Biomedical subjects

F García-Carmona

Publications and source records attributed to F García-Carmona.

At least 19 recordsLinked to original sources

PubMed2006

A colorimetric assay for S-adenosylhomocysteine hydrolase.

A colorimetric method for S-adenosyl-L-homocysteine hydrolase (SAHase) which uses S-adenosyl-L-homocysteine (SAH) as substrate is described. This method involves the hydrolytic conversion of SAH into adenosine (ADO) and L-homocysteine (HCY). The formation of HCY is quantified using Ellman's reagent and spectrophotometrical measured at 412 nm. Under these assay conditions, the product was followed continuously in a facile and quantitative manner until substrate conversion was complete. This method is an easy, cheap and shorter alternative to more complex methods and it is applicable to routine clinical analysis and in the assay and development of new S-nucleosidylhomocysteines to be used as therapeutic compounds.

Adenosine Deaminase↗

PubMed2004

Kinetic analysis of catechin oxidation by polyphenol oxidase at neutral pH.

Catechin oxidation by peach polyphenol oxidase was performed in a pH range of 3.5-8.0. At acidic pH, maximal spectral changes were observed at 390nm and at pH 7.5, at 430nm. Catechin oxidation was studied at pH 7.5 to avoid the formation of free radicals. The results obtained allowed us to propose a pathway for the enzymatic oxidation of catechin, according to which enzymatic oxidation produces the corresponding catechin-o-quinone, which suffers the nucleophilic attack of another catechin unit, leading to the formation of a dimer. This dimer is then oxidized by the enzymatically generated o-quinone. The progress curves obtained for catechin oxidation by PPO showed a lag period, whose length changed with enzyme and substrate concentrations, and which must have been caused by the chemical reactions taking place after the enzymatic reaction. The results obtained by simulation of the model produced the same qualitative dependences as obtained experimentally.

Animals↗

PubMed2003

A continuous spectrophotometric assay for phospholipase A(2) activity.

This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2).

Deoxycholic Acid↗

PubMed2003

Determination of the phospholipase activity of patatin by a continuous spectrophotometric assay.

Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported to serve as a storage protein and also to exhibit lipid phospholipase activity. This paper describes a simple continuous spectrophotometric method for assaying patatin phospholipase activity. The procedure is based on a coupled enzymatic assay using [1,2-dilinoleoyl] PC as the phospholipase substrate and lipoxygenase as the coupling enzyme. In the procedure developed in this work, lipoxygenase oxidizes the linoleic acid released by the phospholipase activity of patatin. This activity can then be followed spectrophotometrically by recording the increase in absorbance at 234 nm that results from the formation of the corresponding hydroperoxide from linoleic acid by the action of lipoxygenase. The optimal assay concentrations of patatin and lipoxygenase were established. Phospholipase activity varied with pH, reaching its optimal value at pH 9.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3 mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (Vm = 9.8 x 10(-3) micromol/min x microg protein, Km = 7.8 microM, Vm/Km = 1.3 min(-1) x microg protein). This method proved to be specific since there was no activity in the absence of patatin. It also had the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase.

Carboxylic Ester Hydrolases↗

PubMed2002

Microencapsulation of Aerococcus viridans with catalase and its application for the synthesis of dihydroxyacetone phosphate.

Dihydroxyacetone phosphate is essential for the synthesis of polyhydroxylated compounds used as components or precursors of active pharmaceutical substances, such as antibiotics or glycosidase inhibitors. Dihydroxyacetone phosphate was produced by enzymatic oxidation of L-alpha-glycerophosphate in the presence of glycerophosphate oxidase or Aerococcus viridans coimmobilized with a hydrogen peroxide-decomposing enzyme. The microencapsulation of A. viridans with catalase in sodium alginate showed a conversion of 98.5%; the conversion percentage remained constant in all five runs. Liquid chromatography of the product revealed that the product peak corresponded to that of the dihydroxyacetone phosphate internal standard. This indicated a high degree of product purity.

Alginates↗

PubMed2001

Hysteresis and positive cooperativity of iceberg lettuce polyphenol oxidase.

