Delia Spanò

Euphorbia latex biochemistry: Complex interactions in a complex environment

Abstract Plant latex is a complex environment. Occurring in hundreds of plant species and contained in a tube system called laticifers, latex is a milky sap with a diverse composition that includes alkaloids, terpenoid compounds, other secondary metabolites and a number of enzymes. These substances are collectively believed to provide an important contribution to plant defence mechanisms by repelling and killing phytopathogens, and sealing wounded areas. This review provides insights of what is currently known about the biochemistry and molecular biology of plant latex, as studied in various model systems, above all the economically important rubber tree, Hevea brasiliensis. Selecting the Mediterranean shrub Euphorbia characias as a complementary experimental model, we have recently begun to disclose the properties of several components of the enzymatic machinery present in its latex. Although the scheme of multi‐enzymatic interactions taking place in the E. characias latex depicted to date is certainly incomplete, the emerging scenario suggests that the role played by latex in plants might be significantly less passive than previously believed.

Tyramine oxidation by copper/TPQ amine oxidase and peroxidase from Euphorbia characias latex

Tyramine, an important plant intermediate, was found to be a substrate for two proteins, a copper amine oxidase and a peroxidase from Euphorbia characias latex. The oxidation of tyramine took place by two different mechanisms: oxidative deamination to p-hydroxyphenylacetaldehyde by the amine oxidase and formation of di-tyramine by the peroxidase. The di-tyramine was further oxidized at the two amino groups by the amino oxidase, whereas p-hydroxyphenylacetaldehyde was transformed to di-p-hydroxyphenylacetaldehyde by the peroxidase. Data obtained in this study indicate a new interesting scenario in the metabolism of tyramine.

Antityrosinase activity of Euphorbia characias extracts

Tyrosinase is a well-known key enzyme in melanin biosynthesis and its inhibitors have become increasingly important because of their potential use as hypopigmenting agents. In the present study, the anti-melanogenic effect of aqueous and ethanolic extracts from Euphorbia characias leaves, stems, and flowers in cell-free and cellular systems was examined. All the extracts showed inhibitory effects against mushroom tyrosinase with leaf extracts exhibiting the lowest IC50 values of 24 and 97 µg/mL for aqueous and ethanolic extracts respectively. Enzyme kinetic analysis indicated that leaf aqueous extract acts as a mixed type inhibitor, while ethanolic extract shows a competitive inhibition effect on mushroom tyrosinase using L-DOPA as substrate. In addition, the inhibitory effect of leaf extracts on tyrosinase activity and melanin production was examined in murine melanoma B16F10 cells. Cellular tyrosinase activity as well as levels of melanin synthesis are reduced in a dose-dependent manner by extracts in cells treated with α-melanocyte stimulating hormone (α-MSH). The effects are comparable, and sometimes even better, than that of kojic acid, a well known tyrosinase inhibitor used for reference. All these results suggest that E. characias could be a great source of the natural inhibitors from tyrosinase and has the potential to be used as a whitening agent in therapeutic fields.

Extraction and characterization of a natural rubber from Euphorbia characias latex

A natural rubber was identified and characterized for the first time in the latex of the perennial Mediterranean shrub Euphorbia characias. Four different methods, i.e., acetone, acetic acid, trichloroacetic acid, and Triton® X-100, followed by successive treatments with cyclohexane/ethanol, were employed to extract the natural rubber. The rubber content was shown to be 14% (w/v) of the E. characias latex, a low content compared with that of Hevea brasiliensis (30-35%) but a similar content to other rubber producing plants. E. characias rubber showed a molecular weight of 93,000 with a M(w) /M(n) of 2.9. (1) H NMR, (13) C NMR, and FTIR analysis revealed the characteristic of the cis-1,4-polyisoprene typical of natural rubber. These results provided novel insight into latex components and will ultimately benefit the broader understanding of E. characias latex composition.

Calcium Ions and a Secreted Peroxidase in Euphorbia characias Latex are Made for Each Other

This minireview deals of a protein, a class III secreted peroxidase, present as unique isoform in the latex of the perennial Mediterranean shrub Euphorbiacharacias. The paper reports on the molecular properties, on the structures (primary, secondary and tertiary), and on the catalytic mechanism of this enzyme. Here is also reported the extraordinary effect of calcium ions on the structure and on the enzyme activity of Euphorbia peroxidase. These ions can either enhance the catalytic efficiency of the enzyme toward some substrates or can regulate the ability of the enzyme to execute different metabolic pathways toward the same substrate. This review will give a valuable reference to the peroxidase fans and the general readers will find many thorough suggestions for future researches giving birth to new studies and important discoveries.

