Báo cáo khoa học: "Excretion of laccase by sycamore (Acer pseudoplatanus cambial cells: effect of copper deficiency, reversible removal of type 2 Cu 2+ "

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Excretion of laccase by sycamore (Acer pseudoplatanus L.) cambial cells: effect of copper deficiency, reversible removal of type 2 Cu 2+ J. Gaillard J. Jordanov 2 2 J. Quinton-Tulloch R. Bligny and R. Douce’ R. Douce 1 CEN-G et Université Joseph-Fourier, DRFlPCV !and , 2 DRFISCPM 85X, F 3804i Grenoble Cedex, France Introduction 97 000) was calculated from the sedimen- tation coefficient (s 6.1 S), the diffu- w , 2o = sion coefficient (D 5.3), and the par- ,"y 2o Cambial cells of sycamore excrete a lac- = tial specific volume (v=0.71The specific polyphenol oxidase (EC case-type activity of the purified enzyme measured 1.10.3.1 ) (Bligny and Douce, 1983). This at pH 6.6 (optimum pH) and in the pres- type of enzyme, which utilizes molecular ence of 20 mM 4-methylcatechol (opti- oxygen to oxidize phenolic substrates, is mum substrate conditions) corresponds to involved in lignin degradation probably an oxygen uptake of at least 32 pmol of (Mayer and Harel, 1979). This processes consumed 0 of protein. The ,/min/mg 2 polyphenol oxidase is excreted by many structure of the protein is stabilized by mycorrhizal fungi (for a review, see Mayer, asparagine-link;ed oligosaccharides, which 1987) and is present in some higher plant are a series of recurrent xylose-containing cells including the genus Rhus (Rhus ver- biantennary complexes that share at the nicifera, see Reinhammar, 1970), Aescu- core a common structural unit, i.e., XyIJ3 lus (Wosilait et aG, 1954) and Prunus 1 2(Mana 1 !6)Manf3 1 ! 4GlcNAcf3 (Lehman et al., 1974). The enzyme was 1 ! 4(Fuca 1 --t 3)GIcNAc (Takahashi et purified from the nutrient medium, into aL,1986). The laccase excreted by the which it was excreted by suspension-cul- cells corresponded to ca 2% of the total tured sycamore cells, using classical puri- protein synthesized during cell growth. fication techniques including a concanava- The molecules are synthesized at the lin A-Sepharose affinity column. The level of the endoplasmic reticulum where sycamore cell laccase is a monomeric Cu atoms are probably incorporated and blue copper protein containing 45% carbo- in the Golgi ciisternae where the protein hydrate and 4 copper atoms (one type 1 matures (glycosylation). The excretion and 2 C and two type 3 Cu per + 2 U +) 2 process is inhibited by 1 ,uM monensin. molecule. The molecular mass (M r =
Effects of deficiency a copper The total amount of active laccase ex- by sycamore cells was closely pro- creted portional to the amount of copper initially present in the culture medium (in the range of 2-100 !g of copper/! of nutrient medium, Bligny et al., 1986). Copper- cells excreted the apolaccase deprived (laccase without copper, inactive) at the copper-supplied cells ex- same rate as crete the active laccase (hololaccase). The concentrated apolaccase (100 mg/ml) has a slightly yellow color contrasting with the deep blue color of hololaccase. As shown in Fig. 1, the absorption spectra of apolaccase showed a striking loss of absorption at 612 and 330 nm cor- responding, respectively, to a strong decrease of type 1 and type 3 copper atoms. In addition, the EPR spectra (Fig. 2) show that the type 2 copper decreased in the same proportion. Addition of 2 ,uM copper to copper-deficient cultures trig- gers the excretion of hololaccase after a 5 h lag phase, corresponding to the time for
