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M Medina

Publications and source records attributed to M Medina.

At least 91 records · Page 5Linked to original sources
PubMed1999

Ferredoxin-NADP(+) reductase uses the same site for the interaction with ferredoxin and flavodoxin.

The enzyme ferredoxin-NADP(+) reductase (FNR) forms a 1 : 1 complex with ferredoxin (Fd) or flavodoxin (Fld) that is stabilised by both electrostatic and hydrophobic interactions. The electrostatic interactions occur between acidic residues of the electron transfer (ET) protein and basic residues on the FNR surface. In the present study, several charge-reversal mutants of FNR have been prepared at the proposed site of interaction of the ET protein: R16E, K72E, K75E, K138E, R264E, K290E and K294E. All of these mutants have been assayed for reactivity with Fd and Fld using steady-state and stopped-flow kinetics. Their abilities for complex formation with the ET proteins have also been tested. The data presented here indicate that the mutated residues situated within the FNR FAD-binding domain are more important for achieving maximal ET rates, either with Fd or Fld, than those situated within the NADP(+)-binding domain, and that both ET proteins occupy the same region for the interaction with the reductase. In addition, each individual residue does not appear to participate to the same extent in the different processes with Fd and Fld.

Anabaena↗
PubMed1998

Role of Arg100 and Arg264 from Anabaena PCC 7119 ferredoxin-NADP+ reductase for optimal NADP+ binding and electron transfer.

Previous studies and the crystal structure of Anabaena PCC 7119 FNR suggest that the side chains of Arg100 and Arg264 may be directly involved in the proper NADP+/NADPH orientation for an efficient electron-transfer reaction. Protein engineering on Arg100 and Arg264 from Anabaena PCC 7119 FNR has been carried out to investigate their roles in complex formation and electron transfer to NADP+ and to ferredoxin/flavodoxin. Arg100 has been replaced with an alanine, which removes the positive charge, the long side chain, as well as the ability to form hydrogen bonds, while a charge reversal mutation has been made at Arg264 by replacing it with a glutamic acid. Results with various spectroscopic techniques indicate that the mutated proteins folded properly and that significant protein structural rearrangements did not occur. Both mutants have been kinetically characterized by steady-state as well as fast transient kinetic techniques, and the three-dimensional structure of Arg264Glu FNR has been solved. The results reported herein reveal important conceptual information about the interaction of FNR with its substrates. A critical role is confirmed for the long, positively charged side chain of Arg100. Studies on the Arg264Glu FNR mutant demonstrate that the Arg264 side chain is not critical for the nicotinamide orientation or for nicotinamide interaction with the isoalloxazine FAD moiety. However, this mutant showed altered behavior in its interaction and electron transfer with its protein partners, ferredoxin and flavodoxin.

Alanine↗
PubMed1998

A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3,N4-ethenocytosine and the G/T mismatch.

Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,N6-ethenoadenine (epsilonA) and 3,N4-ethenocytosine (epsilonC) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises epsilonA. The present work is directed toward identifying and purifying the human glycosylase activity releasing epsilonC. HeLa cells were subjected to multiple steps of column chromatography, including two epsilonC-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS/polyacrylamide gel showed that the epsilonC activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G/T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having epsilonC-DNA glycosylase activity. In addition, oligonucleotides containing epsilonC.G or G/T(U), could compete for epsilonC protein binding, further indicating that the epsilonC-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for epsilonC also was observed in a recombinant G/T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF.

Animals↗
PubMed1998

Lys75 of Anabaena ferredoxin-NADP+ reductase is a critical residue for binding ferredoxin and flavodoxin during electron transfer.

Previous studies, and the three-dimensional structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR), indicate that the positive charge of Lys75 might be directly involved in the interaction between FNR and its protein partners, ferredoxin (Fd) and flavodoxin (Fld). To assess this possibility, this residue has been replaced by another positively charged residue, Arg, by two uncharged residues, Gln and Ser, and by a negatively charged residue, Glu. UV-vis absorption, fluorescence, and CD spectroscopies of these FNR mutants (Lys75Arg, Lys75Gln, Lys75Ser, and Lys75Glu) indicate that all the mutated proteins folded properly and that significant protein structural rearrangements did not occur. Steady-state kinetic parameters for these FNR mutants, utilizing the diaphorase activity with DCPIP, indicate that Lys75 is not a critical residue for complex formation and electron transfer (ET) between FNR and NADP+ or NADPH. However, steady-state kinetic activities requiring complex formation and ET between FNR and Fd or Fld were appreciably affected when the positive charge at position of Lys75 was removed, and the ET reaction was not even measurable if a negatively charged residue was placed at this position. These kinetic parameters also suggest that it is complex formation that is affected by mutation. Consistent with this, when dissociation constants (Kd) for FNRox-Fdox (differential spectroscopy) and FNRox-Fdrd (laser flash photolysis) were measured, it was found that neutralization of the positive charge at position 75 increased the Kd values by 50-100-fold, and that no complex formation could be detected upon introduction of a negative charge at this position. Fast transient kinetic studies also corroborated the fact that removal of the positive charge at position 75 of FNR appreciably affects the complex formation process with its protein partners but indicates that ET is still achieved in all the reactions. This study thus clearly establishes the requirement of a positive charge at position Lys75 for complex formation during ET between FNR and its physiological protein partners. The results also suggest that the interaction of this residue with its protein partners is not structurally specific, since Lys75 can still be efficiently substituted by an arginine, but is definitely charge specific.

