Biomedical subjects

Francisco García-Carmona

Publications and source records attributed to Francisco García-Carmona.

At least 19 recordsLinked to original sources

PubMed2006

A kinetic study of a ternary cycle between adenine nucleotides.

In the present paper, a kinetic study is made of the behavior of a moiety-conserved ternary cycle between the adenine nucleotides. The system contains the enzymes S-acetyl coenzyme A synthetase, adenylate kinase and pyruvate kinase, and converts ATP into AMP, then into ADP and finally back to ATP. L-Lactate dehydrogenase is added to the system to enable continuous monitoring of the progress of the reaction. The cycle cannot work when the only recycling substrate in the reaction medium is AMP. A mathematical model is proposed whose kinetic behavior has been analyzed both numerically by integration of the nonlinear differential equations describing the kinetics of the reactions involved, and analytically under steady-state conditions, with good agreement with the experimental results being obtained. The data obtained showed that there is a threshold value of the S-acetyl coenzyme A synthetase/adenylate kinase ratio, above which the cycle stops because all the recycling substrate has been accumulated as AMP, never reaching the steady state. In addition, the concept of adenylate energy charge has been applied to the system, obtaining the enabled values of the rate constants for a fixed adenylate energy charge value and vice versa.

Adenine Nucleotides↗

PubMed2005

Characterization of the activity of tyrosinase on betaxanthins derived from (R)-amino acids.

The activity of tyrosinase (EC 1.14.18.1) on selected (R)-betaxanthins is characterized in depth, demonstrating that the activity of the enzyme is not restricted to betaxanthins derived from (S)-amino acids. Conversion of (R)-tyrosine-betaxanthin [(R)-portulacaxanthin II] to the pigment (R)-dopaxanthin and its further oxidation to a series of products is described. Compound identity was studied by high performance liquid chromatography and electrospray ionization-mass spectrometry. The reaction rate on the (R)-isomer of dopaxanthin is 1.9-fold lower than that obtained for the (S)-isomer in previous studies. Tyrosinase showed stereospecificity in its affinity toward betaxanthins. The characterization of the activity of tyrosinase on (R)-betaxanthins reinforces the role of the enzyme in the biosynthetic scheme of betalains.

Amino Acids↗

PubMed2005

Betaxanthins as pigments responsible for visible fluorescence in flowers.

Betalains are water-soluble nitrogen-containing pigments present in flowers and fruits of plants of the order Caryophyllales, where they replace anthocyanins. This article describes how flowers containing yellow betaxanthins are fluorescent. Betaxanthins exhibit spectra with excitation maxima between 463 nm and 474 nm and emission maxima between 509 nm and 512 nm. Thus, betaxanthins are able to absorb blue light and emit green light. Relations between fluorescence and the structural properties of the pigments are discussed. For the first time, pictures of flowers naturally emitting light are presented. Yellow flowers of the ornamental plant Portulaca grandiflora were chosen as a model for the studies in fluorescence due to the existence of the white phenotype, which was used as a control. Studies were also performed in Lampranthus productus flowers, which contain dopaxanthin as a single pigment. The visible fluorescence of betaxanthins inside the petal cells was detected in a confocal microscope after laser excitation.

Aizoaceae↗

PubMed2005

Oxidation of the flavonoid eriodictyol by tyrosinase.

A pathway is proposed for the oxidation of the flavonoid eriodictyol by mushroom tyrosinase. In it, the enzymatic oxidation of eriodictyol leads to the formation of eriodictyol-o-quinone, which undergoes the nucleophilic attack of another eriodictyol unit to yield a dimer. This dimer is then oxidized by the eriodictyol-o-quinone. The reaction was followed by recording the time course of formation of this second o-quinone at 475 nm. Progress curves at this wavelength showed the appearance of a lag, the length of which varied with enzyme and substrate concentrations, and which must have been caused by the chemical reactions taking place after the enzymatic reaction. When eriodictyol oxidation was studied in the presence of 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH), which competes with the substrate in the reaction with eriodictyol-o-quinone, the lag disappeared. The kinetic parameters were similar with and without MBTH. Eriodictyol oxidation was inhibited by tropolone, which behaved as a slow-binding inhibitor.

