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G Battistuzzi

Publications and source records attributed to G Battistuzzi.

At least 37 records · Page 2Linked to original sources
PubMed1995

Cyclic voltammetry and 1H-NMR of Rhodopseudomonas palustris cytochrome c2 pH-dependent conformational states.

The pH-induced protein conformational transitions and changes in the ligation state of the heme iron in cytochrome c2 from Rhodopseudomonas palustris were monitored by electrochemical and spectroscopic measurements. In the pH range 1.5-11, the E degree values (and/or the peak potentials) determined by cyclic voltammetry, the electronic spectra and the hyperfine-shifted 1H-NMR resonances of the protein are sensitive to a number of acid/base equilibria. In particular, four equilibria have been determined for the oxidized protein with pKa values of 2.5, 5.5, 6.6 and 9.0. The lowest pKa most probably involves disruption of both axial heme iron bonds and protein unfolding. The subsequent pKa is associated with a low-pH oxidation of the protein by dioxygen, which is accompanied by a conformational change. The equilibrium with an apparent pKa of 6.6 modulates the E degree values without determining any detectable spectral change and most likely involves the acid/base equilibrium of an histidine residue in close vicinity of the heme (possibly His53). Finally, the alkaline ionization is due to the replacement of the methionine axially bound to the heme iron with a stronger (most probably N-donor) ligand. The reduced alkaline form is unstable and spontaneously converts to the neutral reduced form with a kinetic constant of 0.98 s-1 at pH 9.2.

Cytochrome c Group↗
PubMed1995

Magnetic resonance of Fe-S clusters: isolation and characterization of a 7Fe ferredoxin from Rhodopseudomonas palustris.

A novel iron-sulfur protein from the photosynthetic purple bacterium Rhodopseudomonas palustris was purified to homogeneity and identified as a ferredoxin on the basis of its physicochemical properties. Based on the uv/vis spectrum, iron quantitation, cyclic voltammetry, EPR, and 1H NMR data, the ferredoxin is found to contain two iron-sulfur clusters, one [3Fe-4S] and one [4Fe-4S], which places this protein in the class of 7Fe ferredoxins. The voltammetric peak potentials of the two clusters are -0.260 and -0.560 V at pH 8.0. The molecular mass around 19 kDa makes this protein the heaviest known in this class. This paper further demonstrates the diagnostic power of magnetic resonance spectroscopies in recognition of the two types of clusters in iron-sulfur proteins.

Electrochemistry↗
PubMed1995

Metal-induced conformational heterogeneity of transferrins: a spectroscopic study of indium(III) and other metal(III)-substituted transferrins.

The conformation in solution of three different metal(III)-transferrins, namely aluminum(III), gallium(III) and indium(III) transferrin, was investigated by absorption, CD, 1H NMR and 13C NMR spectroscopies. The formation of the respective metal-transferrin complexes and the characteristic 2:1 metal-to-protein binding stoichiometry were unambiguously demonstrated, in all cases, through UV difference studies. The 13C NMR spectra of these metallotransferrins in the carbonyl region are very similar to one another pointing out that the arrangement of the synergistic anion in the binding site must be essentially the same. However, the CD spectra in the near UV (aromatic region) reveal the occurrence of significant differences between indium transferrin, on one side, and the other two derivatives, on the other. Also, the 1H NMR spectra exhibit a number of different features suggesting the occurrence of metal-induced conformational heterogeneity around the metal sites. Such metal-induced conformational heterogeneity probably affects the transferrin-receptor recognition process, resulting in a different metabolic fate of these metals in the organisms.

Carbon Isotopes↗
PubMed1994

Influence of surface charges on redox properties in high potential iron-sulfur proteins.

The pH-dependence of the reduction potential determined through differential pulse voltammetry for the high potential iron sulfur proteins (HiPIP) from R. globiformis, C. vinosum, R. gelatinosus, E. vacuolata (I and II), E. halophila (I and II) is reported. A decrease in reduction potential with pH is invariably observed in the pH range where deprotonation of the imidazolium nitrogen of histidine residue(s) occurs. No pH dependence is observed for the only protein lacking histidines. It appears that surface charges like the His imidazolium groups are capable of influencing the reduction potential despite the known quencing of the electrostatic interactions due to solvent effects.

Bacterial Proteins↗
PubMed1992

Fe3+ binding to ovotransferrin in the presence of alpha-amino acids.

