Biomedical subjects

J L Iborra

Publications and source records attributed to J L Iborra.

At least 37 records · Page 2Linked to original sources

PubMed1987

The existence of apotyrosinase in the cytosol of Harding-Passey mouse melanoma melanocytes and characteristics of enzyme reconstitution by Cu(II).

This paper reports the effect of Cu(II) supplementation on the tyrosinase isozymes from Harding-Passey mouse melanoma. The dopa-oxidase activity of the microsomal and soluble isozymes is increased by incubation with Cu(II), whereas the activity of the unique 'in vivo' melanin-forming isozyme, bound to melanosomes, is not. Other divalent cations are ineffective in increasing the dopa-oxidase activity of tyrosinases. These results indicate the existence of a mixture of tyrosinase and apotyrosinase in the cytosol of melanocytes before reaching the melanosome. The paucity of Cu(II) in the cytosol could be one of the mechanisms of regulation contributing to avoid the formation of melanin outside the melanosome. Some kinetic characteristics of the enzymatic reconstitution of soluble and microsomal isozymes by Cu(II) are also studied, and the results suggest that the glycosylation of apotyrosinase during its maturation yields a conformational change favouring the binding of Cu(II) at the enzyme active site, by lowering the activation energy of the reconstitution reaction.

Animals↗

PubMed1986

Study of alpha-methyldopa oxidation by tyrosinase.

The pathway for alpha-methyldopa oxidation to alpha-methyldopachrome, by mushroom tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometry determination have both been undertaken. The steps for alpha-methyldopa transformation into its aminochrome would be: alpha-methyldopa----o-alpha-methyldopaquinone-H+----o-alpha- methyldopaquinone----leuko-alpha-methyldopachrome----alpha- methyldopachrome. The stoichiometry for this conversion corresponded to the equation: 2 o-alpha-methyldopaquinone-H+----alpha-methyldopa + alpha-methyldopachrome. At very acid pH values, another route implying the addition of water to the quinonic ring, competes with the first one. Two chemical pathways can be proposed from alpha-methyldopaquinone-H+, the relative importance of which is determined by the pH. A theoretical and experimental kinetic approach was applied to this oxidative reaction. Rate constants and thermodynamic activation parameters of the chemical steps, have been evaluated. The results obtained confirmed that alpha-methyldopa oxidation by tyrosinase followed a scheme similar to that established for L-dopa and alpha-methylnoradrenaline.

Catechol Oxidase↗

PubMed1986

Irreversible inhibition of trypsin by TLCK. A continuous method for kinetic study of irreversible enzymatic inhibitors in the presence of substrate.

This work presents a kinetic study on irreversible inhibition of trypsin by TLCK, using a new experimental approach. The method consists of the incubation of the enzyme with an irreversible inhibitor in the presence of a substrate which allows enzyme turnover as well as continuous measurement of the appearance of the product, a simultaneous change in the initial concentrations of the irreversible inhibitor and enzyme being undertaken, though a constant ratio between the latter, is maintained. This new approach enables the kinetic constants for TLCK, k2 and K1, to be determined.

Amino Acid Chloromethyl Ketones↗

PubMed1986

Comparative study of tyrosinases from different sources: relationship between halide inhibition and the enzyme active site.

The inhibition of tyrosinases from frog epidermis (Rana esculenta ridibunda), mushroom (Agaricus bisporus) and Harding-Passey mouse melanoma by halides is compared. In all cases, the inhibition is pH dependent, increasing when the pH decreases. The order of inhibition is I- greater than Br- greater than Cl- much greater than F- for frog epidermis tyrosinase, F- greater than I- greater than Cl- greater than Br- for mushroom tyrosinase and F- greater than Cl- much greater than Br- greater than I- for the mouse melanoma enzyme. These results are discussed in terms of the active site accessibility to exogenous ligands. The activation energies of the enzyme-catalysed L-dopa oxidation were also calculated, being the values 6.86, 17.01 and 20.25 kcal/mol for frog epidermis, mushroom and Harding-Passey mouse melanoma, respectively. A relationship between these values and the evolutionary adaptation of these enzymes is proposed.

Animals↗

PubMed1985

Aggregation equilibria of tyrosinase of Harding-Passey mouse melanoma.

