Giovanni Antonini

Antiviral Activity of Lactoferrin

In 1976, Matthews et al.19 reported the antiviral activity of milk proteins, underlining their possible clinical importance. Only recently, this effect has been ascribed mainly to lactoferrin (Lf). Lf was initially shown to exert a protective influence in mice infected with Friend leukemia virus15. Subsequently, a potent antiviral activity has been attributed to Lf, both in vitro towards cytomegalovirus (HCMV)9,7, herpes simplex virus (HSV)17, human immunodeficiency virus (HIV)7,29 and in vivo towards HSV-16 and HCMV25. Our groups provided evidence on the antiviral activity of Lf towards HSV-218, rotavirus28, and HIV22 infections (Table 1).

Involvement of bovine lactoferrin metal saturation, sialic acid and protein fragments in the inhibition of rotavirus infection

Although the antiviral activity of lactoferrin is one of the major biological functions of this iron binding protein, the mechanism of action is still under debate. We have investigated the role of metal binding, of sialic acid and of tryptic fragments of bovine lactoferrin (bLf) in the activity towards rotavirus (intestinal pathogen naked virus) infecting enterocyte-like cells. The antiviral activity of bLf fully saturated with manganese or zinc was slightly decreased compared to that observed for apo- or iron-saturated bLf. The antiviral activity of differently metal-saturated bLf towards rotavirus was exerted during and after the virus attachment step. The removal of sialic acid enhanced the anti-rotavirus activity of bLf. Among all the peptidic fragments obtained by tryptic digestion of bLf and characterised by advanced mass spectrometric methodologies, a large fragment (86-258) and a small peptide (324-329: YLTTLK) were able to inhibit rotavirus even if at lower extent than undigested bLf.

Catalytic mechanism and role of hydroxyl residues in the active site of theta class glutathione s-transferases investigation of ser-9 and tyr-113 in a glutathione s-transferase from the australian sheep blowfly, lucilia cuprina

Spectroscopic and kinetic studies have been performed on the Australian sheep blowfly Lucilia cuprina glutathione S-transferase (Lucilia GST; EC 2.5.1.18) to clarify its catalytic mechanism. Steady state kinetics of Lucilia GST are non-Michaelian, but the quite hyperbolic isothermic binding of GSH suggests that a steady state random sequential Bi Bi mechanism is consistent with the anomalous kinetics observed. The rate-limiting step of the reaction is a viscosity-dependent physical event, and stopped-flow experiments indicate that product release is rate-limiting. Spectroscopic and kinetic data demonstrate thatLucilia GST is able to lower the pK a of the bound GSH from 9.0 to about 6.5. Based on crystallographic suggestions, the role of two hydroxyl residues, Ser-9 and Tyr-113, has been investigated. Removal of the hydroxyl group of Ser-9 by site-directed mutagenesis raises the pK a of bound GSH to about 7.6, and a very low turnover number (about 0.5% of that of wild type) is observed. This inactivation may be explained by a strong contribution of the Ser-9 hydroxyl group to the productive binding of GSH and by an involvement in the stabilization of the ionized GSH. This serine residue is highly conserved in the Theta class GSTs, so the present findings may be applicable to all of the family members. Tyr-113 appears not to be essential for the GSH activation. Stopped-flow data indicate that removal of the hydroxyl group of Tyr-113 does not change the rate-limiting step of reaction but causes an increase of the rate constants of both the formation and release of the GSH conjugate. Tyr-113 resides on α-helix 4, and its hydroxyl group hydrogen bonds directly to the hydroxyl of Tyr-105. This would reduce the flexibility of a protein region that contributes to the electrophilic substrate binding site; segmental motion of α-helix 4 possibly modulates different aspects of the catalytic mechanism of theLucilia GST.

Structural flexibility modulates the activity of human glutathione transferase P1-1. Influence of a poor co-substrate on dynamics and kinetics of human glutathione transferase.

