Characterization of patatin esterase activity in AOT-isooctane reverse micelles.

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 not only to serve as a storage protein but also to exhibit lipid acyl hydrolase (LAH) activity. In this study patatin is characterized in AOT-isooctane reverse micelles. The influence on the enzymatic activity of characteristic parameters of reverse micelles, w(o) (= H(2)O/AOT), and the percentage of H(2)O, theta, were investigated. The results obtained show that patatin esterase activity varies with w(o) but remains constant throughout the range of theta values studied. The variation with w(o) showed that the activity follows an S-shaped behavior pattern, reaching a maximum at about w(o) = 20 for 2% H(2)O. Patatin esterase activity was compared with p-nitrophenyl (PNP) fatty acid esters of different chain lengths. The activity was much higher for PNP-caprylate. The pH optimum was 6.0, different from the value obtained when patatin esterase activity was measured in mixed micelle systems. The optimal temperature was 35 degrees C, above which the activity decreased to almost zero. The kinetic parameters were also evaluated (K(m) = 10 mM, V(m) = 158 microM/min, V(m)/K(m) = 15.8 x 10(-3) min(-1)). This paper shows the suitability of reverse micelles for measuring patatin esterase activity, since it allows the study of the enzyme in similar conditions to that prevailing in vivo.

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.

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.

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).

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.

Characterization of monophenolase activity of polyphenol oxidase from iceberg lettuce.

Polyphenol oxidase (EC 1.14.18.1), a thylakoid membrane-bound enzyme, was isolated by sonication of osmotically shocked chloroplasts from iceberg lettuce (Lactuca sativa). The enzyme showed monophenolase activity when assayed on (p-hydroxyphenyl)propionic acid with 3-methyl-2-benzothiazolinone hydrazone in a reliable continuous spectrophotometric method, with high sensitivity, accuracy, and precision. The monophenolase activity showed a lag period before the steady-state rate (V(ss)) was reached. Both kinetic parameters, the lag period and the steady-state rate, depended on the pH, the enzyme and substrate concentrations, and the presence of catalytic amounts of o-diphenol. This activity shows inhibition by high substrate concentration. The experimental results correspond with the mechanism previously described for PPO from other sources. Kinetic constants K(m), V(max), and K(i) were determined.

Kinetic study of the suicide inactivation of latent polyphenoloxidase from iceberg lettuce (Lactuca sativa) induced by 4-tert-butylcatechol in the presence of SDS.

In plants, polyphenoloxidase undergoes an irreversible inactivation during the oxidation of o-diphenol to o-quinones. In the present paper, using latent polyphenoloxidase from iceberg lettuce (Lactuca sativa), in the presence of an activating agent, SDS, the kinetic parameters that characterize the enzyme during its action on the suicide substrate 4-tert-butylcatechol have been determined. The effect of pH has also been considered. It was seen that the presence of SDS in the reaction medium changed the kinetic parameters of the enzyme during suicide inactivation, this phenomenon depended on the SDS concentration up to saturating concentrations. Variations were also observed in the kinetic parameters at pH values below 5 where SDS provoked inactivation of the enzyme. This differing kinetic behaviour during suicide inactivation in the presence of SDS may be caused by the conformational changes provoked by SDS in the enzyme in latent state. Thus, polyphenoloxidases showing suicide inactivation would present an enzymatic activity resulting from the balance between the activation process and suicide inactivation.

Kojic acid, a cosmetic skin whitening agent, is a slow-binding inhibitor of catecholase activity of tyrosinase.

It was found that kojic acid, which is used in cosmetics for its excellent whitening effect, inhibits catecholase activity of tyrosinase in a non-classical manner. A decrease in the initial velocity to a steady-state inhibited velocity can be observed over 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. The kinetic data obtained correspond to those for a postulated mechanism that involves the rapid formation of an enzyme inhibitor complex that subsequently undergoes a relatively slow reversible reaction. Kinetic parameters characterizing this type of inhibition were evaluated by means of nonlinear regression of product accumulation curves.