A kinetic study of the diphenolase activity of latent polyphenol oxidase (PPO), purified from Iceberg lettuce (Lactuca sativa L), revealed a sigmoid relationship between the reaction rate and the substrate concentration with a high Hill coefficient (n(H) = 3.8). This positive cooperativity had not been previously described for any PPO. Furthermore, the enzyme showed a lag phase in the expression of this activity, suggesting a hysteretic nature of the enzyme. The kinetic behavior, the latency and the lag phase varied at different steps of the purification process. PPO showed hyperbolic or cooperative kinetics depending on the pH assay and the sodium dodecyl sulfate (SDS) concentration. Substrate-induced slow conformational change of the oligomeric enzyme is suggested. The conformational change would be toward a more active enzyme form with higher affinity for the substrate and favoured by acid pH and SDS.

Catechol Oxidase↗

PubMed2001

Dimethyl sulfide, a volatile flavor constituent, is a slow-binding inhibitor of tyrosinase.

In this paper, the inhibition of tyrosinase by a volatile compound is kinetically analyzed for the first time. The results obtained show that the volatile flavor constituent dimethyl sulfide (DMS) inhibits the catecholase activity of tyrosinase in a nonclassical manner. A decrease in the initial velocity to a inhibited steady-state velocity can be observed within a few minutes. This time dependence, which is unaltered by prior incubation of the enzyme with the inhibitor, is consistent with a first-order transition. Both the initial and the constant rates decreased with increasing concentrations of inhibitor. The kinetic data obtained correspond to those for a postulated mechanism involving rapid formation of an enzyme-inhibitor complex that subsequently undergoes a relatively slow reversible reaction. These results, together with the high levels of DMS precursor in certain organisms, suggest a physiological role for this compound within plant tissues.

Agaricales↗

PubMed2001

Cyclodextrin biospecific-like displacement in dye-affinity chromatography.

Interactions between Cibacron Blue F3GA (CB F3GA), as a model of triazine dye, and 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD), as a model of cyclodextrin, were investigated by monitoring the spectral shift that accompanies the binding phenomena. Matrix analysis of the difference spectral titration of CB F3GA with HP-beta-CD revealed only two absorbing species, indicating a host-guest ratio of 1:1. The dissociation constant for this HP-beta-CD-CB F3GA complex, Kd, was found to be 0.43 mM. The data for HP-beta-CD forming inclusion complexes with CB F3GA were used to develop the concept of competitive elution by inclusion complexes in dye-affinity chromatography. When this concept was applied to the elution of L-lactate dehydrogenase from a CB F3GA affinity matrix, it was shown to be an effective elution strategy. It provided a 15-fold purification factor with 89% recovery and sharp elution profile (0.8 column volumes for 80% recovery), which is as good as that obtained by specific elution with NADH (16-fold, 78% recovery and 1.8 column volumes). In addition, the new elution strategy showed a better purification factor and sharper elution profile than traditional non-specific elution with KCl (4.5-fold, and 1.4 column volumes). Hence, competitive elution by inclusion complexes may be a promising strategy for eluting proteins with high recoveries and purification factors in dye-affinity chromatography.

Chromatography, Affinity↗

PubMed2001

Highly efficient Aerococcus viridans L-alpha-glycerophosphate oxidase production in the presence of H2O2-decomposing agent: purification and kinetic characterization.

Glycerophosphate oxidase was purified from Aerococcus viridans cells by phase partitioning in Triton X-114, ammonium sulfate fractionation, FPLC ion-exchange chromatography and FPLC hydrophobic-interaction chromatography. The purification achieved from a crude extract of A. viridans was 38-fold with a 32% recovery of activity. Under the growth conditions used, A. viridans strain CECT 978 proved to be an excellent glycerophosphate-oxidase producer, with enzyme production 2,800-fold greater than that described in the literature for the same microorganism. The culture medium used in the present work is that commonly used for cultivation of this microorganism, except that an H2O2-decomposing enzyme was added. The addition of catalase to the growth medium had a clear effect on the growth rate. Furthermore, methylglyoxal, a metabolite that is formed enzymatically from triose phosphates, was found to be an inactivator of glycerophosphate oxidase activity.

Catalase↗

PubMed2001

An octaethylene glycol monododecyl ether-based mixed micellar assay for determining the lipid acyl hydrolase activity of patatin.