Enzyme Inhibitors↗

PubMed2005

Botany: floral fluorescence effect.

The way flowers appear to insects is crucial for pollination. Here we describe an internal light-filtering effect in the flowers of Mirabilis jalapa, in which the visible fluorescence emitted by one pigment, a yellow betaxanthin, is absorbed by another, a violet betacyanin, to create a contrasting fluorescent pattern on the flower's petals. This finding opens up new possibilities for pollinator perception as fluorescence has not previously been considered as a potential signal in flowers.

Animals↗

PubMed2005

Differential activation of a latent polyphenol oxidase mediated by sodium dodecyl sulfate.

A kinetic study of the activity of soluble and membrane-bound latent polyphenol oxidase (PPO) extracted from beet root (Beta vulgaris) was carried out. For the first time, two types of behavior (hyperbolic and sigmoid) are reported in the same enzyme for PPO activation by the surfactant sodium dodecyl sulfate (SDS), depending on substrate nature. A kinetic model based on cooperative systems is developed to describe the activation effect of SDS, enabling the determination of the number of surfactant molecules binding to the enzyme in the activation process. The results indicate that the active site of the enzyme is not affected by SDS and that a stepwise conformational change favors the access of hydrophobic substrates compared to hydrophilic ones. Differential activation of PPO mediated by SDS may be of relevance in the control of PPO activity since the enzyme is able to express activity toward a specific substrate while remaining latent to others.

Beta vulgaris↗

PubMed2005

Kinetic properties of lipoxygenase from desert truffle (Terfezia claveryi Chatin) ascocarps: effect of inhibitors and activators.

There is very little information available on the kinetic characteristics of fungal lipoxygenases (LOXs) because most data on the mechanism of this enzyme concern soybean LOX. In this paper, the kinetic properties of LOX from Terfezia claveryi Chatin ascocarps were studied for the first time. The enzyme did not show the "substrate aggregation-dependent activity" described for other LOXs and presented a K(m) for linoleic acid of 41 microM at pH 7.0. The effect of different inhibitors was also studied. The enzyme presented the characteristic lag phase of other LOXs, and the influence of different factors on its duration was analyzed. The lag period was reduced not only by the product of the reaction (13-HPOD) but also by 9-HPOD. Calculation of the activation constant is proposed for the first time as a useful tool for the characterization of LOX because this method makes it possible to quantify the effectiveness of different hydroperoxides as LOX activators. The activation constants obtained were 0.3 and 6.4 microM for 13- and 9-HPOD, respectively; thus, the product of the reaction was approximately 21-fold more effective than 9-HPOD as a T. claveryi LOX activator.

Ascomycota↗

PubMed2005

Characterization and histochemical localization of nonspecific esterase from ascocarps of desert truffle (Terfezia claveryi Chatin).

An esterase activity from Terfezia claveryi Chatin ascocarps, a mycorrhizal hypogeous fungus, is described for the first time. The enzyme was partially purified using phase partitioning in Triton X-114 (TX-114), achieving a reduction of 87% in the triglyceride content and the removal of 63% of phenols. The enzyme showed maximum activity toward short-chain p-nitrophenyl esters, and no interfacial activation was observed, indicating that the enzyme responsible for this activity is an esterase and not a lipase. This esterase presented its maximum activity at pH 7.4 and 60 degrees C. The values obtained for Km at pH 7.4 were 0.3 mM for p-nitrophenyl butyrate and 0.6 mM for p-nitrophenyl acetate with catalytic efficiencies (Vmax/Km) of 0.23 and 0.32, respectively. T. claveryi esterase was inhibited by phenylboric acid, indicating that serine residues were involved in the enzyme activity. This activity was localized only in the hypothecium and was absent from the peridium and gleba.