Amino Acid Sequence↗
PubMed1998

Synergistic effect of nisin and the lactoperoxidase system on Listeria monocytogenes in skim milk.

Nisin added at 10 or 100 IU/ml to ultra-high temperature processed (UHT) skim milk had no effect on counts of Listeria monocytogenes after 24 h at 30 degrees C, whereas addition of the lactoperoxidase system (LPS) resulted in counts of viable cells three log units lower than those of control milk after 24 h at 30 degrees C. Addition of nisin and LPS showed a synergistic effect and resulted in counts up to 5.6 log units lower than the control milk. When the two preservatives were added to actively growing cells of L. monocytogenes in two steps with a 2 h interval, their synergistic effect was enhanced. Counts of L. monocytogenes Ohio after 24 h at 30 degrees C in milk with nisin and LPS added together after 3 h of growth were 5.7 log units lower than the control milk. The difference in counts increased to 7.4 log units if LPS was added after 3 h and nisin after 5 h of growth. Similar but less pronounced effects were observed for the more resistant strain L. monocytogenes Scott A.

Animals↗
PubMed1998

Involvement of glutamic acid 301 in the catalytic mechanism of ferredoxin-NADP+ reductase from Anabaena PCC 7119.

The crystal structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR) suggests that the carboxylate group of Glu301 may be directly involved in the catalytic process of electron and proton transfer between the isoalloxazine moiety of FAD and FNR substrates (NADPH, ferredoxin, and flavodoxin). To assess this possibility, the carboxylate of Glu301 was removed by mutating the residue to an alanine. Various spectroscopic techniques (UV-vis absorption, fluorescence, and CD) indicate that the mutant protein folded properly and that significant protein structural rearrangements did not occur. Additionally, complex formation of the mutant FNR with its substrates was almost unaltered. Nevertheless, no semiquinone formation was seen during photoreduction of Glu301Ala FNR. Furthermore, steady-state activities in which FNR semiquinone formation was required during the electron-transfer processes to ferredoxin were appreciably affected by the mutation. Fast transient kinetic studies corroborated that removal of the carboxylate at position 301 decreases the rate constant approximately 40-fold for the electron transfer process with ferredoxin without appreciably affecting complex formation, and thus interferes with the stabilization of the transition state during electron-transfer between the FAD and the iron-sulfur cluster. Moreover, the mutation also altered the nonspecific reaction of FNR with 5'-deazariboflavin semiquinone, the electron-transfer reactions with flavodoxin, and the reoxidation properties of the enzyme. These results clearly establish Glu301 as a critical residue for electron transfer in FNR.

Anabaena↗
PubMed1998

Expression of Kin, a nuclear protein binding to curved DNA, in mammal and avian brains.

Kin is a nuclear protein which presents cross-immunoreactivity with the bacterial RecA protein and which efficiently binds to curved DNA. This genomic interaction could be implied in DNA repair and illegitimate recombination in eukaryotic cells. Using immunocytochemistry with anti-RecA antibodies, we report the ubiquitous presence of the Kin protein in the CNS of mice and quails. However, some brain structures such as the hippocampal area, the locus coeruleus and Purkinje cells are preferentially immunolabelled and show some homologies between the two species. In conclusion, the expression of the Kin protein is preserved in the phylogeny of the brain of higher vertebrates.

Animals↗
PubMed1998

Inhibitory activity of a nisin-producing starter culture on Listeria innocua in raw ewes milk Manchego cheese.

The inhibitory activity of nisin-producing Lactococcus lactis subsp. lactis ESI 515 on the survival of Listeria innocua during ripening of raw ewes milk Manchego cheese was investigated. After 60 days of ripening, counts of L. innocua in cheese were 4.08 log units lower than the control when Lc. lactis subsp. lactis ESI 515 was used as a single-strain starter. Nisin activity was detected in cheeses manufactured with Lc. lactis subsp. lactis ESI 515 throughout the ripening period.

Animals↗
PubMed1998

Protein-protein interaction in electron transfer reactions: the ferredoxin/flavodoxin/ferredoxin:NADP+ reductase system from Anabaena.

