Plant Physiol.-1992-Valero-774-6

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					Plant Physiol. (1992) 98, 774-776                                                                            Received for publication July 3, 1991
0032-0889/92/98/0774/03/$01 .00/0                                                                                   Accepted November 6, 1991

       Hysteresis and Cooperative Behavior of a Latent Plant
                               Edelmira Valero and Francisco Garcia-Carmona*
       Departamento de Qu(mica-Fisica, E. U. Politecnica de Albacete, Universidad de Castilla-La Mancha,
    E-02006 Albacete, Spain (E. V.); Departamento de Bioquimica y Biologia Molecular, Facultad de Veterinaria,
                            Universidad de Murcia, E-30001 -Murcia, Spain (F.G. -C.)

                             ABSTRACT                                              It has been shown that some enzymes located in cell enve-
   Appearance of a lag period dependent on pH in the expression                 lopes display different kinetic behavior depending on the pH
of the catecholase activity of a polyphenoloxidase extracted in a               of the medium, which may represent a regulatory device
latent state from Airen grape (Vitis vinifera L.) berries, is revealed,         operative in vivo (3). Furthermore, a kinetic model that ex-
suggesting the hysteretic nature of the enzyme. The lag time was                plains pH-induced cooperative effects in hysteretic enzymes
independent of enzyme concentration, indicating that slow pH-                   has been proposed to account for this behavior of enzymes
induced conformational changes in the protein must occur during                 attached to biological polyelectrolytes ( 13). The basis of this
assay. Results obtained by varying substrate concentration show                 model is that, upon ionization or protonation of a strategic
that the system presents hyperbolic or cooperative kinetics de-                 ionizable group, the protein undergoes a "slow" conforma-
pending on the pH of the assay.                                                 tional transition.
                                                                                   In the present paper, the appearance of a lag period in the
                                                                                expression of catecholase activity of a PPO is reported for the
                                                                                first time. The enzyme shows a hysteretic nature and coop-
                                                                                erative kinetics depending on the pH of the assay, these results
   PPo2 (EC is a copper-containing monooxygen-                      being consistent with the above mentioned kinetic model
ase widely distributed in nature that is responsible for melan-                 (13).
ization in animals and browning in plants. The enzyme
catalyzes two distinct reactions involving molecular oxygen,                                   MATERIALS AND METHODS
namely (a) the o-hydroxylation of monophenols to o-diphen-
ols, or cresolase activity, and (b) the subsequent oxidation of                 Materials
o-diphenols to o-quinones, or catecholase activity.                                4-tert-Butylcatechol was purchased from Aldrich and used
   PPO is located in the chloroplast thylakoid membranes                        without further purification. Triton X-1 14 was obtained from
(10), and in some species it exists in a latent form; the latent                Fluka AG (Bucks, Switzerland) and condensed as described
enzyme was recently isolated and characterized from broad                       by Bordier (2) by using 100 mm sodium phosphate buffer (pH
bean (1 1). A new method has been developed that permits                        7.3). The detergent phase of the third condensation had a
the extraction of PPO in this latent state by using temperature-                concentration of 25% Triton X-1 14 (w/v) and was used as
induced phase separation in Triton X-1 14 (14). The latent                      the stock solution of detergent for all the experiments.
enzyme can be activated by different treatments such as
trypsin (14, 17), fatty acids (4), aging (8), acid and base shock               Methods
(5, 6), detergents (1 1, 15), and cations (16). As regards acti-
vation of the enzyme by pH changes in the medium, this                            Airen grape berries (Vitis vinifera L.) used in this study
phenomenon was first studied by Kenten (5) in Viciafaba, in                     were harvested and processed as previously described (18).
which he ascribed the process to the removal of an inhibitory                   PPO was extracted from grape berries in a latent form by
protein attached to the enzyme. More recently, Lerner et al.                    using temperature-induced phase separation in Triton X- 1 14
(6) found irreversible activation of the enzyme from grape                      at pH 7.3 (14). The solution thus obtained, after dialysis
berries following long exposure to acid pH, and Lerner and                      against 1 mm sodium phosphate buffer (pH 7.3), was used as
Mayer (7) further showed that the process was accompanied                       enzyme source. To avoid any possible activation of the
by a change in the Stokes' radius of the protein, indicating                    enzyme by endogenous proteases, leupeptin and N-ethylmal-
the involvement of a conformational change.                                     eimide were added before and after dialysis to give a final
                                                                                concentration of 0.04 and 5 mm, respectively. This did not
   ' This work was partially supported by a grant from the Comisi6n             significantly affect polyphenoloxidase activity.
Interministerial de Ciencia y Tecnologia (Spain), Proyecto No. AGR-               The catecholase activity of the enzyme was determined at
89-0296.                                                                        25°C by following spectrophotometrically at 400 nm the
   2 Abbreviation: PPO, polyphenoloxidase.                                      appearance of the o-benzoquinone product of the reaction
                                      HYSTERESIS OF A LATENT PLANT POLYPHENOLOXIDASE                                                   775