Ascomycota↗

PubMed2005

Characterization of the monophenolase activity of tyrosinase on betaxanthins: the tyramine-betaxanthin/dopamine-betaxanthin pair.

Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is the key enzyme responsible for melanin biosynthesis and for the enzymatic browning of fruits and vegetables. Although the function of tyrosinase in the secondary metabolism of plants remains unclear, it has been proposed that the enzyme plays a role in the betalain biosynthetic pathway. Betalains are an important class of water-soluble pigments, characteristic of plants belonging to the order Caryophyllales. In the present work, the betaxanthins tyramine-betaxanthin (miraxanthin III) and dopamine-betaxanthin (miraxanthin V) are reported as new natural substrates for tyrosinase. The result of the diphenolase activity of the enzyme on dopamine-betaxanthin was a series of products identified by HPLC and ESI-MS as quinone-derivatives. Data indicate that dopamine-betaxanthin-quinone is obtained and evolves to more stable species by intramolecular cyclization. The kinetic parameters evaluated for the diphenolase activity were V(m) = 74.4 microM min(-1), K(m) = 94.7 microM. Monophenolase activity on tyramine-betaxanthin yielded the same compounds in the absence of a reducing agent, but when ascorbic acid was present enzymatic conversion to dopamine-betaxanthin could be found. For the first time, kinetic characterization of the monophenolase activity of tyrosinase on betaxanthins is provided (V(m) = 10.4 microM min(-1) and K(m) = 126.9 microM) and a lag period is described and analyzed according to the mechanism of action of the enzyme. The high affinity shown by tyrosinase for these substrates may be indicative of a previously unconsidered physiological role in betalain metabolism. A possible mechanism for the formation of 2-descarboxy-betacyanins from tyramine-betaxanthin by tyrosinase is also discussed.

Agaricales↗

PubMed2005

A novel method using high-performance liquid chromatography with fluorescence detection for the determination of betaxanthins.

Betaxanthins are natural water-soluble yellow pigments present in plants of the order Caryophyllales. The native fluorescence of these compounds is extensively characterized in this work, with study of the fluorescent properties of 14 different betaxanthins. All the species showed a similar behavior, with excitation maxima between 463 and 475 nm and emission maxima between 506 and 515 nm. Thus, betaxanthins absorb light corresponding to the blue color and emit visible green light. Similarities in excitation and emission spectra point to the responsibility of betalamic acid in fluorescence. The influence of the amine moiety is discussed. For the first time fluorescent properties of betaxanthins are applied to the detection of these pigments after separation by high-performance liquid chromatography. Wavelengths used were 460 nm for excitation and 510 nm for emission, which were suitable for detecting the native fluorescence of all the pigments assayed. Calibration was performed in each case and it exhibited linearity within the range considered, at least 20 microM. The lowest detection limit was 100 nM, corresponding to betaxanthins derived from methionine sulfoxide and leucine. Fluorescence detection was applied to the quantification of betaxanthins present in Carpobrotus acinaciformis. The present work opens up new possibilities for the analysis of betaxanthins by improving existing protocols through fluorescence detection.

Betacyanins↗

PubMed2005

Purification and partial characterization of lipoxygenase from desert truffle (Terfezia claveryi Chatin) ascocarps.

A lipoxygenase from Terfezia claveryi Chatin ascocarp, a mycorrhizal hypogeous fungus, is described for the first time. The higher proportion of PUFA in T. claveryi ascocarps makes lipid rancidity the main factor limiting its storage life. Thus, the studies on LOX from T. claveryi are important because this enzyme, among other roles, may be involved in an alteration of lipids leading to consumer rejection. The enzyme has been purified to apparent homogeneity by phase partitioning in the presence of Triton X-114, followed by two steps of cation-exchange chromatography. The purified T. claveryi LOX preparation consisted of a single major band with an apparent molecular mass of 66 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymic activity exhibited a strong specificity toward linoleic and linolenic acids as substrates, while only 32% activity was observed using gamma-linolenic acid. The pH optimum of this enzyme was pH 7.0. When the enzyme reacted with linoleic acid, it produced a single peak, which comigrated with standard 13-hydroperoxy-octadecadienoic acid; 13-hydroperoxy-octadecatrienoic acid was produced during the reaction with linolenic acid.