Electron transfer reactions involving protein-protein interactions require the formation of a transient complex which brings together the two redox centres exchanging electrons. This is the case for the flavoprotein ferredoxin:NADP+ reductase (FNR) from the cyanobacterium Anabaena, an enzyme which interacts with ferredoxin in the photosynthetic pathway to receive the electrons required for NADP+ reduction. The reductase shows a concave cavity in its structure into which small proteins such as ferredoxin can fit. Flavodoxin, an FMN-containing protein that is synthesised in cyanobacteria under iron-deficient conditions, plays the same role as ferredoxin in its interaction with FNR in spite of its different structure, size and redox cofactor. There are a number of negatively charged amino acid residues on the surface of ferredoxin and flavodoxin that play a role in the electron transfer reaction with the reductase. Thus far, in only one case has charge replacement of one of the acidic residues produced an increase in the rate of electron transfer, whereas in several other cases a decrease in the rate is observed. In the most dramatic example, replacement of Glu at position 94 of Anabaena ferredoxin results in virtually the complete loss of ability to transfer electrons. Charge-reversal of positively charged amino acid residues in the reductase also produces strong effects on the rate of electron transfer. Several degrees of impairment have been observed, the most significant involving a positively charged Lys at position 75 which appears to be essential for the stability of the complex between the reductase and ferredoxin. The results presented in this paper provide a clear demonstration of the importance of electrostatic interactions on the stability of the transient complex formed during electron transfer by the proteins presently under study.

Amino Acids↗
PubMed1998

Detection of specific bacteriocin-producing lactic acid bacteria by colony hybridization.

A colony hybridization method for detecting lactic acid bacteria encoding specific bacteriocins was developed. Specific PCR-generated probes were used to detect colonies of pediocin PA-1, lactococcin A, enterocin AS-48, nisin A and lacticin 481 producing strains. The probes were shown to be sensitive and specific for sequences belonging to the structural genes of the respective bacteriocins.

Bacteriocins↗
PubMed1998

Enhanced bradycardia induced by beta-adrenoceptor antagonists in rats pretreated with isoniazid.

High doses of isoniazid increase hypotension induced by vasodilators and change the accompanying reflex tachycardia to bradycardia, an interaction attributed to decreased synthesis of brain gamma-aminobutyric acid (GABA). In the present study, the possible enhancement by isoniazid of bradycardia induced by beta-adrenoceptor antagonists was determined in rats anaesthetised with chloralose-urethane. Isoniazid significantly increased bradycardia after propranolol, pindolol, labetalol and atenolol, as well as after clonidine, but not after hexamethonium or carbachol. Enhancement was not observed in rats pretreated with methylatropine or previously vagotomised. These results are compatible with interference by isoniazid with GABAergic inhibition of cardiac parasympathetic tone. Such interference could be exerted centrally, possibly at the nucleus ambiguus, or peripherally at the sinus node.

Adrenergic beta-Antagonists↗
PubMed1998

Involvement of blood serum in amphibian fertilization.

A proteolytic enzyme secreted by the first portion of amphibian oviduct, pars recta, called oviductin in Xenopus laevis, causes ultrastructural alterations on the extracellular matrix of coelomic eggs, turning them susceptible to fertilization. Although great advances have been made in the field of reproduction, the molecular mechanisms responsible for the fusion between the egg and the sperm are yet to be understood. We have recently demonstrated the presence of proteins from pars recta fluid in blood serum and extracellular matrix of coelomic eggs in Bufo arenarum. Here we show, using immunofluorescence procedures, that blood serum components are present in the extracellular matrix of coelomic and pars recta fluid-conditioned eggs in Bufo arenarum. Furthermore, by assessing the neutralizing effect on the conditioning activity of pars recta fluid on coelomic eggs we found that antibodies against pars recta secretions and blood serum inhibited the effect of sperm-lysin on the vitelline envelope of conditioned oocytes and impaired fertilization by sperm. Thus, serum proteins appear to be implicated in the molecular events that lead to amphibian fertilization.

Animals↗
PubMed1997

One- and two-dimensional ESEEM spectroscopy of flavoproteins.

