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Properties of polyphenol oxidases produced in sweet potato tissue after wounding
ARCHIVES
OF
Properties
BIOCHEMISTRY
AND
BIOPHYSICS
of Polyphenol
Oxidases after
HIROSHI Laboratory
of Metabolic
122, 299-309 (1967)
Produced
in Sweet
H‘T-ODO
February
Tissue
Wounding’ Ai’TD
IKUZO
URITAXI
Regulation, Institute for Biochemical Regulation, Nagoya University, Nagoya, Japan Received
Potato
21, 1967; accepted
May
Faculty
of Bgriculture,
18, 1967
Two polyphenol oxidases, component IIIa and IIIb, produced in the sweet potato roots tissue in response to infection or cutting, have been isolated from cut injured t.issue and separated from other polyphenol oxidase components. Their enaymic properties were investigated and compared with those of component IIb in healthy root t,issue. Components IIIa and IIIb were shown to be distinct from either component IIb or other kinds of polyphenol oxidase in sweet potato roots by elect.rophoretic, chromatographic, and enzymological behaviors.
Numerous observations on the increase in polyphenol oxidase activity in plant tissues following infection or cut injury (wounding) have been reported, as reviewed by Farkas and Kirsly (1). When sweet potato roots were infected by Ceratocystis jimbriata or injured by cutting, accumulation of polyphenolic compounds and enhancement of polyphenol oxidase activity occurred in the injured tissues (2-4). Besides polyphenol oxidases detect’ed in the healthy root tissue, other kinds of polyphenol oxidase were found in response to either infection or cutting by means of column chromatography, electrophoresis, and antigen-antibody reaction. The former was designated as component II and the latter as components I and III (3). The increase in pol;yphenol oxidase activity was investigated m slices excised from sweet potato roots, namely in cut injured Gssue. After a lag phase during the first 30 hours, a rapid increase in enzyme activity occurred in a sigmoidal fashion over the next TO hours. The increment of polyphenols cccurred over a shorter period of time and was evident before an increase in polyphenol 1 This paper constitutes part 58 of the phytopathological chemistry of sweet potato with black rot.
oxidase activity could be detected (4). The enhancement of polyphenol oxidase activity in cut injured tissue was inferred to be derived from de novo synthesis of the enzyme protein, since by administration of antibiotics such as actinomycin D, puromycin, and blasticidin S, the increase in the enzyme activity was markedly suppressed (5). Component II was found to be a mixture of three subcomponents (components IIa, IIb, and 11~) which were separated by DEAEcellulose column chromatography. Among them, the major component, component IIb, was further purified to the extent that the specific activity increased about 500-fold over that’ of the crude extract, and the ultracentrifugal pattern of the enzyme showed a single peak. Its copper content was determined to be 0.27% by t’he neutron activation analysis (6, 7). All of the three components were specific for o-diphenolic compounds, and their enzyme activities were inhibited by copper chelating agents. This paper deals with the isolation and the properties of some subcomponents const,ituting component III, such as components IIIa and IIIb (3), which are formed in cut injured tissue. Comparative enzymic studies of components IIIa and IIIb with component IIb are also reported.
300
HYODO MATERIALS
AND
AND
METHODS
