Alessandra Padiglia

Plant copper-amine oxidases

In this review, the widely distributed plant copper-amine oxidases are described. The purification procedures, molecular features, substrate specificities, inhibitors, the stoichiometry of the catalysed reaction, spectroscopic features, the prosthetic groups and reaction mechanisms, are all reviewed.

Sensitivity to 6-n-propylthiouracil is associated with gustin (carbonic anhydrase VI) gene polymorphism, salivary zinc, and body mass index in humans

BACKGROUND The individual ability to taste 6-n-propylthiouracil (PROP) may be correlated with body mass index (BMI) and differences in the salivary proteins involved in taste function, such as the zinc-dependent enzyme gustin, which is a trophic factor of taste buds. OBJECTIVE We investigated the possible association of PROP taste responsiveness with gustin gene polymorphism rs2274333 (A/G), salivary ionic zinc concentrations, and BMI. DESIGN We measured cognitive eating behaviors and BMI in 75 volunteers (28 men and 47 women; mean plusmn SEM age: 25 plusmn 3 y). The intensity of taste perception evoked by PROP and sodium chloride solutions was estimated to evaluate PROP taster status. Salivary ionic zinc concentrations were measured, and molecular analyses of the gustin gene polymorphism were performed in individuals classified by PROP status by using polymerase chain reaction techniques. RESULTS We classified subjects as PROP supertasters (n = 27), medium tasters (n = 28), or nontasters (n = 20). Salivary ionic zinc concentrations and BMI were greater in nontasters than in supertasters (P = 0.003 and P = 0.042, respectively). Molecular analyses of gustin DNA showed that allele A and genotype AA were significantly more frequent in supertasters, whereas allele G and genotype GG were significantly more frequent in nontasters (P lt 0.001). CONCLUSIONS These data showed that responsiveness to PROP is inversely related to BMI and salivary ionic zinc concentrations. The gustin gene dimorphism rs2274333 observed in supertaster and nontaster subjects may influence the protein conformation and, thereby, affect zinc ion binding. Our data showed a direct association between PROP sensitivity and a polymorphism in the gustin gene that is hypothesized to affect its function. This trial was registered at clinicaltrials.gov as UNICADBSITB-1.

Polymorphisms in TAS2R38 and the taste bud trophic factor, gustin gene co-operate in modulating PROP taste phenotype

The PROP taste phenotype varies greatly among individuals, influencing eating behavior and therefore may play a role in body composition. This variation is associated with polymorphisms in the bitter receptor gene TAS2R38 and the taste-bud trophic factor gustin gene. The aim of this study was to examine the relationship between TAS2R38 haplotypes and the gustin gene polymorphism rs2274333 in modulating PROP taste phenotype. PROP phenotype was determined in seventy-six volunteers (29 males, 47 females, age 25±3 y) by scaling methods and threshold measurements. TAS2R38 and gustin gene genotyping was performed using PCR techniques. The lowest responsiveness in PROP nontasters is strongly associated with the AVI nontasting TAS2R38 variant and the highest responsiveness in supertasters is strongly associated to allele A and genotype AA of the gustin gene. These data support the hypothesis that the greater sensitivity of supertasters could be mediated by a greater taste-bud density. Polymorphisms in TAS2R38 and gustin gene, together, accounted for up to 60% of the phenotypic variance in PROP bitterness and to 40% in threshold values. These data, suggest that other unidentified factors may be more relevant for detecting low concentrations of PROP. Moreover, the presence of the PAV variant receptor may be important for detecting high concentrations of PROP, whereas the presence of allele A in gustin polymorphism may be relevant for perceiving low concentrations. These data show how the combination of the TAS2R38 and gustin gene genotypes modulate PROP phenotype, providing an additional tool for the evaluation of human eating behavior and nutritional status.

The physiopathological significance of ceruloplasmin: A possible therapeutic approach

This article reviews and comments on the physiological roles of ceruloplasmin (Cp). We show that, in addition to its ascertained involvement in iron homeostasis, the protein, by virtue of its unique structure among multicopper oxidases, is likely involved in other processes of both an enzymatic and a nonenzymatic nature. In particular, based on the analysis of the kinetic parameters, on the one hand, and of the side-products of the oxidation, on the other, we propose that the long-recognized ability of Cp to interact with and oxidize non-iron substrates may be of physiological relevance. The striking example of 6-hydroxydopamine oxidation is presented, where we show that the catalytic action is carried out readily under physiological conditions, without release of potentially toxic oxygen intermediates.

Responsiveness to 6-n-propylthiouracil (PROP) is associated with salivary levels of two specific basic proline-rich proteins in humans.

