Maria Victoria Rossetti

S-Adenosyl-L-methionine: its effect on aminolevulinate dehydratase and glutathione in acute ethanol intoxication

The effect of disulfiram and S-adenosyl-L-methionine (SAM) administration to acute ethanol intoxicated mice on the hepatic glutathione (GSH) concentration and aminolevulinic and dehydratase (ALA-D) activity was investigated. It was found that both GSH levels and ALA-D activity were decreased, and evidence suggested that the toxic action of ethanol was due to its conversion into acetaldehyde. Administration of SAM reverses the effects of acute alcohol abuse by increasing liver GSH availability. In vitro, hepatic ALA-D activity was not modified by ethanol; instead it was non-competitively inhibited by acetaldehyde. This inhibition was efficiently reversed by GSH and cysteine (CySH). Therefore, a mechanism for the action of ethanol on ALA-D, based on the inhibitory effect of acetaldehyde, is proposed.

Genetic and biochemical studies in Argentinean patients with variegate porphyria

BackgroundA partial deficiency in Protoporphyrinogen oxidase (PPOX) produces the mixed disorder Variegate Porphyria (VP), the second acute porphyria more frequent in Argentina. Identification of patients with an overt VP is absolutely important because treatment depends on an accurate diagnosis but more critical is the identification of asymptomatic relatives to avoid acute attacks which may progress to death.MethodsWe have studied at molecular level 18 new Argentinean patients biochemically diagnosed as VP. PPOX gene was amplified in one or in twelve PCR reactions. All coding exons, flanking intronic and promoter regions were manual or automatically sequenced. For RT-PCR studies RNA was retrotranscripted, amplified and sequenced. PPOX activity in those families carrying a new and uncharacterized mutation was performed.ResultsAll affected individuals harboured mutations in heterozygous state. Nine novel mutations and 3 already reported mutations were identified. Six of the novel mutations were single nucleotide substitutions, 2 were small deletions and one a small insertion. Three single nucleotide substitutions and the insertion were at exon-intron boundaries. Two of the single nucleotide substitutions, c.471G>A and c.807G>A and the insertion (c.388+3insT) were close to the splice donor sites in exons 5, 7 and intron 4 respectively. The other single nucleotide substitution was a transversion in the last base of intron 7, g.3912G>C (c.808-1G>C) so altering the consensus acceptor splice site. However, only in the first case the abnormal band showing the skipping of exon 5 was detected. The other single nucleotide substitutions were transversions: c.101A>T, c.995G>C and c.670 T>G that result in p.E34V, p.G332A and W224G aminoacid substitutions in exons 3, 10 and 7 respectively. Activity measurements indicate that these mutations reduced about 50% PPOX activity and also that they co-segregate with this reduced activity value. Two frameshift mutations, c.133delT and c.925delA, were detected in exons 3 and 9 respectively. The first leads to an early termination signal 22 codons downstream (p.S45fsX67) and the second leads to a stop codon 5 codons downstream (p.I309fsX314). One reported mutation was a missense mutation (p.G232R) and 2 were frameshift mutations: c.1082insC and 1043insT. The last mutation was detected in six new apparently unrelated Argentinean families.ConclusionMolecular analysis in available family members revealed 14 individuals who were silent carriers of VP. Molecular techniques represent the most accurate approach to identify unaffected carriers and to provide accurate genetic counselling for asymptomatic individuals. The initial screening includes the insertion search.

Different porphobilinogen-deaminase forms in wild and mutant strains of Saccharomyces cerevisiae, a possible correlation with its segregants behaviour

1. Different porphobilinogen-deaminase (PBG-D) enzyme forms were found for D 27 and D 27/C6 (HEM R+) strains of Saccharomyces cerevisiae. 2. PBG-D was partially purified and chromatographed on Sephadex G-100 in either the presence or absence of a protease inhibitor. For D 27 only one active peak was observed while for D 27/C6 strain two active peaks were found. 3. A correlation between this differential behaviour and the presence of HEM R+ gene was looked for employing two segregants of one tetrad from D 27 and D 27/C6 mating.

Human red cell porphobilinogen deaminase. a simpler method of purification and some unusual properties

A simpler method for purifying human red cell deaminase, using a mixture of n-butanol and chloroform, which denatures hemoglobin, followed by ammonium sulphate fractionation, heat treatment, Sephadex G-100 and DEAE-cellulose chromatography, yielding a 3400 fold purified enzyme is described. Some properties of purified deaminase were studied. The enzyme seems to have a strict requirement for oxygen, neither PBG consumption nor uroporphyrinogens formation were measured under anaerobiosis. Uroporphyrinogens formation was linear with both protein and time over a wide range of enzyme concentration and up to 2 h. The optimum pH was 7.4 and the mol. wt was 40,000 +/- 4000. The enzyme was heat-stable and increased its activity by heating. Ammonium and hydroxylamine ions inhibited the reaction. K+ and Na+ ions did not greatly affect activity, while most divalent cations tested significantly diminished uroporphyrinogen formation and to a lesser degree PBG consumption. Direct plots of velocity against PBG concentration were hyperbolic, however double-reciprocal plots were non-linear, Hill plots gave an n value of 2 and Eadie plots were bell-shaped, indicating the existence of weakly positive cooperative effect between 2 binding sites for PBG per molecule of deaminase.