maturation and excretion of the enzyme sium phosphate + 1 mM EDTA, pH 6.0, including the time necessary for incorpora- and concentrated by ultrafiltration (Diaflo tion of Cu into the catalytic center. XM50 membrane). As shown in Fig. 2, the type 2 Cu atom was reincorporated into + 2 the T2D-lacca,se. Measurements of enzy- mic activity showed, therefore, that the Preparation of type 2 Cu2+ (T2D) syca- specific activity of the reconstituted en- cell laccase; reconstitution more zyme (35 J of 0 consumed/min/mg 2 tmol assays protein) was fully recovered. The type 2 copper atom was removed from the hololaccase according to the in syca- Copper introduction assays method of Morpurgo et aL (1980). 75 JIM more cell apolaccase laccase was dialyzed for 12 h under anaerobiosis, against solution A, con- taining 2 mM dimethylglyoxime, 2 mM In order to introduce the type 2 Cu (and + 2 potassium ferrocyanide and 50 mM the type 1 and 3 Cu into the +) 2 possibly sodium acetate buffer, pH 5.2. After the copper-free laiccase, we subjected the first 8 h, 1 mM EDTA was added to solu- apolaccase to the same experiments as tion A. The sample was then dialyzed described above for the T2D-laccase. Sur- anaerobically 3 times for 5 h against a prisingly, it was not possible to introduce rinsing solution containing 0.1 M phos- the type 2 C into the copper-free lac- :2+ U phate buffer, pH 6.0. case. The only modification observed on After the first 50% of the EPR spectra c;ould correspond to copper experiment, 2 Cu + 2 removed. Then the ex- atoms bound to the protein at non-specific type was periment was repeated and the type 2 sites. No enzymatic activity was detected + 2 Cu was reduced to ca 20%. Optical and in solutions of apolaccase subjected to EPR spectra showed that the type 1 and 3 copper introduction experiments. + 2 Cu were not removed. Under these In conclusion, since type 2 Cu could + 2 conditions, it was observed that the spe- be incorporaten into T2D-laccase and not cific activity of this T2D-laccase was re- into apolacca!;e, it is possible that the duced to 6.5 pmol of 0 consumed/ 2 presence of type 1 and 3 Cu is neces- + 2 min/mg protein, i.e., to about 20% of the sary for the type 2 Cu to be incorporated + 2 normal value. This indicates that the spe- into the glycosylated apoprotein. cific activity of sycamore cell laccase strictly depends upon the presence of the type 2 Cu This result was confirmed by . 2+ reconstitution assays. The type 2 Cu + 2 References was reintroduced into the molecules of T2D-laccase in assays adapted from the R. & Douce R. (1983) Excretion of lac- Bligny method of Malkin et aL (1969). 25 pM sycamore (Acer pseudoplatanus L.) by case T2D-laccase was anaerobically incubated cells - purification and properties of the en- for 1 h in solution B, containing 30 mM zyme. Biochem. ,J. 209, 489-496 ascorbic acid, 50 JI copper sulfate and M Bligny R., Gaillard J. & Douce R. (1986) Excre- 10 mM sodium citrate, pH 6.0. The tion of laccase by sycamore (Acer pseudopla- samples were then dialyzed for 20 h at tanus L.) cells - effects of a copper deficiency. Biochem. J. 237, 583-588 4°C against 2 changes of 25 mM potas-
Lehman E., Harel E. & Mayer A.M. (1974) Cop- ties of Japanese lacquer tree (Rhus vernicife- per content and other characteristics of purified ra) laccase depleted of type 2 copper (II). peach laccase. Phytochemistry 13, 1713-1717 7 Biochem. J. 187, 361-366 Malkin R., Malmstrom B.G. & Vanngard T Reinhammar B. (1970) Purification and proper- (1969) The reversible removal of one specific ties of laccase and stellacyanin from Rhus ver copper (II) from fungal laccase. Eur. J. Bio- nicifera. Biochim. Biophys. Acta 205, 35-47 chem. 7, 253-259 Takahashi N., Hotta T., lshihara H., Mori M., Bli- Mayer A.M. (1987) Polyphenol oxidases in gny R., Akazawa T, Endo S. & Arata Y. (1986) plants - recent progress. Phytochemistry 26, Xylose-containing common structural unit in N- 11-20 linked oligosaccharides of laccase from syca- more cells. Biochemistry 25, 388-395 Mayer A.M. & Harel E. (1979) Polyphenol oxi- dases in plants. Phytochemistry 18, 193-215 5 Wosilait W., Nason A. & Terrell A.J. (1954) Pyri- dine nucleotide-quinone reductase li. J. Biol. Morpurgo L., Graziani M.T., Finazzi-Agr6 A., Chem. 206, 271-282 Rotilio G. & Mondovi B. (1980) Optical proper-