Patatin was extracted from potato tubers (Solanum tuberosum L. cv. Spunta) and purified to homogeneity by ammonium sulfate salt fractionation and one sole chromatographic step. A spectrophotometric mixed micellar assay for patatin lipid acyl hydrolase (LAH) activity was designed with the detergent octaethylene glycol monododecyl ether (C12E8). Patatin LAH used p-nitrophenyl butyrate (PNP-butyrate) as substrate when solubilized in (C12E8) micelles. In the mixed micellar system, patatin LAH responds to the PNP-butyrate surface concentration expressed as mol% (= [PNP-butyratel x 100/([detergentl critical micellar concentration)) and not to the molarity of PNP-butyrate. The kinetic parameters were determined; Vmax was independent of the mixed micelle concentration, as was Km, when expressed as mol%. However, Km was dependent on C12E8 concentration when expressed in molar concentration. C12E8/PNP-butyrate proved to be a reliable system for assaying patatin LAH activity and is superior to the commonly used Triton X-100 and SDS methods. It permits investigation of the substrate requirements of patatin LAH activity because the concentration-independent Km can be determined both in mol% and as the absolute number of substrate molecules per micelle. In addition, the detergent did not affect the enzyme activity.

Carboxylic Ester Hydrolases↗

PubMed2001

Partial purification, characterization, and histochemical localization of fully latent desert truffle (Terfezia claveryi Chatin) polyphenol oxidase.

In the present paper, a fully latent polyphenol oxidase (PPO) from desert truffle (Terfezia claveryi Chatin) ascocarps is described for the first time. The enzyme was partially purified by using phase partitioning in Triton X-114 (TX-114). The achieved purification was 2-fold from a crude extract, with a 66% recovery of activity. The interfering lipids were reduced to 13% of the original content. In addition, the purification gave rise to a reduction of phenolic compounds to only 37.5%, thus avoiding the postpurification tanning of the enzyme. Latent PPO was activated by the anionic surfactant sodium dodecyl sulfate (SDS) or by incubation with trypsin. The amount of SDS necessary to obtain a maximum activation was dependent on the nature of the substrate. The use of SDS also permitted the histochemical localization of the latent enzyme within the ascocarp. Terfezia polyphenol oxidase was kinetically characterized using two phenolic substrates (L-DOPA and tert-butylcatechol). The latter substrate presented inhibition at high substrate concentration with a K(si) of 6.3 mM. Different inhibiting agents (kojic and cinnamic acid, mimosine and tropolone) were also studied, tropolone being the most effective.

Ascomycota↗

PubMed2001

Proteolytic activation of latent Paraguaya peach PPO. Characterization of monophenolase activity.

The kinetics of the activation process of latent peach PPO by trypsin was studied. By coupling this activation process to the oxidation of 4-tert-butylcatechol (TBC) to its corresponding quinone, it was possible to evaluate the specific rate constant of active PPO formation, k(3), which showed a value of 0.04 s(-1). This proteolytic activation of latent peach PPO permitted us to characterize the monophenolase activity of peach PPO for the first time using p-cresol as substrate, and it showed the characteristic lag period of the kinetic mechanism of monophenols hydroxylation, which depended on the enzyme and substrate concentration, the pH and the presence of catalytic amounts of o-diphenol (4-methylcatechol). The enzyme activation constant, k(act), was 2 microM.

Catechol Oxidase↗

PubMed2001

Eggplant lipoxygenase (Solanum melongena): product characterization and effect of physicochemical properties of linoleic acid on the enzymatic activity.

Lipoxygenase (LOX) from eggplant (Solanum melongena L. cv. Belleza negra) was partially purified, and the products and kinetics of the enzyme were studied. Linoleic acid (LA) was the best substrate for this enzyme. Product analysis by HPLC and GC/MS revealed that, at its pH optimum (pH 7.0), the enzyme converted LA almost totally into the 9-hydroperoxy isomer, whereas the 13-hydroperoxy isomer was only a minor product. At this pH, the enzyme had K(m) and V(max) values for LA of 1.4 microM and 2.2 micromol min(-1) (mg of protein)(-1), respectively, when the monomeric form of LA was used as substrate. The dependence of eggplant LOX activity on the physicochemical properties of LA was also studied. Experiments revealed that LA aggregates were used more efficiently than monomeric LA as substrate. The apparent substrate cooperativity observed may be due to the different activities exhibited toward monomers and aggregates. This result can be interpreted as a substrate-aggregation dependent activity.

Chemical Phenomena↗

PubMed2001

Evidence for a tetrameric form of iceberg lettuce (Lactuca sativa L.) polyphenol oxidase: purification and characterization.

