Manuela Pérez-Gilabert

Triton X-114 phase partitioning in plant protein purification

Abstract A brief overview is given of how Triton X-114 can be used not only to solubilize plant membranes but also as an excellent reagent in a bulk fractionation method to purify enzymes compared with the classical drastic methods using acetone powder or ammonium sulphate fractionation. Triton X-114 removes the tenacious phenols and chlorophylls on centrifugation. There is no need to use insoluble synthetic resins or organic solvents as Triton X-114 is so mild that the enzymes can be extracted in their natural form without activating them. The methods developed with Triton X-114 are easily reproducible and sufficiently cheap to be used in large-scale purification procedures. The classical topological use of Triton X-114 in plant membranes is also discussed.

A continuous spectrophotometric assay for phospholipase A2 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).

Lipoxygenase-catalyzed oxidation of chlorpromazine by hydrogen peroxide at acidic pH.

The hydroperoxidase activity of soybean lipoxygenase, a non-heme protein, oxidizes chlorpromazine using H2O2 at acidic pHs ranging from 3.0 to 4.0. The enzyme is assayed at pH 3.5, at which the half-life is 2 h (lower pHs cause higher inactivation rates). This oxidation is enzymatical since boiled enzyme or even iron ions both with H2O2 failed to produce any increase in absorbance. In addition, the concentration of CPZ radical cation formed and the concomitant enzyme activity directly depends on the enzyme concentration up to 0.23 microM. The Vmax value is 125 mumol/min per mg protein and the Km for chlorpromazine and H2O2 are 2.1 mM and 0.25 mM, respectively. Similar results were obtained when linoleic acid hydroperoxide was used instead of H2O2 with a Km value of 95 microM. The radical cation obtained in the oxidation of chlorpromazine by lipoxygenase decays by a disproportionation reaction. This permits to consider the overall reaction as a sum of an enzymatic reaction coupled with a chemical second order reaction with substrate regeneration, similar to those produced by peroxidases from different sources.

Enzymatic oxidation of phenothiazines by lipoxygenase/H2O2 system

Lipoxygenase (LOX) (EC 1.13.11.12) oxidized a wide range of phenothiazine (Pt) tranquillizers to their corresponding radical cations in the presence of H2O2 by means of an enzymatic chemical second-order mechanism with substrate regeneration similar to that of horseradish peroxidase. The optimum pH of LOX for this hydroperoxidase activity was in the acid range (pH 3.0-4.0), as has been shown for other Pt oxidizing systems, such as peroxidase/H2O2 and haemoglobin. LOX showed Michaelis constants for Pt ranging from 1.4 to 8.5 mM and which, in some cases, e.g. trifluoperazine, displayed substrate inhibition. By contrast, it had a high affinity for H2O2 in the microM to mM range. A new, previously undescribed plot, which relates the enzymatic affinity and the apparent second-order decay of the cation radical, was developed to study the influence of the 2- and 10-substituents in the Pt ring. The implications of this new plot and the LOX-mediated Pt oxidation are also discussed.

HYDROPEROXIDASE ACTIVITY OF LIPOXYGENASE : A KINETIC STUDY OF ISOPROTERENOL OXIDATION

The hydroperoxidase activity of soybean lipoxygenase, a non-heme protein, oxidized isoproterenol using H2O2 at pH 6.0. This oxidation was enzymatic, since neither heat-denaturated enzyme or iron ions in the presence of H2O2 produced an increase in absorbance. The initial rate was not linear and showed a characteristic lag period whose length depended on the enzyme and substrate concentration. The lag was decreased if the enzyme and isoproterenol concentration were increased, whereas it increased if the H2O2 concentration was increased. Lipoxygenase showed the typical low specificity for electron donor characteristic of this hydroperoxidase activity (26 mM), but a high affinity for H2O2 (94 microM), although with substrate inhibition (ksi = 3.6 mM). The chemical intermediates produced during the oxidation of isoproterenol were characterized in order to determine the origin of the lag period. A plausible kinetic mechanism is proposed to explain the observed lag period and inhibition by high concentrations of H2O2.