The ability of L-alpha-amino acids as synergistic anions for iron binding to ovotransferrin was investigated through electronic spectroscopy. Glycine and glutamic acid were found to form by far the most stable ternary Fe(3+)-ovotransferrin-amino acid complexes. Less stable adducts were formed by amino acids with a hydroxy, amide or sulphur-containing group in the side chain, while the complexes with leucine, isoleucine, valine, lysine, arginine, tyrosine and tryptophan failed to form. Evidence is obtained that the synergistic effectiveness of the H2N-CH-COO- moiety is determined not only by the isoelectric point of the amino acid and the steric hindrance of its side chain, but a significant role is also played by interactions of the side chain itself with residues in the metal binding domains. Zn2+, Cd2+ and Co2+ are found to bind to ovotransferrin in the presence of glycine. 113Cd-NMR spectra on the Cd-derivative indicate that, according to the interlocking-sites model, the amino group of glycine directly binds to the metal ion.

Amino Acids↗
PubMed1990

Genetic heterogeneity at the glucose-6-phosphate dehydrogenase locus in southern Italy: a study on a population from the Matera district.

Glucose-6-phosphate dehydrogenase (G6PD) has been analyzed by gel electrophoresis and by quantitative assay in an unselected sample of 1524 schoolboys from the province of Matera (Lucania) in southern Italy. We have identified 43 subjects with a G6PD variant. Of these, 31 had severe G6PD deficiency, nine had mild to moderate deficiency, and three had a non-deficient electrophoretic variant. The overall rate of G6PD deficiency was 2.6%. The frequency of G6PD deficiency, ranging from 7.2% on the Ionian Coast to zero on the eastern side of the Lucanian Apennines, appears to be inversely related to the distance of each town examined from the Ionian Coast, suggesting that this geographic distribution may reflect, at least in part, gene flow from Greek settlers. Biochemical characterization has shown that most of the G6PD deficiency in this population is accounted for by G6PD Mediterranean. In addition, we have found several examples of two other known polymorphic variants (G6PD Cagliari and G6PD A-); three new polymorphic variants, G6PD Metaponto (class III), G6PD Montalbano (class III), and G6PD Pisticci (class IV); and two sporadic variants, G6PD Tursi (class III) and G6PD Ferrandina (class II). These data provide further evidence for the marked genetic heterogeneity of G6PD deficiency within a relatively narrow geographic area and they prove the presence in the Italian peninsula of a gene (GdA-) regarded as characteristically African.

Child↗
PubMed1990

Common glucose-6-phosphate dehydrogenase (G6PD) variants from the Italian population: biochemical and molecular characterization.

By biochemical characterization of glucose-6-phosphate dehydrogenase (G6PD) from the red cells of seventeen subjects of the population of Matera (Southern Italy) we have identified six genetically determined common variants. Among these, G6PD Metaponto and G6PD A(-) Matera had been already fully characterized. We have now found that A(-) Matera is genetically heterogeneous since one of two subjects examined had the two mutations at codons 68 and 126 characteristic of a typical A(-) variant, while the other subject had only the codon 126 mutation. G6PD Pisticci and G6PD Tursi are two new variants whose molecular lesion is not yet known. G6PD Cagliari-like has biochemical characteristics reminiscent of G6PD Cagliari, isolated in Sardinia, and was found to have the same nucleotide substitution as G6PD Mediterranean. G6PD Montalbano is a new variant, with nearly normal properties, due to a G----A transition which causes an Arg----His amino acid replacement at position 285.

Alleles↗
PubMed1989

Common and rare genetic variants of human red blood cell enzymes in Italy.

In the present paper we report on new data of the frequency of common and rare variants in the Italian population for ADA, AK-1, 6-PGD, EsA, EsB, EsD, PGM-1, PGM-2, SOD-A, AcP, GPT, and PGI. Moreover we present a comprehensive review of the available data on the electrophoretic variants of red cell enzymes in Italians. We find a considerable degree of genetic heterogeneity between the various populations living in the Peninsula and between the population of the Peninsula and of Sardinia. We also find that the estimates of the average heterozygosity are considerably smaller for the population of Sardinia as compared to Peninsula and Sicily. Finally, we report on the occurrence of several uncommon enzyme variants, which overall frequency is very similar to previously reported estimates for North European populations (Harris et al. 1974).

Acid Phosphatase↗
PubMed1988

Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia.