Presteady-state and steady-state kinetics of human glutathione transferase P1-1 (EC) have been studied at pH 5.0 by using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a poor co-substrate for this isoenzyme. Steady-state kinetics fits well with the simplest rapid equilibrium random sequential bi-bi mechanism and reveals a strong intrasubunit synergistic modulation between the GSH-binding site (G-site) and the hydrophobic binding site for the co-substrate (H-site); the affinity of the G-site for GSH increases about 30 times at saturating co-substrate and vice versa. Presteady-state experiments and thermodynamic data indicate that the rate-limiting step is a physical event and, probably, a structural transition of the ternary complex. Similar to that observed with 1-chloro-2,4-dinitrobenzene (Ricci, G., Caccuri, A. M., Lo Bello, M., Rosato, N., Mei, G., Nicotra, M., Chiessi, E., Mazzetti, A. P., and Federici, G. (1996) J. Biol. Chem. 271, 16187-16192), this event may be related to the frequency of enzyme motions. The observed low, viscosity-independent kcat value suggests that these motions are slow and diffusion-independent for an increased internal viscosity. In fact, molecular modeling suggests that the hydroxyl group of Tyr-108, which resides in helix 4, may be in hydrogen bonding distance of the oxygen atom of this new substrate, thus yielding a less flexible H-site. This effect might be transmitted to the G-site via helix 4. In addition, a new homotropic behavior exhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole is found in Cys-47 mutants revealing a structural intersubunit communication between the two H-sites.

Microbiological safety and quality of Mozzarella cheese assessed by the microbiological survey method

Dairy products are characterized by reduced shelf life because they are an excellent growth medium for a wide range of microorganisms. For this reason, it is important to monitor the microbiological quality of dairy products and, in particular, the total viable count and concentration of Escherichia coli, as they are indicators of the hygienic state of these products. In addition, in dairy products such as Mozzarella cheese, it is important to monitor the concentration of lactic acid bacteria (LAB), as they are the major components of starter cultures used in cheese production, contributing to the taste and texture of fermented products and inhibiting food spoilage bacteria by producing growth-inhibiting substances. For these reasons, to ensure the quality and safety of their products, cheese makers should monitor frequently, during fresh cheese production, the concentration of LAB and spoilage bacteria. However, usually, small- to medium-size dairy factories do not have an internal microbiological laboratory and external laboratories of analysis are often too expensive and require several days for the results. Compared with traditional methods, the microbiological survey (MBS) method developed by Roma Tre University (Rome, Italy) allows faster and less-expensive microbiological analyses to be conducted wherever they are necessary, without the need for a microbiological laboratory or any instrumentation other than MBS vials and a thermostat. In this paper, we report the primary validation of the MBS method to monitor LAB concentration in Mozzarella cheese and the analysis, using the MBS method, of total viable count, E. coli, and LAB concentrations in the production line of Mozzarella cheese as well as during the shelf life of the product stored at 20°C. The results obtained indicate that the MBS method may be successfully used by small- to medium-size dairy factories that do not have an internal microbiological laboratory. Using the MBS method, these dairy factories can monitor autonomously the microbiological safety and quality of their products, saving both time and money.

The extended catalysis of glutathione transferase

Glutathione transferase reaches 0.5–0.8 mM concentration in the cell so it works in vivo under the unusual conditions of, [S] ≪ [E]. As glutathione transferase lowers the pK a of glutathione (GSH) bound to the active site, it increases the cytosolic concentration of deprotonated GSH about five times and speeds its conjugation with toxic compounds that are non‐typical substrates of this enzyme. This acceleration becomes more efficient in case of GSH depletion and/or cell acidification. Interestingly, the enzymatic conjugation of GSH to these toxic compounds does not require the assumption of a substrate–enzyme complex; it can be explained by a simple bimolecular collision between enzyme and substrate. Even with typical substrates, the astonishing concentration of glutathione transferase present in hepatocytes, causes an unusual “inverted” kinetics whereby the classical trends of v versus E and v versus S are reversed.