The purification of two isoenzymes of tyrosinase has been carried out in Harding-Passey mouse melanoma. One is found in the cytosol and the other one bound to melanosomes. Both migrate as single bands on sodium dodecyl sulphate/polyacrylamide gels, having an apparent Mr of 58 000. Solubilized particulate tyrosinase showed an aggregation equilibrium involving a monomer, tetramer, octamer and a high-Mr micellar form with Brij 35, the solubilizing agent. H.p.l.c. studies indicated a interconversion between those species, the monomer contribution increasing with the sample dilution. The tetramer and the octamer probably represent the predominant forms in vivo. Soluble tyrosinase showed a simpler aggregation equilibrium, involving two forms, monomer and tetramer, with the same interconversion pattern. Fluorescence studies suggested that tryptophan residues were exposed to the aqueous environment when tyrosinase was dissociated by dilution. Tyrosinase shows a tendency to aggregate, at low protein concentration, and a resistance to dissociation by urea or SDS so remarkable that gel-permeation chromatography in 4M-urea does not affect the equilibrium, and the band obtained on SDS/polyacrylamide-gel electrophoresis is a dimer.

Animals↗

PubMed1985

A method for assaying the rhamnosidase activity of naringinase.

The use of the p-nitrophenyl-alpha-L-rhamnopyranoside for the specific measurement of the alpha-rhamnosidase activity of naringinase, by colorimetrically following the appearance of p-nitrophenolate anion, is proposed. Use of this synthetic substrate did not change the pH, temperature, or ionic strength optima of the enzyme. It did, however, result in (a) a decrease of the Michaelis constant of the enzyme, allowing the Vmax to be measured, this being impossible to accomplish with naringin, (b) an increase in the sensitivity of the assay to the presence of inhibitors in the reaction media, (c) an increase in the sensitivity which enabled measurement of low levels of naringinase due to the high absorptivity of p-nitrophenolate, and (d) a quick and cheap method of evaluating the alpha-rhamnosidase activity of naringinase.

Chromatography, High Pressure Liquid↗

PubMed1985

Regulation of the cytosolic and melanosome-bound tyrosinase activities in Harding-Passey mouse melanoma.

Mouse melanoma tyrosinases exist in the cytoplasm of melanocytes and also in a particulate form, bound to melanosomes. The cytosolic isoenzyme activity is not expressed in the melanocytes in vivo. One of the mechanisms for the activity regulation is the existence of a soluble inhibitor. This inhibition is non-competitive with regard to L-dopa. Particulate tyrosinase can be solubilized from the melanosome by several agents, Brij 35 and Triton X-100 being the most effective ones. Melanin accumulation in the organelle produces a competitive inhibition of the activity.

Animals↗

PubMed1985

Isoproterenol oxidation by tyrosinase: intermediates characterization and kinetic study.

The present work deals with isoproterenol oxidation by mushroom tyrosinase and sodium metaperiodate. Intermediates produced at short reaction time were characterized by scanning repetitive spectrophotometry and the stoichiometry of the respective aminochrome appearance was established. The oxidation pathway from isoproterenol to aminochrome is parallel to the previously proposed for L-dopa oxidation by mushroom tyrosinase, whose steps are as follow: Isoproterenol----o-quinone-H+----o-quinone----leukoaminochrome---- aminochrome. The stoichiometry for the conversion of o-quinone-H+ into the aminochrome of isoproterenol followed the equation: 2 o-quinone-H+----isoproterenol + aminochrome. The kinetics of chemical reactions that take place from the o-quinone-H+ to aminochrome has been studied as a system of various chemical reactions coupled to an enzymatic reaction (EzCC: Enzymatic-Chemical-Chemical mechanism).

Catechol Oxidase↗

PubMed1985

The involvement of histidine at the active site of Harding-Passey mouse melanoma tyrosinase.

The nature of the essential residues at the active site of Harding-Passey mouse melanoma tyrosinase has been explored by kinetic and photochemical modification studies. Km for L-dopa depends strongly on pH, so that acidic pH prevents the formation of the enzyme-substrate complex because the protonation of an enzyme group with a pKa of 6.6. Halide ions inhibit competitively the enzyme activity, being F the more potent one. This inhibition is also pH-dependent, showing the involvement of a protonatable group of the enzyme with apparent pKa ranging from 5.9 to 7.0. Tyrosinase has also been modified with visible light using Rose Bengal as photosensitizer, yielding a pH-dependent photoinactivation, characteristic of histidyl residues. All these results strongly support that histidine plays an important role in the dopa-oxidase activity of the enzyme, very probably acting as the ligand of copper at the active site of the enzyme.

Animals↗

PubMed1985

FT-IR spectroscopy of natural melanins isolated from Harding-Passey mouse melanoma.

The alterations caused on the structure of melanins by acid and alkaline extraction methods were studied by means of techniques such as Fourier Transform Infrared spectroscopy and thermogravimetry. Acid extraction results in the hydrolysis of aromatic monomers of the pigment, leading to uncyclicized residues and to a less conjugated polymer. Alkaline methods catalyse the covalent binding of proteins present in the melanin extract, thus leading to melanoprotein artifacts. A new method based on the delipidation with ether and deproteinization with SDS of previously isolated melanosomes is proposed. This method shows that melanins present in Harding-Passey mouse melanoma have a bound protein moiety, so that they should exist "in vivo" as melanoprotein complexes. Thermogravimetric data suggest that the chromogen moiety of the melanoproteins in Harding-Passey mouse melanoma is a mixture of eu- and pheomelanins.