Norin No. 1, a variety of sweet potato, was used for the experiments. The roots were harvested in 1965 at the Kariya farm, Aichi, and were stored at l&11” until used. They were scrubbed in water, immersed in 0.1 ‘% sodium hypochlorite solution for 30 minutes to sterilize the outer parts, and rinsed in running tap water. The roots from which the cortices has been removed were crosssectionally cut into slices 2 mm thick by a ham slicer. The slices were incubated in the dark at 2&30” under high humidity for 3 days. Preparation of acetone powder. Six hundred-gm portions of the incubated slices (cut injured tissue) were chopped and homogenized in a Homomixer (Nihonseiki Co. Ltd.) with 2.4 liters of cold acetone containing 0.1% ascorbic acid at -30” for 2 minutes. The homogenate was filtered on a Buchner funnel. The residue was washed with 600 ml of cold acetone and 600 ml of cold ethyl et,her, and then dried to powder in a desiccator over P205 in 2racuo. Finally 3.2 kg of acetone powder was obtained from 9.0 kg of the incubated slices by repitition of the same process 15 times. The powder was stored in a deep freezer at -20” until used for enzyme extraction. Enzyme extraction from acetone powder. Unless otherwise noted, all subsequent operations were performed in a cold room at 2-4”. The above dried powder (2.8 kg) was suspended in 22 liters of 0.02 M potassium phosphate buffer, pH 7.0, containing 1% sodium isoascorbate. The suspension was stirred for 30 minutes and then squeezed through chesse cloth. The filtrate was left standing overnight to precipitate starch. The resultant yellowish brown supernatant fluid (20 liters) was subjected to ammonium sulfate fractionation. Ammonium sulfate fractionation. To the crude extract (20 liters) 3580 gm of ammonium sulfate was added, with constant stirring. During the addition of ammonium sulfate, the pH of the solution was adjusted to between 6 and 7 with 5 N NHaOH. The resultant suspension, which was 25yo saturated with respect to ammonium sulfate, was mixed with standard Super-Cel (Johns-Manville) and filtered on a Buchner funnel with the aid of a suction pump. To the filtrate were added 7160 gm of ammonium sulfate to make 75% saturation, and the suspension was again mixed with SuperCel and filtered on a Buchner funnel. The residue was suspended in 0.02 M potassium phosphate buffer, pH 7.0, containing 1% sodium isoascorbate and ammonium sulfate of 200% saturation. The suspension was stirred for 1 hour and filtered on a Buchner funnel to remove undissolved materials. To the filtrate (4.4 liters) was added 1575 gm of ammonium sulfate (about 70% saturation). The
URITANI resultant suspension was centrifuged at 809Og for 10 minutes. The precipitate was dissolved in 400 ml of 0.02 M potassium phosphate buffer, pH 7.0, containing 1% sodium isoascorbate. This solution was dialyzed 4 times against 5-liter portions of the same buffer for 40 hours. To the dialyzate (520 ml) was added 107 gm of ammonium sulfate (30% saturation), and the suspension was centrifuged 8000g for 10 minutes. The precipitate was dissolved in 100 ml of 0.02 M Tris-HCl buffer, pH 7.6, and the solution was dialyzed against the same buffer (30y0 saturated fraction). To the supernatant solution was added 90 gm of ammonium sulfate, and the suspension was centrifuged at 8OOOgfor 10 minutes. The precipitate was dissolved in 100 ml of 0.02 M Tris-HCl buffer, pH 7.6, and the solution was dialyzed against the same buffer (3&55$& saturated fraction). To the supernatant fluid was added a further 54 gm of ammonium sulfate, and centrifugation, dissolving, and dialysis were performed as described above (5570 ‘% saturated fraction). The analytical results on polyphenol oxidase activity in the above three
FIG. 1. Polyacrylamide gel electrophoretic pattern of polyphenol oxidases. IIa, IIb, IIc, IIIa, IIIb, and IV represent component IIa, IIb, IIc, IIIa, IIIb and IV, respectively. H indicates an extract from acetone powders of healthy tissue of sweet potato roots; C, an extract from acetone powders of cut injured tissue of sweet potato roots.
POLYPHENOL
OXIDASE
IN WOUNDED
IO0 EFFLUENT
SWEET
POTATO
150 (tubs number)
FIG. 2. DEAE-cellulose column chromatogram of polyphenol oxidases. The solid line shows polyphenol oxidase activity in units, and the dotted line, optical density at 280 mp. The abscissa shows effluent (tube numbers). The points labeled with an arrow indicate the fractions which were subjected to polyacrylamide gel electrophoresis.
