Papers by Matilde Maiorino
Purification and characterization of phospholipid hydroperoxide glutathione peroxidase from rat testis mitochondrial membranes
Biochimica Et Biophysica Acta-protein Structure and Molecular Enzymology, 1994
Phosphorus Sulfur and Silicon and The Related Elements, 1988
Human glutathione transferases (GSTs) from Alpha (A), Mu (M) and Theta (T) classes exhibited glut... more Human glutathione transferases (GSTs) from Alpha (A), Mu (M) and Theta (T) classes exhibited glutathione peroxidase activity towards phospholipid hydroperoxide. The specific activities are in the order : GST A1-1 GST T1-1 GST M1-1 GST A2-2 GST A4-4. Using a specific and sensitive HPLC method, specific activities towards the phospholipid hydroperoxide, 1-palmitoyl-2-(13-hydroperoxy-cis-9,trans-11octadecadienoyl)--3-phosphatidylcholine (PLPC-OOH) were determined to be in the range of 0.8-20 nmol\min per mg of protein. Two human class Pi (P) enzymes (GST P1-1 with Ile or Val at position 105) displayed no activity towards the phospholipid hydroperoxide. Michaelis-Menten kinetics were followed only for glutathione, whereas there was a linear dependence of rate with PLPC-OOH concentration. Unlike the selenium-
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1989
Human glutathione transferases (GSTs) from Alpha (A), Mu (M) and Theta (T) classes exhibited glut... more Human glutathione transferases (GSTs) from Alpha (A), Mu (M) and Theta (T) classes exhibited glutathione peroxidase activity towards phospholipid hydroperoxide. The specific activities are in the order : GST A1-1 GST T1-1 GST M1-1 GST A2-2 GST A4-4. Using a specific and sensitive HPLC method, specific activities towards the phospholipid hydroperoxide, 1-palmitoyl-2-(13-hydroperoxy-cis-9,trans-11octadecadienoyl)--3-phosphatidylcholine (PLPC-OOH) were determined to be in the range of 0.8-20 nmol\min per mg of protein. Two human class Pi (P) enzymes (GST P1-1 with Ile or Val at position 105) displayed no activity towards the phospholipid hydroperoxide. Michaelis-Menten kinetics were followed only for glutathione, whereas there was a linear dependence of rate with PLPC-OOH concentration. Unlike the selenium-
Selenium
Handbook of Antioxidants, 2001
Oxidative stress, spermatogenesis and fertility
Biological chemistry
Reactive oxygen species production and glutathione depletion in mammalian male germ cells are phy... more Reactive oxygen species production and glutathione depletion in mammalian male germ cells are physiological events that are requisite to the functional maturation and capacitation of spermatozoa. In relation to this oxidative stress, an oxidation of the bulk of protein sulfydryl groups takes place during the final phases of male germ cell maturation. The selenoenzyme phospholipid hydroperoxide glutathione peroxidase catalyzes this reaction, and accounts for both the assembly of the mid-piece of spermatozoa and chromatin condensation. This process highlights the role of H2O2 and selenium in spermatogenesis and provides a mechanism for coupling a 'physiologically controlled' oxidative stress to a specialized phenotypic function.
Measurement of lipid hydroperoxides in human plasma and lipoproteins by kinetic analysis of photon emission
Methods in enzymology, 1999
... Plasma and L ipoproteinsby K inetic A nalysisof Photon Emission By ANTONIO M. PASTORINO, ADRI... more ... Plasma and L ipoproteinsby K inetic A nalysisof Photon Emission By ANTONIO M. PASTORINO, ADRIANA ZAMBURLINI, LucIo ZENNARO, MATILDE ... 13 and Rosen and Rauckman TM suggested a Fenton-like reaction, where hydroperoxides reduce ferric hematin to ferrous ...