One- and two-dimensional (1D and 2D) electron spin echo envelope modulation (ESEEM) spectroscopy was applied to study the flavin cofactors in the neutral semiquinone states of flavodoxin and ferredoxin-NADP+ reductase (FNR) from the cyanobacterium Anabaena PCC 7119, and the anionic semiquinone state of cholesterol oxidase from Brevibacterium sterolicum. High-resolution crystal structures are available for all these proteins. Three- and 4-pulse ESEEM and hyperfine sublevel correlation spectroscopy (HYSCORE) techniques at X-band were used. HYSCORE spectra showed correlations between transitions caused by interaction of the isoalloxazine unpaired electronic spin present in the semiquinone state with several nitrogen and hydrogen nuclei. Measurements of isotopic labeled samples ([15N]FMN flavodoxin and [2H]flavodoxin) allowed the assignment of all the detected transitions to nuclei belonging to the FMN cofactor group. Interactions of nitrogens in positions 1 and 3 of the isoalloxazine ring were determined to have isotropic hyperfine coupling constants in the 1-2 and 0.5-1 MHz ranges for all the different flavoprotein semiquinones studied. Information about the quadrupolar term of these nuclei was also obtained. An intense correlation in the negative quadrant was detected. It has been associated to the strongly interacting N(10) nucleus. The complete hyperfine term parameters (including the sign) were obtained from detailed analysis of this signal, being the quadrupolar parameter, K, also estimated. Another correlation in the HYSCORE spectra, corresponding to hydrogen bound to the N(5) position in neutral flavin semiquinones, was detected. Its interaction parameters were also determined. This study demonstrates that ESEEM spectroscopy, and in particular the HYSCORE technique, are of particular utility for detecting and assigning nuclear transition frequencies in flavoprotein semiquinones. Moreover, the results reported here are complementary to ENDOR studies, and both techniques together provide an important tool for obtaining information about spin distribution in the flavin ring of flavoproteins in the semiquinone state.

Anabaena↗
PubMed1997

Presenilin 1 interaction in the brain with a novel member of the Armadillo family.

One approach to understanding the function of presenilin 1 (PS1), is to discover those proteins with which it interacts. Evidence for a function in developmental patterning came from C. elegans, in which a PS homologue was identified by screening for suppressors of a mutation in Notch/lin-12, a gene which specifies cell fate. However, this genetic experiment cannot determine which proteins directly interact with PS1. Therefore, we utilized the two hybrid system and confirmatory co-immunoprecipitations to identify a novel catenin, termed delta-catenin, which interacts with PS1 and is principally expressed in brain. The catenins are a gene family related to the Armadillo gene in Drosophila, some of which appear to have dual roles-they are components of cell-cell adherens junctions, and may serve as intermediates in the Wingless (Wg) signaling pathway, which, like Notch/lin-12, is also responsible for a variety of inductive signaling events. In the non-neuronal 293 cell line, PS1 interacted with beta-catenin, the family member with the greatest homology to Armadillo. Wg and Notch interactions are mediated by the Dishevelled gene, which may form a signaling complex with PS1 and Wg pathway intermediates to regulate the function of the Notch/lin-12 gene.

Amino Acid Sequence↗
PubMed1997

Presenilin 1 interaction in the brain with a novel member of the Armadillo family.

One approach to understanding the function of presenilin 1 (PS1), is to discover those proteins with which it interacts. Evidence for a function in developmental patterning came from C. elegans, in which a PS homologue was identified by screening for suppressors of a mutation in Notch/lin-12, a gene which specifies cell fate. However, this genetic experiment cannot determine which proteins directly interact with PS1. Therefore, we utilized the two hybrid system and confirmatory co-immunoprecipitations to identify a novel catenin, termed gamma-catenin, which interacts with PS1 and is principally expressed in brain. The catenins are a gene family related to the Armadillo gene in Drosophila, some of which appear to have dual roles-they are components of cell-cell adherens junctions, and may serve as intermediates in the Wingless (Wg) signaling pathway, which, like Notch/lin-12, is also responsible for a variety of inductive signaling events. In the non-neuronal 293 cell line, PS1 interacted with gamma-catenin, the family member with the greatest homology to Armadillo. Wg and Notch interactions are mediated by the Disheveled gene, which may form a signaling complex with PS1 and Wg pathway intermediates to regulate the function of the Notch/lin-12 gene.

Amino Acid Sequence↗
PubMed1997

Electron spin echo envelope modulation studies of the semiquinone anion radical of cholesterol oxidase from Brevibacterium sterolicum.

The electron spin echo envelope modulation (ESEEM) technique of pulsed EPR spectroscopy was applied to the anionic semiquinone of the cholesterol oxidase flavin cofactor, formed when the enzyme was photoreduced in the presence of 5-deazariboflavin and EDTA. Fourier transforms of the three-pulse ESEEM spectra showed the presence of 14N nuclei magnetically coupled to the paramagnet. In 2H2O buffer the surroundings of the flavin ring were shown to be accessible to solvent exchange, with a deuterium population in close proximity to the paramagnetic centre. Upon binding of the pseudosubstrate, dehydroisoandrosterone, subtle changes were observed in the coupling to nitrogen nuclei, which are interpreted as changes in the electron density distribution of the flavin ring system. The results are discussed in terms of the three-dimensional structure reported for the protein and the flavin ring architecture.

Bacterial Proteins↗