Ultrastructural localization of acid phosphatase in arbusculate coils of mycorrhizal Phoenix canariensis roots

Acid phosphatase (ACP) activity has been detected in roots of mycorrhizal and non-mycorrhizal Phoenix canariensis. This enzyme was ultrastructurally localized in arbusculate coils for the first time. This localization was carried out using a cerium-based method, which minimizes non-specific precipitation. The ACP was localized in inter- and intracellular hyphae, in the fungal cytoplasm as well as at the interface and the fungal cell wall and the periarbuscular membrane limiting it. The novel localization of an ACP in the arbuscular mycorrhizal (AM) interface of arbusculate coils suggests that this enzyme may be involved in the phosphorus efflux from the mycorrhizal fungus to the host. The results presented in this article indicate that the role played by ACP in AM symbiosis may be more important than was previously thought and that arbusculate coils are highly relevant when considering nutrient transfer through AM symbiosis.

Partial purification, characterisation and histochemical localisation of alkaline phosphatase from ascocarps of the edible desert truffle Terfezia claveryi Chatin.

In the present paper, we confirmed that alkaline phosphatase (ALP) is the main phosphatase present in ascocarps of the edible mycorrhizal fungus Terfezia claveryi. The enzyme was partially purified by precipitation with polyethylene glycol. The purification achieved from a crude extract was fivefold, with 53% of the activity recovered, and acid phosphatase, most of the lipids and phenolic compounds were eliminated. Alkaline phosphatase was kinetically characterised at pH 10.0, the optimum for this enzyme, using p-nitrophenyl phosphate as substrate. The V(max) and K(m) values were 0.3 micromol.min(-1).mg(-1) protein and 9.0 mM, respectively. Orthovanadate was a competitive inhibitor of ALP, with a K(i) of 42.5 microM. The enzyme was histochemically localised in the peridium, the hypothecium and in the ascogenic hyphae of the gleba using both colour and fluorescent reactions. The results presented suggest that the ascocarp of T. claveryi, at some stages of its development, may become nutritionally autonomous and independent of the host plant.

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=9.8×10−3 μmol/min·μg protein, Km=7.8 μM, Vm/Km=1.3 min−1·μg 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.

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, wo (= H2O/AOT), and the percentage of H2O, θ, were investigated. The results obtained show that patatin esterase activity varies with wo but remains constant throughout the range of θ values studied. The variation with wo showed that the activity follows an S‐shaped behavior pattern, reaching a maximum at about wo = 20 for 2% H2O. 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 °C, above which the activity decreased to almost zero. The kinetic parameters were also evaluated ( Km = 10 mM, Vm = 158 μM/min, Vm/ Km = 15.8 × 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.

Rapid reversed-phase high-performance liquid chromatographic determination of the regiospecificity of lipoxygenase products on linoleic acid

A new reversed-phase high-performance liquid chromatography method for the separation of regioisomeric products from lipoxygenase acting on linoleic acid was studied. The addition of salts to the mobile phase improved the retention and separation behaviour of 13-hydroperoxy-9,11-octadecadienoic acid and 9-hydroperoxy-10,12-octadecadienoic acid with respect to the results obtained with other mobile phases reported in the literature. The effect of the pH and ionic strength of the buffer on the retention times, capacity factor and separation factor of these lipoxygenase products were also studied. The pH optimum coincided with the pKa of linoleic acid (close to 7 depending on the fatty acid concentration). Phosphate concentrations close to 100 mM considerably reduced the retention times and led to better separation of the mixture of both products. Finally, this method was applied to the identification and separation of two linoleic acid hydroxides (13-hydroxy-9,11-octadecadienoic acid and 9-hydroxy-10,12-octadecadienoic acid) obtained by the reduction of their corresponding hydroperoxides.