Euphorbia peroxidase catalyzes thiocyanate oxidation in two different ways, the distal calcium ion playing an essential role

The oxidation of the pseudohalide thiocyanate (SCN(-)) by Euphorbia peroxidase, in the presence or absence of added calcium, is investigated. After incubation of the native enzyme with hydrogen peroxide, the formation of Compound I occurs and serves to catalyze the thiocyanate oxidation pathways. The addition of a stoichiometric amount of SCN(-) to Compound I leads to the native enzyme spectrum; this process clearly occurs via two electron transfers from pseudohalide to Compound I. In the presence of 10 mM calcium ions, the addition of a stoichiometric amount of SCN(-) to Compound I leads to the formation of Compound II that returns to the native enzyme after addition of a successive stoichiometric amount of SCN(-), indicating that the oxidation occurs via two consecutive one-electron transfer steps. Moreover, different reaction products can be detected when the enzyme-hydrogen peroxide-thiocyanate reaction is performed in the absence or presence of 10 mM Ca(2+) ions. The formation of hypothiocyanous acid is easy demonstrated in the absence of added calcium, whereas in the presence of this ion, CN(-) is formed as a reaction product that leads to the formation of an inactive species identified as the peroxidase-CN(-) complex. Thus, although monomeric, Euphorbia peroxidase is an allosteric enzyme, finely tuned by Ca(2+) ions. These ions either can enhance the catalytic efficiency of the enzyme toward some substrates or can regulate the ability of the enzyme to exploit different metabolic pathways toward the same substrate.

Purification, Primary Structure, and Properties of Euphorbia characias Latex Purple Acid Phosphatase

A purple acid phosphatase was purified to homogeneity from Euphorbia characias latex. The native protein has a molecular mass of 130 ± 10 kDa and is formed by two apparently identical subunits, each containing one Fe(III) and one Zn(II) ion. The two subunits are connected by a disulfide bridge. The enzyme has an absorbance maximum at 540 nm, conferring a characteristic purple color due to a charge-transfer transition caused by a tyrosine residue (Tyr172) coordinated to the ferric ion. The cDNA nucleotide sequence contains an open reading frame of 1392 bp, and the deduced sequence of 463 amino acids shares a very high degree of identity (92–99%) to other purple acid phosphatases isolated from several higher plants. The enzyme hydrolyzes well p-nitrophenyl phosphate, a typical artificial substrate, and a broad range of natural phosphorylated substrates, such as ATP, ADP, glucose-6-phosphate, and phosphoenolpyruvate. The enzyme displays a pH optimum of 5.75 and is inhibited by molybdate, vanadate, and Zn2+, which are typical acid phosphatase inhibitors.

Chitinase III in Euphorbia characias latex: Purification and characterization

This paper deals with the purification of a class III endochitinase from Euphorbia characias latex. Described purification method includes an effective novel separation step using magnetic chitin particles. Application of magnetic affinity adsorbent noticeably simplifies and shortens the purification procedure. This step and the subsequently DEAE-cellulose chromatography enable to obtain the chitinase in homogeneous form. One protein band is present on PAGE in non-denaturing conditions and SDS-PAGE profile reveals a unique protein band of 36.5 ± 2 kDa. The optimal chitinase activity is observed at 50 °C, pH 5.0. E. characias latex chitinase is able to hydrolyze colloidal chitin giving, as reaction products, N-acetyl-D-glucosamine, chitobiose and chitotriose. Moreover, we observed that calcium and magnesium ions enhance chitinase activity. Finally, we cloned the cDNA encoding the E. characias latex chitinase. The partial cDNA nucleotide sequence contains 762 bp, and the deduced amino acid sequence (254 amino acids) is homologous to the sequence of several plant class III endochitinases.

Euphorbia characias latex: micromorphology of rubber particles and rubber transferase activity.

We have recently characterized a natural rubber in the latex of Euphorbia characias. Following that study, we here investigated the rubber particles and rubber transferase in that Mediterranean shrub. Rubber particles, observed by scanning electron microscopy, are spherical in shape with diameter ranging from 0.02 to 1.2 μm. Washed rubber particles exhibit rubber transferase activity with a rate of radiolabeled [(14)C]IPP incorporation of 4.5 pmol min(-1)mg(-1). Denaturing electrophoresis profile of washed rubber particles reveals a single protein band of 37 kDa that is recognized in western blot analysis by antibodies raised against the synthetic peptide whose sequence, DVVIRTSGETRLSNF, is included in one of the five regions conserved among cis-prenyl chain elongation enzymes. The cDNA nucleotide sequence of E. characias rubber transferase (GenBank JX564541) and the deduced amino acid sequence appear to be highly homologous to the sequence of several plant cis-prenyltransferases.

Bioseparation of Four Proteins from Euphorbia characias Latex: Amine Oxidase, Peroxidase, Nucleotide Pyrophosphatase/Phosphodiesterase, and Purple Acid Phosphatase

This paper deals with the purification of four proteins from Euphorbia characias latex, a copper amine oxidase, a nucleotide pyrophosphatase/phosphodiesterase, a peroxidase, and a purple acid phosphatase. These proteins, very different in molecular weight, in primary structure, and in the catalyzed reaction, are purified using identical preliminary steps of purification and by chromatographic methods. In particular, the DEAE-cellulose chromatography is used as a useful purification step for all the four enzymes. The purification methods here reported allow to obtain a high purification of all the four proteins with a good yield. This paper will give some thorough suggestions for researchers busy in separation of macromolecules from different sources.