Glucose-6-phosphate dehydrogenase (G6PD; EC 1.1.1.49) deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. We have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A we have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. In one of the commonest, G6PD Mediterranean, which is associated with favism among other clinical manifestations, a single amino acid replacement was found (serine----phenylalanine): it must be responsible for the decreased stability and the reduced catalytic efficiency of this enzyme. Single point mutations were also found in G6PD Metaponto (Southern Italy) and in G6PD Ilesha (Nigeria), which are asymptomatic, and in G6PD Chatham, which was observed in an Indian boy with neonatal jaundice. In G6PD "Matera," which is now known to be the same as G6PD A-, two separate point mutations were found, one of which is the same as in G6PD A. In G6PD Santiago, a de novo mutation (glycine----arginine) is associated with severe chronic hemolytic anemia. The mutations observed show a striking predominance of C----T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency.

Amino Acid Sequence↗
PubMed1985

Genetic heterogeneity at the glucose-6-phosphate dehydrogenase locus in southern Italy: a study on the population of Naples.

Glucose-6-phosphate dehydrogenase (G6PD) electrophoretic phenotype was determined in red cells from 979 male subjects born in Naples (Southern Italy). In 0.7% of the cases no activity could be detected in haemolysates, while in 1.3% of the cases G6PD activity was approximately 20% of normal and electrophoretic mobility was altered. Moreover in two subjects a G6PD with altered mobility and normal activity was shown. G6PD was characterized in 10 subjects with variant phenotype. We conclude that the G6PD(-) phenotype in the population of Naples consists of at least six different G6PD variants associated with mild deficiency and at least one, G6PD Mediterranean, associated with severe deficiency.

Erythrocytes↗
PubMed1985

Tissue-specific levels of human glucose-6-phosphate dehydrogenase correlate with methylation of specific sites at the 3' end of the gene.

Glucose-6-phosphate dehydrogenase (G6PD) is a ubiquitous enzyme that supplies the cell with NADPH required for a variety of reductive reactions and biosynthetic processes. Therefore, the gene G6PD, located in mammals on the X chromosome, that specifies G6PD can be regarded as a typical housekeeping gene. We have investigated the expression of human G6PD in eight different fetal and adult tissues by determining the level of enzyme activity, the level of G6PD mRNA, and the methylation pattern of the 3' end of the gene, for which we have nucleic acid probes. By combining sequence information with results of Southern blot analysis of DNA samples digested with the methylation-sensitive restriction enzyme Hpa II, we have identified five specific sites that are unmethylated in all tissues examined, a number of sites that are uniformly methylated, and a number of sites that are sometimes methylated. A subset of Hpa II sites, designated on our restriction map as H37-H55, exhibit positive correlation between degree of methylation, level of mRNA, and level of G6PD activity. A comparison of these methylation patterns with those we previously have observed in the G6PD gene on the inactive X chromosome [Toniolo, D., D'Urso, M., Martini, G., Persico, M.G., Tufano, V., Battistuzzi, G. & Luzzatto, L. (1984) EMBO J. 3, 1987-1995] indicates that different sites are associated with X-inactivation and with the regulation of G6PD on the active X chromosome. We conclude that this housekeeping gene is subject to tissue-specific transcriptional regulation, which in turn correlates with methylation of specific sites located at and near the 3' end of the gene.

Gene Expression Regulation↗
PubMed1984

Specific methylation pattern at the 3' end of the human housekeeping gene for glucose 6-phosphate dehydrogenase.

During detailed restriction enzyme mapping of the human X-linked gene Gd, specifying the enzyme glucose 6-phosphate dehydrogenase (G6PD), we have observed the presence, over a 14-kb DNA region spanning across the 3' end of the G6PD transcript, of a large number of methylatable sites. These include 60 HpaII sites, 13 SmaI sites, 22 AvaI sites and 46 HhaI sites. In male leukocyte DNA the majority of HpaII sites are resistant to digestion, indicating that they are in the Cm5CGG form. However, a few sites are found reproducibly unmethylated in 24 samples analyzed. By double and triple digestions we have mapped five unmethylated sites, four of which are within the gene transcript and one distal to the end of transcription. We have also identified a number of sites which are fully methylated, whereas for others the methylation status could not be positively assessed. Thus, in a housekeeping gene expressed in leukocytes, the 3' end is extensively methylated, but some specific sites are unmethylated. In female leukocyte DNA, we found that all sites methylated in males were also methylated. However, of the five sites that are unmethylated in males two are partly methylated in females. This additional site-specific methylation involves approximately 50% of the female leukocyte DNA, and we show evidence that it is associated with the inactive X-chromosome.