Thiourea tasting can be predictive of individual differences in bitter taste responses, general food preferences and eating behavior, and could be correlated with saliva chemical composition. We investigated the possible relationship between PROP bitter taste responsiveness and the salivary proteome in subjects genotyped for TAS2R38 and gustin gene polymorphisms. Taste perception intensity evoked by PROP and NaCl solutions was measured in sixty-three volunteers (21 males, 42 females, age 25±3 y) to establish their PROP taster status, and 24 PROP super-tasters and 21 nontasters were selected to participate in the study. TAS2R38 and gustin gene molecular analysis were performed using PCR techniques. Qualitative and quantitative determination of salivary proteins was performed by HPLC-ESI-MS before and after PROP taste stimulation. PROP super-tastings was strongly associated with the ‘taster’ variant (PAV haplotype) of TAS2R38 and the A allele of rs2274333 polymorphism in the gustin gene and nontasting was associated with the minor alleles at both loci. ANOVA revealed that basal levels of II-2 and Ps-1 proteins, belonging to the basic proline-rich protein (bPRPs) family, were significantly higher in PROP super-taster than in nontaster un-stimulated saliva, and that PROP stimulation elicited a rapid increase in the levels of these same proteins only in PROP super-taster saliva. These data show for the first time that responsiveness to PROP is associated with salivary levels of II-2 peptide and Ps-1 protein, which are products of the PRB1 gene. These findings suggest that PRB1, in addition to TAS2R38 and gustin, could contribute to individual differences in thiourea sensitivity, and the expression of the PROP phenotype as a complex genetic trait.

The Gustin (CA6) Gene Polymorphism, rs2274333 (A/G), as a Mechanistic Link between PROP Tasting and Fungiform Taste Papilla Density and Maintenance

Taste sensitivity to PROP varies greatly among individuals and is associated with polymorphisms in the bitter receptor gene TAS2R38, and with differences in fungiform papilla density on the anterior tongue surface. Recently we showed that the PROP non-taster phenotype is strongly associated with the G variant of polymorphism rs2274333 (A/G) of the gene that controls the salivary trophic factor, gustin. The aims of this study were 1) to investigate the role of gustin gene polymorphism rs2274333 (A/G), in PROP sensitivity and fungiform papilla density and morphology, and 2) to investigate the effect of this gustin gene polymorphism on cell proliferation and metabolic activity. Sixty-four subjects were genotyped for both genes by PCR techniques, their PROP sensitivity was assessed by scaling and threshold methods, and their fungiform papilla density, diameter and morphology were determined. In vitro experiments examined cell proliferation and metabolic activity, following treatment with saliva of individuals with and without the gustin gene mutation, and with isolated protein, in the two iso-forms. Gustin and TAS2R38 genotypes were associated with PROP threshold (p=0.0001 and p=0.0042), but bitterness intensity was mostly determined by TAS2R38 genotypes (p<0.000001). Fungiform papillae densities were associated with both genotypes (p<0.014) (with a stronger effect for gustin; p=0.0006), but papilla morphology was a function of gustin alone (p<0.0012). Treatment of isolated cells with saliva from individuals with the AA form of gustin or direct application of the active iso-form of gustin protein increased cell proliferation and metabolic activity (p<0.0135). These novel findings suggest that the rs2274333 polymorphism of the gustin gene affects PROP sensitivity by acting on fungiform papilla development and maintenance, and could provide the first mechanistic explanation for why PROP super-tasters are more responsive to a broad range of oral stimuli.

CUI-SEMIQUINONE RADICAL SPECIES IN PLANT COPPER-AMINE OXIDASES

The intermediate CuI‐semiquinone radical species in the catalytic mechanism of copper‐amine oxidase from Lens esculenta and Pisum sativum seedlings has been studied by optical, Raman resonance and ESR spectroscopies and by stopped‐flow and temperature‐jump measurements. Treatment of highly purified enzyme preparations with good, poor or suicide substrates, under anaerobic and aerobic conditions, at different pH values and temperatures, makes it possible to generate, detect and characterize this free radical intermediate.

Purification and characterization of Opuntia peroxidase

Abstract Peroxidase from Opuntia ficus indica fruits was purified with chromatographic methods. The enzyme had a characteristic spectrum in the visible region and R z ( A 403 A 275 ) value of 2.56. It showed a single band in SDS-PAGE electrophoresis. The peroxidase had a Mr of 58 000±2000, an isoelectric point of 7.2 and contained an ironprotoporphyrin IX as prosthetic group. The pH optimum was at 5.75 in 100 mM Na acetate buffer using o-dianisidine as substrate. The activation energy was estimated to be 16 kcal mol−1 and 50% inactivation occurred after 60 min at 60°.

Effect of metal substitution in copper amine oxidase from lentil seedlings

Abstract The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the kc value is different from that of native or Cu-fully-reconstituted enzyme, while Km is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism.

Modulation of 6-hydroxydopamine oxidation by various proteins

The spontaneous autoxidation of the neurotoxin 6-hydroxydopamine proceeds by a free radical chain reaction involving the superoxide anion radical and produces the corresponding chromogen 6-hydroxydopamine quinone and hydrogen peroxide. The rate of this reaction is increased in the presence of ceruloplasmin and peroxidase, and reduced by superoxide dismutase, catalase, and DT-diaphorase. We report some explanations of why these proteins may increase or reduce the rate of autoxidation of 6-hydroxydopamine.