Biochimica et biophysica acta, Jan 17, 1995
The lipid hydroperoxide content of isolated, native human plasma lipoproteins, was measured, by t... more The lipid hydroperoxide content of isolated, native human plasma lipoproteins, was measured, by the luminol-based chemiluminescent reaction, using a highly sensitive single photon counting instrument. The reaction was specific for lipid hydroperoxides since the signal completely disappeared after treatment with the selenoperoxidase specific for lipidic substrates. In this analytical procedure the whole kinetic of photon emission induced by lipid hydroperoxides and hemin in the presence of luminol is integrated, taking advantage of the mono-exponential fitting of the decay of photon emission. The addition of a detergent to the reaction mixture improved the precision of the measurements apparently by preventing oxidative chain reactions affecting the shape of the decay of photon emission. The sensitivity of the instrument allowed measurements on samples containing just a few picomoles of hydroperoxides, small enough to minimize the effect of antioxidants and quenchers possibly present...
Biochimica et biophysica acta, Jan 15, 1982
The cell sap from pig liver contains a protein which protects phosphatidylcholine liposomes and b... more The cell sap from pig liver contains a protein which protects phosphatidylcholine liposomes and biomembranes from peroxidative degradation in the presence of glutathione. The activity of this protein has been assayed by measuring the inhibition of aged phosphatidylcholine liposome peroxidation induced by the Fe3+-triethylenetetramine complex. The peroxidation-inhibiting protein from pig liver has been purified 585-fold to homogeneity with overall recovery of activity of 12%. (NH4)2SO4 precipitation, ion-exchange chromatography on DEAE-Sepharose CL-6B and CM23-cellulose, affinity chromatography on glutathione-bromosulfophthalein-Sepharose and gel filtration on Sephadex G-50 were used. Gel filtration and SDS- polyacrylamide gel electrophoresis indicated a molecular weight of approximately 20 000. The protein inhibited peroxidation by Fe3+-triethylenetetramine following a 15 min preincubation of phosphatidylcholine liposomes in the presence of 5mM glutathione or 2-mercapthoethanol. The...
Biochimica et biophysica acta, Jan 6, 1990
Lipid hydroperoxides (LOOHs) in various lipid assemblies are shown to be efficiently reduced and ... more Lipid hydroperoxides (LOOHs) in various lipid assemblies are shown to be efficiently reduced and deactivated by phospholipid hydroperoxide glutathione peroxidase (PHGPX), the second selenoperoxidase to be identified and characterized. Coupled spectrophotometric analyses in the presence of NADPH, glutathione (GSH), glutathione reductase and Triton X-100 indicated that photochemically generated LOOHs in small unilamellar liposomes are substrates for PHGPX, but not for the classical glutathione peroxidase (GPX). PHGPX was found to be reactive with cholesterol hydroperoxides as well as phospholipid hydroperoxides. Kinetic iodometric analyses during GSH/PHGPX treatment of photoperoxidized liposomes indicated a rapid decay of total LOOH to a residual level of 35-40%; addition of Triton X-100 allowed the reaction to go to completion. The non-reactive LOOHs in intact liposomes were shown to be inaccessible groups on the inner membrane face. In the presence of iron and ascorbate, photoperoxi...
Phospholipid hydroperoxide glutathione peroxidase is a monomeric Se-peroxidase highly expressed i... more Phospholipid hydroperoxide glutathione peroxidase is a monomeric Se-peroxidase highly expressed in mammalian male germ cells. Its nuclear form, sperm nuclei glutathione peroxidase (snGPx), has been originally identified in maturating spermatozoa as a transcription product containing an alternative exon within the phospholipid hydroperoxide glutathione peroxidase gene. In this paper, we show that this form is inconstantly detectable in rat spermatozoa where a 20.0 and 25.9 kDa major forms are detected instead. These have been conclusively characterized. The N-terminus sequence of the 20.0 kDa form confirmed that the protein is identical to cytosolic form, suggesting diffusion into the nucleus. The 25.9 kDa protein represented a truncated form of the previously described nuclear snGPx, lacking the basic nuclear localization signal. This protein is present in two forms differing from each other by the presence of an N-terminal methionine. The presence of traces of the larger snGPx form suggests that exhaustive proteolytic processing of the precursor produces the 25.9 kDa enzyme, although the alternate use of a downstream ATG, at least in rodents, could not be unequivocally ruled out.