The Nutraceutical Properties of Ovotransferrin and Its Potential Utilization as a Functional Food.

Ovotransferrin or conalbumin belong to the transferrin protein family and is endowed with both iron-transfer and protective activities. In addition to its well-known antibacterial properties, ovotransferrin displays other protective roles similar to those already ascertained for the homologous mammalian lactoferrin. These additional functions, in many cases not directly related to iron binding, are also displayed by the peptides derived from partial hydrolysis of ovotransferrin, suggesting a direct relationship between egg consumption and human health.

Is the Internal Electron Transfer the Rate‐Limiting Step in the Catalytic Cycle of Cytochrome c Oxidase?

Pulsed cytochrome c oxidase, obtained by reaction of the fully reduced enzyme with dioxygen,’-’ has been characterized functionally and spectroscopically by comparison to the resting state of the protein. The increase in turnover number, which is the primary functional feature of the pulsed enzyme, is not associated with a change in the bimolecular rate constant for electron transfer from reduced cytochrome c, but it is accounted for by an enhancement of the rate of internal electron transfer from cytochrome a and/or Cua to the binuclear center, cytochrome a3/Cua3. This electrontransfer step was proposed by Wilson et aL4*’ to represent the rate-limiting step of the overall catalytic process, and a minimal reaction scheme was found to account quantitatively for transient and steady-state kinetic data.5 More recent kinetic studiesb” have confirmed the crucial role of conformational changes in controlling the redox and proton-pumping functions of cytochrome oxidase, and in particular have indicated that a two-electron reduced enzyme (a2+/Cua+a:+/Cua:+) undergoes a closed-to-open transition, which accounts for the rapid binding of cyanide at the binuclear center. Similar events have also been described for cytochrome oxidase reconstituted into phospholipid vesicles,” and Malmstrom” has proposed that a closed-to-open conformational transition is coupled to the expression of the proton-pumping function of the enzyme. It is well known (see Wikstrom et al.I4 and Brunori et al.”) that the functional properties of cytochrome oxidase are affected by solvent conditions, including pH,

The Glu216/Ser218 pocket is a major determinant of spermine oxidase substrate specificity

SMO (spermine oxidase) and APAO (acetylpolyamine oxidase) are flavoenzymes that play a critical role in the catabolism of polyamines. Polyamines are basic regulators of cell growth and proliferation and their homoeostasis is crucial for cell life since dysregulation of polyamine metabolism has been linked with cancer. In vertebrates SMO specifically catalyses the oxidation of spermine, whereas APAO displays a wider specificity, being able to oxidize both N¹-acetylspermine and N¹-acetylspermidine, but not spermine. The molecular bases of the different substrate specificity of these two enzymes have remained so far elusive. However, previous molecular modelling, site-directed mutagenesis and biochemical characterization studies of the SMO enzyme-substrate complex have identified Glu²¹⁶-Ser²¹⁸ as a putative active site hot spot responsible for SMO substrate specificity. On the basis of these analyses, the SMO double mutants E216L/S218A and E216T/S218A have been produced and characterized by CD spectroscopy and steady-state and rapid kinetics experiments. The results obtained demonstrate that mutation E216L/S218A endows SMO with N¹-acetylspermine oxidase activity, uncovering one of the structural determinants that confer the exquisite and exclusive substrate specificity of SMO for spermine. These results provide the theoretical bases for the design of specific inhibitors either for SMO or APAO.

Hypotaurine protection on cell damage by singlet oxygen.

Singlet oxygen (1O2), generated by irradiating methylene blue, is toxic to melanoma cell cultures. Hypotaurine is known to scavenge efficiently singlet oxygen; the addition of hypotaurine (800 microM) to the medium during irradiation of the dye produces a greater protective effect on cells than taurine added at the same concentration. The assay of some detoxifying enzymatic activities indicate a different mechanism of protection of the two molecules: taurine induces an efficient detoxifying enzymatic action with respect to the control; hypotaurine exerts its effect greatly by specifically scavenging singlet oxygen.