(e = 1150 m-'cm-') (19). Steady-state rate was defined as the
slope of the linear zone of the product accumulation curve.                  0.15
The lag period was estimated by extrapolation of the linear
portion of the product accumulation curve to the abscissa             LU
axis. Unless otherwise stated, the reaction media contained           z
4.5 mM 4-tert-butylcatechol at the indicated pH in 50 mM              m      0.10
sodium acetate (pH 3.5-5.3) or sodium phosphate (pH 7.0)              0
buffers and 0.0 16 units of enzymatic activity, in a final volume     U)
of 1 mL. One unit of enzyme activity is the amount of enzyme                 0.05
that produces 1 Mmol of 4-tert-butyl-o-benzoquinone/min as
measured at pH 3.5 under the above experimental conditions.
   SDS-PAGE was carried out as described by Angleton and
Flurkey (1). Samples were mixed with glycerol and brom-
phenol blue before being applied to 7.5 or 12.5% polyacryl-                                     5           10           15
amide gels. Electrophoresis was carried out for 6 h at room
temperature. Gels were stained for PPO activity in 100 mL                                              time (min)
of 10 mm sodium acetate buffer (pH 4.5) containing 5 mm               Figure 2. Progress curves for catecholase activity of latent PPO
L-dopa.                                                               previously preincubated (0.02 units of enzyme activity) at pH 3.3 at
                                                                      250C with 50 mm acetic acid for 10 min. The assay was performed
                 RESULTS AND DISCUSSION                               at (a) pH 5.3 and (b) pH 7.0. Once the steady-state was reached in
                                                                      curve b, (c) 20 uL of 1 M acetic acid was added to the reaction
   When catecholase activity of latent grape PPO was assayed          medium.
at pH 3.5, a steady-state rate was immediately attained (Fig.
1, curve a), in accordance with the results obtained with the
active enzyme purified at the same pH by (NH4)2SO4 frac-
tionation (18). However, when the assay was performed at              catalysis (12). The lag observed in a hysteretic enzyme can be
pH 5.3, the enzyme activity increased with time, reaching a           abolished by preincubation with ligands that cause the slow
steady-state after a discernible lag phase (Fig. 1, curve b). This    transition (for example, protons in this case) (12). The results
lag period in the expression of catecholase activity of PPO has       presented in Figure 2 (curve a) show that the lag observed in
never been previously reported, and neither has any slow              the expression of catecholase activity of latent PPO when the
transition phenomenon affecting catecholase activity been             assay is performed at pH 5.3 was indeed abolished on prein-
described. This cannot be an artifact of the enzyme assay             cubating the enzyme with a sufficient amount of acetic acid;
because the reaction is followed by measuring the appearance          in addition, a progress curve in the opposite direction, i.e. a
of the first product of catalytic activity, 4-tert-butyl-o-benzo-     burst in this case, is seen when, after preincubation with the
quinone, which is very stable (19). The presence of only one          ligand responsible for the slow transition, the enzyme is
catecholase enzyme was indicated by gel electrophoresis.              returned back to its previous experimental conditions (Fig. 2,
   This response of the enzyme to pH changes in the medium            curve b). The reaction rate decreases with time, reaching a
is a characteristic property of a hysteretic enzyme undergoing        steady-state rate lower than the one seen before (Fig. 2, curve
slow transition to another form kinetically different during          a). This indicates slow pH-induced conformational changes
                                                                      in the enzyme to a catalytically less active form. We can also
                                                                      see that the process is reversible, because on adding new

,U   0.10
                                                                       c                                                           2    c
m                                                                      E 20
0                                                                                                                                       E
<s   0.051                                                              IA


                          2               4              6                                 20             40            60
                              time     (min)                                                    ENZYME (il)
Figure 1. Progress curves for catecholase activity of latent PPO at   Figure 3. Effect of enzyme concentration on steady-state rate (0)
(a) pH 3.5 and (b) pH 5.3.                                            and lag period (A) expressed at pH 5.3.
776                                                  VALERO AND GARCIA-CARMONA                                    Plant Physiol. Vol. 98, 1992

                                                                        domain of the protein that undergoes the "slow" conforma-
      30                                                                tional transition.
                                                                          Hysteretic enzymes are important in metabolic regulation.
 E                                                                c     The pH response of latent PPO shown in this paper may
                                                                        represent a mechanism of regulation of its activity in vivo.
                                                                                                LITERATURE CITED
      15                                                          -j