Animals↗

PubMed1984

Kinetic study and intermediates identification of noradrenaline oxidation by tyrosinase.

Characterization of intermediates formed in the noradrenaline oxidation by mushroom tyrosinase and sodium periodate has been performed by rapid scanning spectrophotometry and graphical analysis of obtained spectra. In a pH range from 5.0 to 6.0, it has been possible to detect o-noradrenalinequinone-H+ as the first intermediate in these oxidations. The following steps for noradrenaline transformation into noradrenochrome would be: noradrenaline----o-noradrenalinequinone-H+----o- noradrenalinequinone----leukonoradrenochrome----noradreno chrome. It has been also verified that o-noradrenalinequinone-H+ is transformed into noradrenochrome at a constant ratio. The stoichiometry for this converstion followed the equation: 2-noradrenalinequinone-H+----noradrenaline + noradrenochrome. The pathway between noradrenaline and noradrenochrome has been studied as a system of various chemical reactions coupled to an enzymatic reaction. We have denominated this type of mechanism as an enzymatic-chemical-chemical mechanism, (E2CC). Whole rate constants for the implicated chemical steps at different pH and temperature values have been evaluated from measurement of the lag period arising from the accumulation of noradrenochrome that takes place when noradrenaline was oxidized at pH 5-6. The lag period was independent on enzyme concentration, but was increased when pH and/or temperature were increased. Rate constants pH independent for the deprotonation of noradrenalinequinone-H+ into noradrenalinequinone and for the internal cyclization of noradrenalinequinone into leukonoradrenochrome have been obtained. We conclude that this minor pathway of noradrenaline oxidation by tyrosinase follows a scheme similar to that established for L-dopa.

Basidiomycota↗

PubMed1984

Kinetic analysis of chemical reactions coupled to an enzymic step. Application to acid phosphatase assay with Fast Red.

Acid phosphatase assay with alpha-naphthyl phosphate as substrate and the use of diazonium salt (Fast Red TR) for chromophore formation was kinetically analysed as a system of two chemical reactions coupled to an enzymic reaction. This system follows a mechanism defined as enzymic-chemical-chemical (EzCC). The accumulation of chromophore with reaction time presented a marked lag period, which was only dependent on the rate constants of the chemical reactions and was independent of the enzymic step. The specific rate constants of each chemical step were determined in 3.8-5.0 pH and 10-35 degrees C temperature ranges. Thermodynamic parameters of the chemical steps were also obtained. Measurement of acid phosphatase activity can be carried out in the pH range 3.8-5.0 (4.8 was optimal pH) without the need to eliminate the lag period.

Acid Phosphatase↗

PubMed1984

Kinetic study on the slow inhibition of epidermis tyrosinase by m-coumaric acid.

The inhibition by m-coumaric acid of oxidation of L-dopa by epidermis tyrosinase (monophenol,dihydroxy-L-phenylalanine:oxygen oxidoreductase, EC 1.14.18.1) is characterized by a prolonged transient phase. Kinetic data correspond to that for a postulated mechanism that involves rapid formation of a reduced enzyme-m-coumaric acid complex that subsequently undergoes a relatively slow reversible reaction. An overall inhibition constant for m-coumaric acid of 0.05 mM was calculated. The value of the Ki for the dissociation of m-coumaric acid from the rapidly formed complex was calculated as 0.53 mM. The first-order rate constants for the slow isomerization of the enzyme-inhibitor complex were calculated as 3.0 +/- 0.1 min-1 for the forward step and 0.31 +/- 0.06 min-1 for the reverse step.

Animals↗

PubMed1984

Kinetic study of the interaction between frog epidermis tyrosinase and chloride.

The effect of halide ions on frog epidermis tyrosinase has been characterized with the trypsin-activated enzyme. At pH 7, the order of inhibition is I- greater than Br- greater than Cl- greater than F-. Chloride, the most extensively studied halide, shows a competitive pattern with respect to the substrate, L-DOPA. Inhibition is strongly pH-dependent, with a pKa of 6.12 for the responsible protonatable group. Other kinetic constants are also calculated using a novel approach. The mechanism of interaction between chloride and the enzyme is discussed, and a model is proposed in which chloride interferes the tyrosinase activity by displacing a catalytically important ligand, probably a histidine residue of the side-chain, from the copper at the enzyme-active site.

Animals↗

PubMed1984

Conformational studies of soluble and immobilized frog epidermis tyrosinase by fluorescence.

