Alberto Vita

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 enzymes 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 Ellmans 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.

Effects of Lyophilization and Use of Probiotics on Donkey's Milk Nutritional Characteristics

Cow milk protein allergy (CMPA) is an abnormal IgE-mediated reaction to cow milk proteins. Donkey’s milk could be considered suitable for feeding young children affected by severe IgE-mediated CMPA because its nutritional properties and composition are very close to human milk. Since donkey’s milk is available during a limited range of months during the year, it may be useful to find better storage conditions for this product. This study investigated the effects of the lyophilization treatment on donkey’s milk nutritional characteristics, and the results were compared with those obtained on fresh and frozen milk. Nutritional properties of lyophilized donkey’s milk remained basically unchanged compared with fresh milk. Two different probiotic strains were added to lyophilized donkey’s milk, and their viability was evaluated after milk reconstitution. The results obtained confirmed the possibility of producing a probiotic infant formula with beneficial properties using donkey’s milk as raw material.

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.

A Proteomic Study on Donkey Milk

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.

Uridine phosphorylase from Escherichia coli B.: Kinetic studies on the mechanism of catalysis

Using a highly purified enzyme preparation of uridine phosphorylase from Escherichia coli B, we have performed detailed kinetic studies which include initial-velocity and product-inhibition experiments in the forward and reverse directions of the reaction. These studies indicate a rapid-equilibrium random mechanism for this enzyme with the formation of an enzyme . uracil phosphate abortive complex. Lack of formation of the enzyme . uridine . ribose-1-phosphate abortive complex suggests that the ribosyl moiety of the two ligands compete for the same binding site. The random mechanism is different from the ordered addition of substrates found for uridine phosphorylase from other sources. All the kinetic constants in the forward and reverse directions and the Keq of reaction for E. coli uridine phosphorylase are reported herein.

Uridine phosphorylase from Escherichia coli B. enzymatic and molecular properties

Uridine phosphorylase (EC 2.4.2.3) from Escherichia coli B is an oligomeric protein composed of four identical subunits of 29,000 mol. wt. The enzyme has four half-cystine residues per subunit titrable only in denaturing condition. No disulphide linkages either inter- or intra-chain are present. The isoelectric point is 5.25. The enzyme shows strict specificity toward uridine and 5-methyluridine and is inhibited by thymine, deoxycytidine and heavy metal ions.

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. Proteins 2008.

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.

UNNATURAL ENANTIOMERS OF 5-AZACYTIDINE ANALOGUES: SYNTHESES AND ENZYMATIC PROPERTIES

2′-Deoxy-β-L-5-azacytidine (L-Decitabine), β-L-5-azacytidine, and derivatives were stereospecifically prepared starting from L-ribose or L-xylose. D- and L-enantiomers of 2′-deoxy-β-5-azacytidine were weak substrates of human recombinant deoxycytidine kinase (dCK), whereas both enantiomers of β-5-azacytidine or the L-xylo-analogues were not substrates of the enzyme. None of the reported derivatives of β-L-5-azacytidine was a substrate of human recombinant cytidine deaminase (CDA).

A one-step procedure for the purification of uridine phosphorylase from Escherichia coli

Adsorption to Matrex Gel Green A and successive elution with uridine provides a one-step procedure for the purification of uridine phosphorylase from Escherichia coli B. The homogeneous preparation of the enzyme can be obtained within a single day with an activity recovery above 80%.