A subclass of LDL described on the basis of its greater electronegativity and oxidative status is... more A subclass of LDL described on the basis of its greater electronegativity and oxidative status is further charac- terized using a new, highly sensitive single photon counting technique to measure lipid hydroperoxides. We describe in this report that these particles, which we refer to as LDL-, are enriched in lipid peroxides and other peroxidation products as compared to the bulk
Selenium deficiency is known to be as- sociated with male infertility, and the selenoprotein PHGP... more Selenium deficiency is known to be as- sociated with male infertility, and the selenoprotein PHGPx has been shown to increase in rat testis after puberty and to depend on gonadotropin stimulation in hypophysectomized rats (Roveri et al. (1992) J. Biol. Chem. 267, 6142-6146). Exposure of decapsulated whole testis, however, failed to reveal any transcrip- tional activation or inhibition of the
Fertility and Sterility, 2005
Objective: To clarify the role of carnitine supplementation in idiopathic asthenozoospermia and t... more Objective: To clarify the role of carnitine supplementation in idiopathic asthenozoospermia and to look for a rationale for its use in asthenozoospermic patients. Design: Blind clinical study. Setting: Academic. Patient(s): Thirty asthenozoospermic patients divided in two groups according to phospholipid hydroperoxide glutathione peroxidase (PHGPx) levels. Intervention(s): Placebo for 3 months, then oral L-carnitine (2 g/day) for 3 months; semen samples were collected at baseline, after placebo, after carnitine administration, and again after 3 months with no drugs. Main Outcome Measure(s): Evaluation of seminal parameters and determination of seminal PHGPx levels, measured as rescued activity.
Trends in Molecular Medicine, 2002
European Journal of Biochemistry, 1996
The 100 OOOXg supernatant of the parasite platyhelminth Schistosoma mansoni exhibits a glutathion... more The 100 OOOXg supernatant of the parasite platyhelminth Schistosoma mansoni exhibits a glutathione peroxidase activity with the substrate phosphatidylcholine hydroperoxide. Purification yielded a protein of 20 kDa molecular mass both on gel filtration column chromatography and SDS/PAGE, thus suggesting that S. mansoni expresses a protein similar to the mammalian selenoenzyme phospholipid-hydroperoxide glutathione peroxidase. Kinetic analysis and substrate specificity corroborated this assumption, the second-order rate constants for the oxidation of the ground-state enzyme ( k , I) being higher with phosphatidylcholine hydroperoxide than with other peroxide substrates, such as cumene hydroperoxide or H,02, and quantitatively similar to those of mammalian phospholipid-hydroperoxide glutathione peroxidase. Partial sequencing of the protein and selenium measurement by neutron activation analysis established that the purified peroxidase corresponded to the product of the S. mansoni gene previously reported and supposed to encode a selenium-containing glutathione peroxidase [Roche, C., Williams, D. L., Khalife, J., LePresle, T., Capron, A. & Pierce, R. J. (1994) Cloning and characterization of gene encoding Schistosoma mansoni glutathione peroxidase, Gene 138, 149-1521. S. munsoni thus contains a selenoperoxidase sharing molecular mass, catalytic efficiency and substrate specificity with phospholipid-hydroperoxide glutathione peroxidase, dismantling the concept that those enzymes are unique to vertebrate organisms.