Fluorescence spectra and soluble quenching of intrinsic protein fluorescence were used as indexes of conformational changes suffered by frog epidermis tyrosinase. The activation process and the immobilization of the enzyme involving either free amino groups or its carbohydrate moiety were studied. The conformational changes resulting from denaturation of each one of the protein derivatives, as well as the effect of active center copper extraction, were followed by fluorescence studies. The results showed that: both activation and immobilization were accompanied by conformational changes of the protein leading to more unfolded states; neither enzyme nor immobilized enzyme were fully unfolded upon denaturation although enzymic activity was lost; the enzyme immobilized through its carbohydrate moiety was more unfolded upon denaturation than the enzyme immobilized through amino groups, thus pointing to a higher conformational stabilization in the last situation; and that tryptophyl residues moved to a localization near the active site upon activation.

Animals↗

PubMed1984

Chemical intermediates in dopamine oxidation by tyrosinase, and kinetic studies of the process.

A minor pathway for dopamine oxidation to dopaminochrome, by tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometric determination have both been undertaken. After oxidizing dopamine with mushroom tyrosinase or sodium periodate in a pH range from 6.0 to 7.0, it was spectrophotometrically possible to detect o-dopaminoquinone-H+ as the first intermediate in this pathway. The steps for dopamine transformation to dopaminochrome are as follows: dopamine----o-dopaminequinone-H+----o-dopaminequinone---- leukodopaminochrome--- - dopaminochrome. No participation of oxygen was detected in the conversion of leukodopaminochrome to dopaminochrome. Scanning spectroscopy and graphical analysis of the obtained spectra also verified that dopaminequinone-H+ was transformed into aminochrome in a constant ratio. The stoichiometry equation for this conversion is 2 o-dopaminequinone-H+----dopamine + dopaminochrome. The pathway for dopamine oxidation to dopaminochrome by tyrosinase has been studied as a system of various chemical reactions coupled to an enzymatic reaction. A theoretical and experimental kinetic approach is proposed for such a system; this type of mechanism has been named "Enzymatic-chemical-chemical" (EzCC). Rate constants for the implied chemical steps at different pH and temperature values have been evaluated from the measurement of the lag period arising from the accumulation of dopaminochrome that took place when dopamine was oxidized at acid pH. The thermodynamic activation parameters of the chemical steps, the deprotonation of dopaminequinone-H+ to dopaminequinone, and the internal cyclization of dopaminequinone to leukodopaminochrome have been calculated.

Basidiomycota↗

PubMed1984

Incorporation of bovine thyroid peroxidase in liposomes.

Bovine thyroid peroxidase (TPO), an enzyme requiring lipids for demonstrating catalytic activity, was incorporated in liposomes made of pure phospholipids. The enzyme did not show high differences in activity when bilayer thickness was changed, but dipalmitoyl phosphatidyl choline (DPPC) seemed to be more appropriate for activity. The perturbation caused on lipid fluidity by enzyme incorporation was studied by differential scanning calorimetry (DSC) and fluorescence polarization of the apolar probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The complexes of TPO with dimyristoyl phosphatidyl choline (DMPC), DPPC, and distearoyl phosphatidyl choline (DSPC) bilayers showed transition temperatures (Tc) which were lower than the characteristic ones shown by liposomes with the respective phospholipids alone. The microsomal fraction from which TPO was extracted was in the fluid state at 37 degrees C, the temperature at which thyroid peroxidase works 'in vivo'. Since the effect of the protein in lowering the transition temperature of the phospholipids was so low, the contribution of phospholipids containing unsaturated fatty acids has to be essential for obtaining a fluid bilayer at body temperature.

Animals↗

PubMed1984

Acid proteinase activity in fish--I. Comparative study of extraction of cathepsins B and D from Mujil auratus.

Acid catheptic activity was measured in crude extracts of muscle, liver, heart, spleen and gonads from the fishes Mujil auratus, Sparus aurata and Lightonatus mormyrus. The spleen was the organ which showed the highest activity. A comparative study of the seven most commonly used extraction methods was made. Some were modified to account for the characteristics of the fish organs and the activity extracted from them. The Siebert method resulted as the best extraction method only if 1 mM EDTA was present in the medium. The activity from Mujil auratus muscle was strongly inhibited by iodoacetate, N-ethylmaleimide, p-hydroxy mercuribenzoate, and diazo-acetyl-DL-norleucine methyl ester. The results indicated the presence of a carboxyl-proteinase and a thiol-proteinase. According to inhibition studies, the levels of proteinase and amidase activities shown by different organs of Mujil auratus were re-examined. The spleen extract showed the maximum activity for both cathepsins, but muscle extract accounted for more than 95% of total catheptic activity.

Animals↗