Base Sequence↗
PubMed1984

Partial purification and characterization of the messenger RNA for human glucose-6-phosphate dehydrogenase.

Glucose-6-phosphate dehydrogenase (G6PD) is a household enzyme that accounts, in many cells, for about 0.03% of cellular protein. We have developed an assay for G6PD-specific mRNA based on in vitro translation of RNA from human fibroblasts and immunoprecipitation of the translation products with an anti-G6PD antiserum. By making use of this assay, G6PD mRNA has been purified 50 to 100-fold in three steps. We estimate that the mRNA encoding G6PD constitutes less than 0.02% of total poly(A)+ RNA in human fibroblasts. The size of the G6PD mRNA has been established in denaturing conditions as being in the range between 2800 and 3200 nucleotides. This has been confirmed by Northern blot analysis. Since the G6PD coding sequence is estimated to be about 1491 nucleotides, the G6PD mRNA has long untranslated sequences, most of which is at the 3' end and which may be heterogeneous in length. The sequence of the last 608 nucleotides of this mRNA has been determined.

Cells, Cultured↗
PubMed1983

A new glucose 6-phosphate dehydrogenase variant (G-6-PD Verona) in a patient with myelodysplastic syndrome.

A 67-year-old woman investigated because of 'myelodysplastic syndrome' was found to have a 4-fold increase in G-6-PD activity in her erythrocytes. The enzyme was partially purified and characterized. On grounds of: (a) reduced electrophoretic mobility, (b) abnormal cathodic band(s) in isoelectrofocusing, (c) increased Michaelis constant for glucose 6-phosphate, (d) abnormal thermostability, and (e) abnormal interaction with the ligand NADPH, we conclude that this is a new structural variant which we designate G-6-PD Verona. G-6-PD Verona was the sole apparent source of G-6-PD activity in the patient's erythrocytes; by contrast, the patient's fibroblasts had only normal G-6-PH (type B). The patient's haematological course terminated into acute myeloid leukaemia. We believe G-6-PD Verona was the result of a somatic mutation in an X-chromosome which took place in a haemopoietic cell clone which subsequently underwent neoplastic transformation.

Aged↗
PubMed1981

Nonrandom patterns of codon usage and of nucleotide substitutions in human alpha- and beta-globin genes: an evolutionary strategy reducing the rate of mutations with drastic effects?

Nucleotide substitutions within a structural gene can cause two principal "drastic" phenotypic effects at the protein level: translatable leads to untranslatable and nonpolar hydrophobic in equilibrium hydrophilic amino acid substitutions. The sequence of nucleotides in the structural human alpha- and beta-globin genes and their variants were examined to determine whether codon usage, patterns of nucleotide substitutions, or both, reduced the relative and absolute rates of these unfavorable mutations. Based on translation of abnormal hemoglobins, it is likely that all 61 nontermination codons are potentially translatable, though only 47 are normally used. Moreover, codons that can mutate to a termination codon are never used whenever the corresponding amino acid is specified also by triplets that cannot mutate to termination by a single-step mutation. Thus, the number of opportunities to mutate to an untranslatable codon is reduced to the minimum compatible with the amino acid composition of these chains. The relative rates of U in equilibrium non-U substitutions were much lower than those of other substitutions. Because U residues must be involved in most termination mutations and in all nonpolar hydrophobic in equilibrium hydrophilic amino acid substitutions, there is a considerable reduction of mutational events, causing drastic phenotypic effects. These findings are likely to be the end result of evolutionary selection by yet unknown mechanisms.

Base Sequence↗
PubMed1981

Activity of adenosine deaminase allelic forms in intact erythrocytes and in lymphocytes.

The distribution of ADA activity has been studied for ADA 1 and ADA 2-1 individuals in intact and lysed erythrocytes and in lymphocyte lysates. ADA activity in intact RBC is 20-30% lower than in lysates, in lymphocytes it is 10 times higher. The difference between ADA 1 and ADA 2-1 mean enzyme activities which we previously described in RBC lysates has been found also in intact RBC and in lymphocyte lysates.

Adenosine Deaminase↗