Jesús D. Galindo

A new spectrophotometric method for the determination of cresolase activity of epidermis tyrosinase

A sensitive, rapid, quantitative method for the determination of the activities of the bifunctional frog epidermis enzyme, tyrosinase (E.C. 1.14.18.1), has been developed. It is a spectrophotometric method using p-coumaric acid and caffeic acid as substrates at pH 7.0. Lineweaver-Burk plots yielded straight lines and the initial velocities for the reactions were proportional to enzyme concentrations. It is simpler, faster, and more economical than radiometric methods and, furthermore, permits continuous monitoring.

Steady-state study of the mechanism of dopa-oxidase activity of tyrosinase

The mechanism of the dopa-oxidase activity of frog epidermis tyrosinase has been studied. Initial reaction rates have been measured as function of substrate concentrations, L-dopa and oxygen, in the presence and absence of an inhibitor, product of the reaction. Initial reaction rates versus substrate concentrations, without inhibitor, show a linear dependence in the double-reciprocal space, that discarded Ordered and Random mechanisms. Initial reaction rates versus substrate concentrations, in the presence of an inhibitor product of the reaction, show a non-linear dependence in the double-reciprocal space. This point, joined to the former one, indicates a Ping-Pong mechanism, different of the Hexa-Uni type. The reaction is discussed for first time taking into account a trisubstrate mechanism. The experimental results lead to an (Uni Uni Bi Uni) Ping-Pong mechanism. On the other hand, they can explain the differences between known data of tyrosinases from several sources. Michaelis constant have been calculated for both substrates. The values are 0.16 and 7.14 mM for oxygen and L-dopa respectively.

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.

Kinetic study of the activation process of frog epidermis pro-tyrosinase by trypsin

1. The rate of tyrosinase formation has been calculated by coupling the activatory process of frog epidermis pro-tyrosinase by trypsin to the oxidation of L-DOPA to dopachrome. Under certain conditions ([trypsin]/[pro-tyrosinase] greater than or equal to 300), the lag period of the coupled reactions, tau, is independent of trypsin concentration. 2. The specific rate constant of tyrosinase formation at different temperatures has been calculated, ranging from 0.025 sec-1, at 5 degrees C to 0.248 sec-1, at 30 degrees C. 3. Thermodynamic parameters of the activatory process (delta G not equal to = + 18.5 kcal/mol; delta H not equal to = + 14.8 kcal/mol; delta S not equal to = -12.4 e.u.; Ea = + 15.3 kcal/mol), have been determined by the study of the system at different temperatures. These values are characteristic for a normal chemical reaction. 4. From these kinetic data, the order of products formation in the proteolytic step, can be determined, active tyrosinase being the last product released.

Opposite sexual dimorphism of 3,4-dihydroxyphenylalanine decarboxylase in the kidney and small intestine of mice

3,4-Dihydroxyphenylalanine decarboxylase (DDC; also known as l-amino acid decarboxylase) is involved in the synthesis of dopamine, norepinephrine, and serotonin, and also acts as an androgen receptor co-regulator protein. In contrast to other amino acid decarboxylases that are modulated by sex hormones, little is known about the influence of these hormones on DDC regulation. In the present work, we studied the influence of gender in the expression of DDC in different mouse tissues. Among the different organs studied, including brain, liver, kidney, intestine, heart, adrenal gland, and skeletal muscle, only kidney and small intestine showed a sex-dependent dimorphism in DDC expression. In the kidney, levels of DDC activity, DDC mRNA, and protein were remarkably higher in females than in males. On the contrary, in the small intestine, male mice displayed higher levels of DDC activity than females but they did not correlate precisely with mRNA levels. This dimorphism was dependent on androgens, since male castration and treatment of female mice with testosterone propionate, oppositely affected DDC levels in kidney and small intestine. However, estrogen ablation or treatment with estradiol did not significantly affect DDC activity in these tissues. Immunocytochemical analysis revealed that DDC was mainly located in the proximal straight tubular cells of the kidney and in the cytoplasm of enterocytes. These data and the fact that renal DDC inversely correlated with renal sodium reabsorption suggest that renal and intestinal gender dimorphism in DDC could be related to sex-related differences in sodium balance observed between males and females.

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.

Antiandrogenic effect of RU-486 in the mouse kidney

RU-486 (mifepristone) is a synthetic steroid with potent antiprogesterone and antiglucocorticoid activity, that is currently used as a contraceptive agent. In the present work we have evaluated the antiandrogenic effect of this compound on mouse kidney, a very well known extragenital model of androgen action by studying the effect of RU-486 on renal parameters that depend on androgens, such as renal ornithine decarboxylase (ODC) activity and kidney hypertrophy, as well as the inhibitory action of mifepristone on the induction of renal ODC and kidney hypertrophy elicited by testosterone treatment in female mice and in castrated male. The results showed that: (1) 48 hr after treatment of male mice with of RU-486 (50 mg/kg, four injections) renal ODC activity decreased from 3.381 +/- 490 nmol CO2/h.g to 605 +/- 163 (SD, n = 5); (2) in female mice or orchidectomized male mice, RU-486 also inhibited the renal ODC induction elicited by exogenous administration of testosterone propionate (TP), the magnitude of the inhibition was dependent on the doses of TP and RU-486 used. While RU-486 at a dose of 25 mg/kg inhibited more than 80% ODC induction produced by treatment with 5 mg/kg TP, the same dose did not significantly affect ODC when the dose of TP was increased up to 100 mg/kg. Higher concentration of RU-486 (200 mg/kg) clearly inhibited the increase in ODC produced by treatment with TP 100 mg/kg; (3) RU-486 was more effective in blocking the anabolic effects produced by stanozolol, a steroidal anabolizing agent, than those produced by testosterone; and (4) RU-486 was less effective than the nonsteroidal antiandrogen flutamide in inhibiting renal ODC activity in male mice. Our results clearly indicate that RU-486 possesses moderate antiandrogenic activity in mouse kidney. The possibility that RU-486 may have similar effects in man should be considered when using this drug.

The effect of glycine administration on taurine concentration in the rat liver.

1. The treatment of rats with glycine (2 mg/g) produced a marked decrease in the hepatic taurine content of neonate rats but not of adult rats. 2. The decrease observed in taurine concentration in the liver of newborn rats was not found in other organs, such as brain or kidney. 3. The results showed that the change in hepatic taurine concentration was dose- and time-dependent, suggesting the existence of an exchange mechanism between taurine and glycine in the rat liver that could participate in regulating the hepatic concentration of these amino acids.

Inactivation of ornithine decarboxylase by intermediates of tyrosinase-catalyzed reaction

1. Intermediates in the process of melanin synthesis formed through oxidation of catechols by tyrosinase produced the inactivation of ornithine decarboxylase (ODC), a key enzyme in the polyamine biosynthesis pathway. 2. The inactivation was dependent on the substrate used (dihydroxybenzylamine > L-3,4-dihydroxyphenylalanine > L-tyrosine) and on the concentration of intermediate produced rather than on the rate of formation. 3. Sulfhydryl compounds (dithiothreitol and glutathione) or quinone-reducing agents (ascorbic acid) prevented the inactivation of ODC; L-ornithine, but not other amino acids, also protected partially ODC. The results suggest that different cysteine residues in ODC molecule are implicated in the inactivatory event. 4. When 14C-labeled catechols were used, numerous polypeptides resulted labeled, showing that the reactive quinones formed as intermediates in the process of melanin biosynthesis bind covalently to many cellular proteins.