Polyphenol oxidase from iceberg lettuce (Lactuca sativa L.) chloroplasts was released from the thylakoid-membrane by sonication, and it was extensively purified to homogeneity as judged by SDS-PAGE. Purification was achieved by ammonium sulfate fractionation, gel-filtration chromatography, and ion-exchange chromatography. Two molecular forms were separated by gel-filtration chromatography with apparent molecular masses of 188 and 49 kDa. Both forms were characterized by sedimentation analysis with S(20,W) values of 10.2 and 4.1 S, respectively. For the high-molecular-weight form purified to homogeneity, denaturing SDS-PAGE indicated a molecular mass of 60 kDa. Thus, from these data we suggest that lettuce polyphenol oxidase is a tetramer of identical subunits.

Ammonium Sulfate↗

PubMed2001

Competitive inhibition of mushroom tyrosinase by 4-substituted benzaldehydes.

A kinetic study of the inhibition of mushroom tyrosinase by 4-substituted benzaldehydes showed that these compounds behave as classical competitive inhibitors, inhibiting the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) by mushroom tyrosinase (o-diphenolase activity). The kinetic parameter (K(I)) characterizing this inhibition was evaluated for all of the seven compounds assayed. Cuminaldehyde showed the most potent inhibitory activity (K(I) = 9 microM). It also inhibited the oxidation of L-tyrosine by mushroom tyrosinase (o-monophenolase activity) in a competitive manner. The corresponding kinetic parameter for this inhibition was evaluated (K(I) = 0.12 mM).

Agaricales↗

PubMed2001

Non-linear slow-binding inhibition of Aerococcus viridans lactate oxidase by Cibacron Blue 3GA.

Lactate oxidase (LOD) was purified from cells of Aerococcus viridans by phase partitioning in Triton X-114 (TX-114), ammonium sulphate fractionation and FPLC ion exchange chromatography. The purification achieved from a crude extract of A. viridans was 32-fold with a 60% recovery of activity. The isolated enzyme was a true FMN-containing LOD in tetrameric form with a subunit molecular weight of 48,000. The KM for L-lactate was 175 microM, a 6-fold less value than described in the literature. Among the inhibitors tested, Cibacron Blue 3GA showed the lowest Ki. At low concentrations, Cibacron Blue 3GA behaved as a dye-, pH- and time-dependent inhibitor. A Dixon plot of the steady-state rate showed the time-dependent inhibition to be non-linear, contrary to that described for other slow-binding inhibitors. A model to explain this phenomenon was proposed. The model implies the binding of Cibacron Blue 3GA to the isomerised form of the initial enzyme-inhibition complex (E'I).

Apoenzymes↗

PubMed2000

Kinetics of a self-amplifying substrate cycle: ADP-ATP cycling assay.

A kinetic study of an ATP-ADP amplification cyclic system involving the enzymes adenylate kinase, pyruvate kinase and L-lactate dehydrogenase has been made. The stoichiometry of the cycle is 2:1, because two molecules of ADP are synthesized from one each of ATP and AMP, and one molecule of ADP is converted back into one of ATP at each turn of the cycle. This results in a continuous exponential increase in the concentrations of ATP and ADP in the reaction medium, according to the equations obtained. This is therefore a substrate cycle that amplifies itself, the cycling rate increasing continuously with time. The background signal of the reagent was reduced by using apyrase to degrade ATP and ADP in the reagent, permitting detection limits as low as 16 pmol of ATP and/or ADP in a continuous spectrophotometric assay.

Adenosine Diphosphate↗

PubMed2000

Reversible sodium dodecyl sulfate activation of latent peach polyphenol oxidase by cyclodextrins.

The reversibility of the SDS-mediated activation of latent peach PPO has been studied using cyclodextrins as strip detergent agent. Cyclodextrins produced a combined inhibitory effect on enzymatic activity of latent peach PPO due to the complexation of detergent and the hydrophobic substrate 4-tert-butylcatechol (TBC) molecules. To study the reversibility of the activation process, this combined effect has to be separated. On the one hand, the enzyme was activated by acid-shocking and the activity was measured in the presence of cyclodextrins, using TBC as substrate. The inhibition curves obtained permitted study of the complexation of TBC into cyclodextrins. On the other hand, the enzyme was activated by SDS and the activity in the presence of cyclodextrins was measured using the highly hydrophilic o-diphenol dopamine as substrate. In this case, the inhibition curves obtained indicated the reversibility of the activation process when SDS was trapped by cyclodextrins. In addition, the complexation constant between SDS and 2-hydroxypropyl-beta-cyclodextrins was calculated by measuring conductivity (K(s) = 3500 M(-1)).

2-Hydroxypropyl-beta-cyclodextrin↗