Ascomycota↗

PubMed2005

Betaxanthins as substrates for tyrosinase. An approach to the role of tyrosinase in the biosynthetic pathway of betalains.

Tyrosinase or polyphenol oxidase (EC 1.14.18.1) is the key enzyme in melanin biosynthesis and in the enzymatic browning of fruits and vegetables. The role of tyrosinase in the secondary metabolism of plants still remains unclear, but its implication in betalain biosynthesis has been proposed. Betalains are an important class of water-soluble pigments, characteristic of plants belonging to the order Caryophyllales. In this article, the betaxanthins, tyrosine-betaxanthin (portulacaxanthin II) and dopaxanthin, are reported to be physiological substrates for tyrosinase. The direct activity of tyrosinase on selected betaxanthins is characterized in depth, and conversion of tyrosine-betaxanthin to dopaxanthin and its further oxidation to a series of compounds are described. Identity of the reaction products was studied by high-performance liquid chromatography and electrospray ionization-mass spectrometry. Masses determined for the reaction products were the same in all cases, 389 m/z ([M + H]+) and equal to that determined for betanidin. Data indicate that dopaxanthin-quinone is obtained and evolves to more stable species by intramolecular cyclization. Kinetic parameters for tyrosinase acting on dopaxanthin were evaluated, showing a high affinity for this substrate (K(m) = 84.3 microM). The biosynthetic scheme of betalains is reviewed and a branch is proposed based on the description of physiological substrates for tyrosinase. Lampranthus productus, Glottiphylum oligocarpum, and Glottiphylum pigmaeum are described as sources of stereopure (2S/S)-dopaxanthin.

Betalains↗

PubMed2005

A kinetic study of p-cresol oxidation by quince fruit polyphenol oxidase.

The monophenolase activity of quince pulp polyphenol oxidase was characterized by extracting samples using a combination of a two-phase partition step in Triton X-114, followed by a PEG 8000/phosphate partition step, and a final ammonium sulfate fractionation between 30 and 75%. The purification method avoids the loss of cresolase activity described in another quince pulp polyphenol oxidase. The activity was characterized by a lag period, whose duration depended on the substrate concentration, the pH, and the presence of catalytic amounts of o-diphenol. By increasing the concentration of o-diphenols, it was possible to evaluate the enzyme activation constant, K(act), which showed a value of 4.5 microM for 4-methylcatechol. A general kinetic mechanism for this enzyme is used to explain the loss of activity that normally occurs during quince pulp polyphenol oxidase purification.

Catechol Oxidase↗

PubMed2005

Lipase-catalyzed preparation of S-propranolol in presence of hydroxypropyl beta-cyclodextrins.

A simple method for the preparation of S-propranolol catalyzed by a Rhizopus niveus lipase in an aqueous medium is described. Hydroxypropyl-beta-cyclodextrin was used for the first time to increase the solubility of (R,S)-O-butyryl propranolol thus permitting the reaction to be carried out in water. The formation of an inclusion complex between (R,S)-O-butyryl propranolol and hydroxypropyl-beta-cyclodextrin was studied and a stoichiometry of 1:1 was determined. The influences of the hydroxypropyl-beta-cyclodextrin concentration, pH and percentage of ethanol on the enzymatic activity were also investigated. Under the conditions presented in this paper, values of ee(s) of 90% and E=48 were obtained.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed2005

Evidence for a common regulation in the activation of a polyphenol oxidase by trypsin and sodium dodecyl sulfate.

