified to the extent of approximately 50%. These allosteric-site lysyl residues are located in the largest cyanogen bromide peptide, designated BrCN1. BrCN1 is linked to the NH 2 -terminal segment whose sequence was reported earlier [Botelho et al. (1977) Arch. Biochem. Biophys. 184 , 535–545]. The sequence analysis of this large cyanogen bromide peptide, comprising residues 80–163 of the fructose-1,6-bisphosphatase subunit, is reported.

ATP, UTP, GTP, and CTP were found to be powerful competitive inhibitors of 5′-nucleotidase partially purified from guinea pig skeletal muscle, the concentrations required for 50% inhibition being 1.25 uM, 5.5 uM, 10 uM and 27.5 uM respectively with 5′-AMP as substrate. The enzyme does not require divalent cations. Furthermore magnesium, calcium and cobalt ions added in large excess with respect to nucleoside triphosphates did not completely relieve the inhibition, indicating that the complexes nucleoside triphosphates-divalent cations are also inhibitors. Using specific optical assays to follow the dephosphorylation of AMP, GMP and IMP it was found that the hydrolysis of each 5′-mononucleotide is competitively inhibited by other 5′-mononucleotides. The regulation of skeletal muscle 5′-nucleotidase supports the hypothesis of its role in the mechanism of muscular contraction.

A homogeneous preparation of yeast NMN adenylyltransferase (EC 2.7.7.1) showed microheterogeneity, which was revealed by FPLC (Fast Protein Liquid Chromatography) ion exchange chromatography. The resolved components have been characterized with respect to electrophoretic behavior and adenine content. The results led to a hypothesis about a possible role of poly(ADP-ribosylation) in modulating the enzyme activity.

Digestion of native rabbit liver fructose-1,6-bisphosphatase (Fru-P 2 ase, EC 3.1.3.11) with a membrane-bound proteinase from rat liver lysosomes yields a fragment of M r = 9850. This peptide contains the COOH terminus of the Fru-P 2 ase polypeptide chain and also the cyanogen bromide peptide (BrCN5) carrying the active site lysyl residue. The sequence of BrCN5 and its location with respect to the COOH terminus of the polypeptide chain have been determined. The active site lysyl residue is located at approximately residue −54 from the COOH terminus. The bond hydrolyzed by the lysosomal proteinase is located between residues −88 and −89 from the COOH terminus.

Summary A new spectrophotometric assay for human erythrocyte pyrimidine 5′-nucleotidase using a continuous spectrophotometric method is described. The activity is detected by measuring uracil, which is produced by a coupled assay including cytidine deaminase and uridine nucleosidase. The results obtained with the continuous spectrophoto-metric method have been confirmed by high pressure liquid chromatography (HPLC) analysis. This procedure, not based upon inorganic phosphate formation and avoiding the use of radioactive material, offers significant advantages with respect to commonly adopted assays.

Synthesis and study of the antimonoamine oxidase activity of some N′-substituted hydrazides of 2-methyl-3-indolizinecarboxylic acid are reported. The activities were generally of about the same order as those of the corresponding hydrazides of 2-indolizinecarboxylic acid.

Uridine nucleosidase (EC 3.2.2.3) was purified from commercial bakers' yeast to homogeneity, as judged by a single band observed on polyacrylamide disc gel electrophoresis. The molecular weight of the enzyme, estimated by gel filtration, was approximately 32,500. Polyacrylamide electrophoresis in 0.2% sodium dodecyl sulfate showed the presence of two apparently identical subunits of 17,000 molecular weight. The amino acid composition indicated a large excess of glutamic acid and aspartic acid over other amino acid residues and a very low content of tyrosine and tryptophan. Th SH groups analysis performed with 5,5'-dithiobis (2-nitrobenzoic acid) on thenative protein as well as in the presence of 1% sodium dodecyl sulfate showed the existence of one sulfhydryl group per mole of enzyme. Uridine nucleosidase is active on uridine and 5-methyluridine (ribosylthymine) resulting inactive toward all other pyrimidine and purine nucleosides tested. The Km values for uridine and 5-methyluridine were 0.86 x 10(-3) M and 1.66x10--3M, respectively. The optimal pH is around 7.0. The isoelectric point is 5.1. Among a variety of compounds tested only ribose and glucose 6-phosphate were inhibitory and Ki values were 7.2 mM and 0.19 mM, respectively. Furthermore, ribosylthymine competitively inhibited the hydrolysis of uridine. The type of all inhibitions was competitive and the n' values of the Hill plots were near 1. The effect of temperature on the enzyme activity plotted accoring to Arrhenius gave a value of E = 4740 cal per mole. The enzyme in 100 mM phosphate, pH = 7.0, is stable at 4 degrees for 15 days without any loss of activity.

Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from baker's yeast crude extract. The purification procedure is relatively simple and consists of high-salt extraction of enzyme activity and precipitation with poly(ethylenimine), followed by ion-exchange and dye ligand chromatography separations. The final enzyme preparation is homogeneous as judged by a single Coomassie blue stainable band when run on nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 200 000, calculated by gel filtration and sucrose gradient centrifugation. The protein possesses quaternary structure and is composed of four apparently identical Mr 50 000 subunits. The absorption spectrum shows a maximum at 280 nm and a minimum at 253 nm. The isoelectric point is 6.2. Amino acid composition analysis shows the presence of 28 half-cystine residues. The same result has been obtained by titrating the enzyme in denaturating conditions with Ellman's reagent after incubation with sodium borohydride. NMN adenylyltransferase is a glycoprotein containing 2% sugar, 2 mol of alkali-labile phosphate per mole of enzyme, and 1 mol of adenine moiety per mole of enzyme. Therefore, the possibility that the enzyme is ADP-ribosylated exists. The Km values for ATP, NMN, and nicotinate mononucleotide are 0.11 mM, 0.19 nM, and 5 mM, respectively. Kinetic analysis reveals a behavior that is consistent with an ordered sequential Bi-Bi mechanism. The pH optimum is in the range 7.2-8.4.

It has been previously demonstrated in our laboratory that uridine nucleosidase (EC 3.2.2.3) is subjected in yeast to inactivation. An inactivating fraction has been isolated and purified to homogeneity with a procedure which includes gel filtration, adsorption chromatography, and electrofocusing techniques. The molecular weight of the enzyme, estimated either by sodium dodecyl sulfate disc gel electrophoresis or by gel filtration is approximately 44,000. No quaternary structure was evidenced. The inactivating activity possesses proteolytic activity against casein and hemoglobin with pH optima of 2.5 and 3.2, respectively. The optimal pH for uridine nucleosidase inactivation is around 4.7. The inactivating activity as well as the proteolytic activity of the preparation can be inhibited by IA but not by IB2 and IC, yeast macromolecular inhibitors for proteinase A (EC 3.4.23.8), B (EC 3.4.22.9), and C (EC 3.4.12.8), respectively. The apparent isoelectric point is pH 4.03. The carbohydrate content is 8.5%. A comparison of the properties of the inactivating protein with those of known yeast proteinases leads to the conclusion that it is identical with the enzyme previously designated as proteinase A, which for the first time has been obtained homogeneous and characterized. It has been shown that proteinase A could play a physiological role in the uridine nucleosidase inactivation process when it is associated, as a complex, with proteinase B.

A new analog of thymosin β 4 has been isolated from tissues of several mammalian species. This peptide, designated thymosin β 10 , is composed of 42 amino acid residues and shows 75% sequence homology with thymosin β 4 . It occurs together with thymoxin β 4 in a variety of tissues including spleen, liver, and thymus and also in several cultured cell lines. In the spleen of rat, mouse, cat, and man, the new peptide accounts for approximately 0.02% by weight of the total protein. In the calf it is replaced by another homologous peptide, designated thymosin β 9 , whose structure has been reported.

The enzymatic properties of homogeneous bakers' yeast uridine nucleosidase, prepared as previously described (G. Magni et al., J. Biol. Chem. 1975 250 , 9–13) have been further investigated, and in addition to glucose-6-phosphate and ribose the enzyme activity was inhibited by ribose-5-phosphate and ribulose-5-phosphate. The curves describing this inhibitions were sigmoidal and when the data were plotted according to Hill, n′ values different from 1 were observed suggesting the existence of interactions among the inhibitory molecules binding sites. Furthermore the percentage of inhibition exerted by glucose-6-phosphate, ribose and ribose-5-phosphate on the enzyme activity varied at different pH values. The addition of various chelating agents to the activity assay mixture caused a strong inhibition of the enzyme activity and metal analysis by atomic absorption spectrophotometry, colorimetric methods and electronic paramagnetic resonance, indicated the presence of 1 copper atom per enzyme molecule. Finally the inhibition exerted by metal ions on the enzyme activity was described.

Uridine 5′-monophosphate pyrophosphorylase was found to be present in baker's yeast. The enzyme preparation, purified about 30-fold, shows a strict specificity toward uracil and requires Mg++ for its activity.

It has been previously demonstrated in our laboratory that uridine nucleosidase (EC 3.2.2.3) is inactivated by yeast protease A (EC 3.4.23.8). A complete purification procedure for protease A from bakers' yeast, which lacks the acidic activation step used by other workers, and the major properties of the enzyme are shown. The enzyme is homogeneous as judged by disc gel electrophoresis. Its molecular weight, calculated from both sodium dodecyl sulfate-disc gel electrophoresis and gel filtration experiments, is around 45,000. The protein does not possess quaternary structure. The isoelectric point is 4.1. Carbohydrate content is around 8%. Amino acids analysis and sulfur analysis reveal the presence of 1-SH group and two disulfide bridges. The free-SH group does not seem to be involved in catalysis. Amino terminal analysis shows that isoleucine is at the amino terminal position. The pH optima are 2.4 for the hydrolysis of azocasein and casein, and 3.3 for the hydrolysis of hemoglobin. The Km value for hemoglobin is 1.7 × 10−5 m . The inhibition exerted by pepstatin on the proteolytic activity of protease A is pH dependent. Among various yest enzyme substrates only uridine nucleosidase is inactivated by protease A.

Summary: 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 enzymeinhibitor 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.