                                                                         1. Angleton EA, Flurkey WH (1984) Activation and alteration of
                                                                              plant and fungal polyphenoloxidase isoenzymes in sodium
                                                                              dodecylsulfate electrophoresis. Phytochemistry 23: 2723-2725
                                                                         2. Bordier C (1981) Phase separation ofintegral membrane proteins
                                                                              in Triton X-1 14 solution. J Biol Chem 256: 1604-1607
                                                                         3. Crasnier M, Ricard J, Noat G (1982) pH-Regulation of acid
                          5                 10            15                  phosphatase of plant cell walls. An example of adaptation to
                                                                              the intracellular milieu. FEBS Lett 144: 309-312
                              [S I   (mM)                                4. Golbeck JH, Cammarata K (1981) Spinach thylakoid polyphenol
                                                                              oxidase: isolation, activation, characterization, and properties
Figure 4. Effect of substrate concentration on steady-state rates             of the native chloroplast enzyme. Plant Physiol 67: 977-984
expressed at pH 3.5 (@) and 5.3 (0), and on the lag period that shows    5. Kenten RH (1957) Latent phenolase in extracts of broad bean
catecholase activity of the enzyme at pH 5.3 (A).                             (Viciafaba L.) leaves. 2. Activation by acid and alkali. Biochem
                                                                              J 67: 300-307
                                                                         6. Lerner HR, Mayer AM, Harel E (1972) Evidence for confor-
                                                                              mational changes in grape catechol oxidase. Phytochemistry
                                                                              11: 2415-2421
amounts of acetic acid to the medium after the steady-state              7. Lerner HR, Mayer AM (1975) Stokes' radius changes of solubi-
has been reached, the activity recovered (Fig. 2, curve c).                   lized grape catechol oxidase. Phytochemistry 14: 1955-1957
                                                                         8. Lieberei R, Biehl B (1978) Activation of latent phenolase from
   We then tested this behavior of latent PPO by measuring                    spinach chloroplast by aging and by frost. Phytochemistry 17:
the lag time as a function of enzyme concentration. It was                    1437-1438
found that lag period remained constant and steady-state rates           9. Marquardt DW (1963) An algorithm for least-squares estimation
were linear with enzyme concentration (Fig. 3), indicating                    of non-linear parameters. J Soc Ind Appl Math 11: 431-441
                                                                        10. Mayer AM (1987) Polyphenol oxidase in plants-recent pro-
that the hysteresis observed is not due to oligomerization of                 gress. Phytochemistry 26: 11-20
the enzyme and must be due to pH-induced isomerization                  11. Moore BM, Flurkey WH (1990) SDS activation of a plant
of the enzyme to another form that has different catalytic                    polyphenoloxidase. Effect of SDS on enzymatic and physical
activity.                                                                     characteristics of purified broad bean polyphenoloxidase. J
                                                                              Biol Chem 265: 4982-4988
   Another interesting aspect of study is the dependence of             12. Neet KE, Ainslie GR (1980) Methods Enzymol 64: 192-226
the lag period and steady-state rates upon substrate concen-            13. Ricard J, Noat G, Nari J (1984) pH-Induced effects in hysteretic
tration. Figure 4 shows that lag period observed at pH 5.3                    enzymes. 1. A theoretical model of a new type of co-operative
                                                                              behaviour controlled by pH. Eur J Biochem 145: 311-317
decreased when 4-tert-butylcatechol concentration was in-               14. Sanchez-Ferrer A, Bru R, Garcia-Carmona F (1989) Novel pro-
creased in the reaction medium. With respect to catalytic                     cedure for extraction of a latent grape polyphenol oxidase using
activity, it can be seen that, whereas at pH 3.5 the enzyme                   temperature-induced phase separation in Triton X-1 14. Plant
followed hyperbolic kinetics with an apparent KM for the           o-
                                                                              Physiol 91: 1481-1487
                                                                        15. Sanchez-Ferrer A, Bru R, Garcia-Carmona F (1990) Partial pu-
diphenolic substrate of 2.5 mM, at pH 5.3 it exhibited kinetic                rification of a thylakoid-bound enzyme using temperature-
cooperativity. These results were fitted to the Hill equation (v              induced phase partitioning. Anal Biochem 184: 279-282
= Vmax[Sjh/(KiH + [SIh)) by using nonlinear regression (9), a           16. Siderhall K, Calberg I, Eriksson T (1985) Isolation and partial
Hill coefficient of0.74 being obtained. This result is consistent             purification of prophenoloxidase from Daucus carota L. cell
                                                                              cultures. Plant Physiol 78: 730-733
with the kinetic model described by Ricard et al. (13) to               17. Tolbert NE (1973) Activation of polyphenol oxidase of chloro-
explain pH-induced cooperative effects of hysteretic enzymes,                 plasts. Plant Physiol 51: 234-244
and which was believed to account for some pH responses of              18. Valero E, Varon R, Garcia-Carmona F (1988) Characterization
enzymes attached to biological membranes. The basis of this                   of polyphenol oxidase from Airen grapes. J Food Sci 53:
model is that cooperativity arises from the ionization or               19. Waite JH (1976) Calculating extinction coefficients for enzy-
protonation of a group located outside the active site in a                   matically produced o-quinones. Anal Biochem 75: 211-218

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