Polyphenol oxidase (PPO) was extracted from beet root, in both soluble and membrane fractions, and in both cases the enzyme was in a latent state. PPO from the membrane fraction showed no diphenolase activity unless it was activated by trypsin or sodium dodecyl sulfate (SDS). The kinetics of the activation process of latent PPO by trypsin was studied and the specific rate constant of active PPO formation, k 3 , showed a value of 0.03 s(-1). The protease-activated form showed a pH optimum (6.5) and kinetic properties identical to those of the SDS-activated enzyme. Evidence is provided for the existence of a common peptide responsible for the regulation of the activity of the enzyme by both proteolysis and SDS detergent. Formation of the active proteolyzate was followed by spectroscopic measurements, Western blotting and partially denaturing SDS-PAGE.

Beta vulgaris↗

PubMed2004

Purification and characterization of a latent polyphenol oxidase from beet root (Beta vulgaris L.).

Polyphenol oxidase (PPO) has been extracted from beet root, in both soluble and membrane fractions. In both cases, the enzyme was in its latent state, and it was activated by sodium dodecyl sulfate. PPO was purified to apparent homogeneity. The soluble PPO purification was achieved by hydrophobic interaction chromatography and gel filtration chromatography, with apparent molecular mass of 55 kDa. The membrane PPO purification was achieved by anion exchange chromatography and gel filtration with apparent molecular mass of 54 kDa. A totally denaturing SDS-PAGE indicated the presence of a single polypeptide with an apparent molecular mass of 60 kDa for both fractions, with the band also revealed by Western blot. A partially denaturing SDS-PAGE stained a single active 36 kDa band for both fractions. Under native isoelectric focusing, a major acidic band of pH 5.2 was detected in both fractions. Kinetic characterization of PPO on the natural substrate l-dopa was carried out.

Beta vulgaris↗

PubMed2003

Tyrosinase inhibitory activity of cucumber compounds: enzymes responsible for browning in cucumber.

The inhibition of mushroom tyrosinase by cucumber extracts was evaluated. The inhibitory effect was measured by both polarographic and spectrophotometric methods. The commercial aldehyde, trans,cis-2,6-nonadienal, described as a major volatile compound of cucumber, was characterized as a noncompetitive inhibitor against 4-tert-butylcatechol oxidation by mushroom tyrosinase. The K(I) obtained was 3.4 mM. Polyphenol oxidase (PPO) activity was not detected in cucumber skin extracts. However, the presence of PPO was revealed by Western blot; a single band was found with a M(r) of 53 kDa. These results support the assumption that the enzyme PPO is present in the cucumber skin, but its activity is inhibited. Peroxidase (PO) was also found in cucumber skin extracts. This enzyme was detected in the soluble fraction but not in the membrane fraction. The kinetic characterization of PO was carried out. Native isoelectric focusing revealed several acidic PO isoenzymes with a pI in the range between 5 and 6, a basic isoenzyme, and one principal neutral isoenzyme of pI = 7.2.

Catechol Oxidase↗

PubMed2003

Catalytic oxidation of acetaminophen by tyrosinase in the presence of L-proline: a kinetic study.

A kinetic study of acetaminophen oxidation by tyrosinase in the presence of a physiological nucleophilic agent such as the amino acid L-proline is performed in the present paper. The o-quinone product of the catalytic activity, 4-acetamido-o-benzoquinone, becomes unstable through the chemical addition of L-proline, in competition with the nucleophilic addition of hydroxide ion from water. In both cases, the catechol intermediate, 3(')-hydroxyacetaminophen, is generated, as can be demonstrated by liquid chromatography. When the effect of the presence of the nucleophilic agent on the time course of the enzymatic reaction was kinetically analyzed, it was seen to decrease the duration of the lag period and increase the steady-state rate. Rate constants for the reaction of 4-acetamido-o-benzoquinone with water and L-proline were also determined. The results obtained in this paper open a new possibility to acetaminophen toxicity, that has been attributed hitherto to its corresponding p-quinone, N-acetyl-p-benzoquinone imine.

Acetaminophen↗