Porphyrin biosynthesis in Euglena gracilis—V. Soluble and particulate PBG-ASE

Abstract 1. 1. Porphobilinogenase (PBG-ase) from soluble and particulate fractions of Euglena gracilis was isolated and purified. 2. 2. A highly efficient method for the dissociation of the membrane-bound enzyme using the chaotropic agent SCN is described. 3. 3. The chromatographic behaviour of both soluble and particulate PBG-ase on Sephadex G-100 and Sepharose 4B was quite similar but not identical. 4. 4. Euglena gracilis PBG-ase appears to exist in an equilibrium mixture of two active species of mol. wt 50,000 and 25,000.

Saccharomyces cerevisiae porphobilinogenase: Some physical and kinetic properties

1. Properties of porphobilinogenase (PBGase), the enzyme complex converting porphobilinogen (PBG) into uroporphyrinogens, were studied in a wild strain, D273-10B and a mutant, B231, of Saccharomyces cerevisiae. 2. A well-defined maximum of enzyme activity was observed for the mutant strain after 20 hr of growth; whilst the activity in the wild strain did not vary significantly during growth. 3. Neither PBG consumption nor uroporphyrinogen formation were modified by the presence of air either in the wild or in the mutant strain. 4. In both the wild and mutant strains uroporphyrinogen formation increased linearly with both protein concentration and incubation time. 5. The addition of a mixture of sodium and magnesium salts to the assay system inhibited the enzyme activity of both strains by 50% without modifying the isomer composition. 6. The same optimum pH (7.4) and mol. wt (50,000 +/- 5000) was found for the enzyme from both strains. 7. The enzyme from both the wild and mutant strains shows Michaelis-Menten kinetics when isolated from cells at either the exponential or the stationary phases of growth. Accumulation of porphyrins and delta-aminolevulinic acid occurring during the exponential phase in the mutant strain, did not modify the kinetics.

Functional associations of genetic variants involved in the clinical manifestation of erythropoietic protoporphyria in the Argentinean population

Backgroundu2002 Combined inheritance of genetic variants in ferrochelatase gene (FECH) are implicated in clinical manifestation of Erythropoietic Protoporphyria (EPP).

Characterization of Variegate Porphyria Mutations Using a Minigene Approach

Porphyrias are a group of metabolic diseases that affect the skin and/or nervous system. In 2008, three unrelated patients were diagnosed with variegate porphyria at the CIPYP (Centro de Investigaciones sobre Porfirinas y Porfirias). Sequencing of the protoporphyrinogen oxidase gene, the gene altered in this type of porphyria, revealed three previously undescribed mutations: c.338+3insT, c.807G>A, and c.808-1G>C. As these mutations do not affect the protein sequence, we hypothesized that they might be splicing mutations. RT-PCRs performed on the patients mRNAs showed normal mRNA or no amplification at all. This result indicated that the aberrant spliced transcript is possibly being degraded. In order to establish whether they were responsible or not for the patients disease by causing aberrant splicing, we utilized a minigene approach. We found that the three mutations lead to exon skipping; therefore, the abnormal mRNAs are most likely degraded by a mechanism such as nonsense-mediated decay. In conclusion, these mutations are responsible for the disease because they alter the normal splicing pathway, thus providing a functional explanation for the appearance of disease and highlighting the use of minigene assays to complement transcript analysis.

Delta-aminolevulinate dehydratase from free-living Rhizobium

Abstract 1. 1. δ-Aminolevulinate-dehydratase (ALA-D) from Rhizobium japonicum and Rhizobium meliloti was isolated and some properties were studied. 2. 2. The enzyme from both strains require DTT to maintain full activity and a concentration of about 8 mM is necessary for its maximum expression. Thiol inactivating compounds and heavy metals ions such as Pb2+ and Cd2+ inactivate ALA-D. 3. 3. The enzyme exhibits Michaelis-Menten kinetics and has an apparent Km of 0.095 and 0.1–0.37 mM for Rhizobium japonicum and Rhizobium meliloti respectively. 4. 4. For both strains the pH profiles show a well denned maximum at about 7.2–7.6 and a second broad peak or shoulder in the range of pH 9–10. 5. 5. ALA-D from Rhizobium does not appear to be a heat stable enzyme as it happens to be in other sources.