PROTEOMICS, 2008
Present proteomic studies increasingly address experimental strategies focused on multiple compar... more Present proteomic studies increasingly address experimental strategies focused on multiple comparisons of proteomic profiles. To accomplish semiautomatic protein separations based on 2-D LC, the Beckman Coulter PF2D has been developed. Here, we present a novel general purpose tool called MPA (multiple peak alignment) able to perform multiple comparisons of proteomic profiles both in a pairwise guided fashion and in a fully automatic mode using a strategy based on dynamic programing and progressive alignment of time series. The tool is available at
Molecular and Cellular Biology, 2005
The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is regarded as the maj... more The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is regarded as the major molecular target of selenodeficiency in rodents, accounting for most of the histopathological and structural abnormalities of testicular tissue and male germ cells. PHGPx exists as a cytosolic form, mitochondrial form, and nuclear form (nPHGPx) predominantly expressed in late spermatids and spermatozoa. Here, we demonstrate that mice with a targeted deletion of the nPHGPx gene were, unlike mice with the full knockout (KO) of PHGPx, not only viable but also, surprisingly, fully fertile. While both morphological analysis of testis and epididymis and sperm parameter measurements did not show any apparent abnormality, toluidine blue and acridine orange stainings of spermatozoa indicated defective chromatin condensation in the KO sperm isolated from the caput epididymis. Furthermore, upon drying and hydrating, KO sperm exhibited a significant proportion of morphologically abnormal heads. Monobromobimane labeling and protein-free thiol titration revealed significantly less extensive oxidation in the cauda epididymis when compared to that in the wild type. We conclude that nPHGPx, by acting as a protein thiol peroxidase in vivo, contributes to the structural stability of sperm chromatin.
An overview of mechanisms of redox signaling
Journal of Molecular and Cellular Cardiology, 2014
A principal characteristic of redox signaling is that it involves an oxidation-reduction reaction... more A principal characteristic of redox signaling is that it involves an oxidation-reduction reaction or covalent adduct formation between the sensor signaling protein and second messenger. Non-redox signaling may involve alteration of the second messenger as in hydrolysis of GTP by G proteins, modification of the signaling protein as in farnesylation, or simple non-covalent binding of an agonist or second messenger. The chemistry of redox signaling is reviewed here. Specifically we have described how among the so-called reactive oxygen species, only hydroperoxides clearly fit the role of a second messenger. Consideration of reaction kinetics and cellular location strongly suggests that for hydroperoxides, particular protein cysteines are the targets and that the requirements for redox signaling is that these cysteines are in microenvironments in which the cysteine is ionized to the thiolate, and a proton can be donated to form a leaving group. The chemistry described here is the same as occurs in the cysteine and selenocysteine peroxidases that are generally considered the primary defense against oxidative stress. But, these same enzymes can also act as the sensors and transducer for signaling. Conditions that would allow specific signaling by peroxynitrite and superoxide are also defined. Signaling by other electrophiles, which includes lipid peroxidation products, quinones formed from polyphenols and other metabolites also involves reaction with specific protein thiolates. Again, kinetics and location are the primary determinants that provide specificity required for physiological signaling although enzymatic catalysis is not likely involved. This article is part of a Special Issue entitled "Redox Signalling in the Cardiovascular System".
Journal of Chromatography B, 2008
The effect of 100 nM sodium selenite supplementation was studied on LNCaP cells by a proteomic ap... more The effect of 100 nM sodium selenite supplementation was studied on LNCaP cells by a proteomic approach, on ProteomeLab TM PF 2D platform. Proteins were separated by liquid phase bi-dimensional chromatography and analyzed by pair-wise alignment of peaks to detect those differentially expressed. Differential expression threshold was set at a twice difference level and proteins matching this criterion were identified by MALDI-TOF and confirmed by ESI-ion trap MS/MS. Not all differentially expressed proteins found by PF 2D could be identified by MS analysis, the sensitivity of which emerging as the limiting factor. Thus, only the most abundant proteins, differently expressed following selenium supplementation, were identified. We positively showed an increase of expression of thioredoxin reductase 1, enolase 1, phosphoglycerate mutase 1, glyceraldehyde-3-phosphate dehydrogenase, heterogeneous nuclear ribonucleoprotein A2/B1, isoform A2, Ras-GTPase-activating protein SH3-domain-binding protein and Keratin 18 and a decrease of expression of peroxiredoxin 1 and heat shock protein 70, protein 8, isoform 1. Results are consistent, at least in part, with the less oxidant environment brought about by the synthesis of Se-dependent peroxidases, keeping low the steady-state concentration of hydrogen peroxide.
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Papers by Matilde Maiorino