fractions showed that the activity was exclusively recovered in 3&55y0 saturated fraction. DEAE-cellulose column chromatography. Diethylaminoethyl (DEAE)-cellulose (0.90 meq/gm, Brown Chem. Co.) in a chromatographic column (6 X 37 cm) was pre-equilibrated with 0.02 M TrisHCl buffer, pH 7.6. One hundred ml of a solution of the fraction precipitated by 3&55% ammonium sulfate saturation was applied to the top of the column. The column was t’hen eluted at 4 ml/ minute with an exponential gradient of sodium chloride by allowing 1.6 liters of 0.02 M Tris-HCl buffer, pH 7.6, containing 1 M sodium chloride to flow with, constant stirring, into 3.1 liters of 0.02 M Tris-HCl buffer, pH 7.6. Fractions, each containing 22 ml, were collected automatically by a G.M.E. (Gilson Medical Electronics) fraction collector. As shown in the chromatographic pattern (Fig. 2), polyphenol oxidases were separated to several fractions containing components IIa, IIb, IIc, IIIa, and IIIb, which were named previously (3, 7). This was ascertained by polyacrylamide gel electrophoresis (Fig. 3). The confirmation will be shown in det,ail in Results. Components IIa, IIb, and IIc in healthy tissue were investigated in the previous experiments (7). Separat,ion of components IIIa and ITIb was carried by the
following procedures, during which another component, component IIIc, was detected in the component IIIb fraction. Polyacrylamide gel electrophoresis. “Cyanogum 41” (Weyerhaeuser Co., Hartford, Connecticut), consisting of a mixture of acrylamide monomer and NJ’-methylenebisacrylamide, was used for gel formation. Five gm of Cyanogum 41 was disbuffer, pH 9.3, solved in 95 ml of 0.02 M Tris-HCl and the solution was filtered to remove a trace of insoluble matter. To the solution were added two catalysts, 1.0 ml of 10% dimethylaminopropionitryle (DMAPN) and 1.0 ml of 10% ammonium persulfate. All these solutions were freshly prepared and mixed just before use. The flask which contained the mixture was evacuated by an aspirator to avoid formation of air bubbles, and the solution was poured into a plastic mould (13 X 30 X 0.15 cm). The solution was covered with a plastic sheet and allowed to stand for 1 hour until gelled. Eight plastic pieces (1 X 0.1 X 0.05 cm) had been set in a row on the plastic cover to make slots for sample insertion. After gel formation was complete, samples were inserted into the slots. The polyacrylamide slab was connected by filter papers to the electrode vessels which contained 0.02 M Tris-HCl buffer, pH 9.3. Electrophoresis was
302
HYODO
AND
FIG. 3. Polyacrylamide gel electrophoretic pattern of polyphenol oxidases. IIa, IIb, IIc, IIIa, IIIb, and IV represent components IIa, IIb, IIc, IIIa, IIIb, and IV, respectively. A, B, C, D, E, F, G, and H indicate the points labeled in Fig. 2. carried out routinely at 16 V/cm and 0.4 mA/cm for 12 hours. The positions of polyphenol oxidases in polyacryalmide gel after electrophoresis were revealed by staining the gel with a solution made up of equal volumes of 0.9% caffeic acid and 0.1% p-phenylenediamine (5). Sephadex G-100 column chromatography. Sephadex G-106 (Pharmacia, Sweden) in a chromatographic column was pre-equilibrated with 0.02 M Tris-HCl buffer, pH 7.6, in the case of component IIIa fraction, or 0.01 M potassium phosphate buffer, pH 6.8, in the case of component IIIb fraction and the fractions obtained by DEAE-cellulose column chromatography. The column was eluted with the same buffer. Effluents were collected automatically by a G.M.E. fraction collector. DEAE-cellulose column chromatography for isolation of components IIIa and IIIb. To a DEAEcellulose column (1.8 X 24 cm) previously equilibrated with 0.02 M Tris-HCl buffer, pH 7.6, was applied 1.8 ml of the solution containing the component IIIa obtained with Sephadex G-100 column chromatography. The column was eluted with an exponential gradient, of sodium chloride from 0 to
URITANI 0.5 M. In the case of component IIIb, 4 ml of the solution obtained with Sephadex G-100 column chromatography was applied to DEAE-cellulose column (1.8 X 24 cm) previously equilibrated with 0.05 M Tris-HCI buffer, pH 7.6, containing 12.57, sucrose. The column was developed by the same buffer with an exponential gradient of sodium chloride from 0 to 1.0 -II. Assay of polyphenol oxidase activity. An oxygen electrode was used for determining polyphenol oxidase activity. The ingredients of the mixture in the reaction cell were as follows: 3.5 ml of 0.1 M potassium phosphate buffer (pH 6.0), 0.1 ml of the enzyme solution, and 0.1 ml of substrate solution in a total volume 3.7 ml. The procedures used in this assay and the unit of the enzyme activity are described in more detail elsewhere (7). Caffeic acid (5 X 1W M) adjusted to pH 6.0 was used as the substrate for routine assay system. In the experiments to measure Michaelis constants or to determine the substrate specificity, the following compounds were used: chlorogenic acid, pyrocatechol, pyrogallol, caffeic acid, dihydroxyphenylalanine, hydroquinone, p-coumaric acid, p-cresol, m-cresol, o-hydroxybenzoic acid, and phydroxyphenylpyruvic acid. Estimation of protein content. The protein content was determined by the method of Lowry et al. (8). The amount of protein was calculated on the basis of a standard curve prepared by using bovine serum albumin (fraction V, Armour & Co.).
Chemicals. Caffeic acid and pyrocatechol were recrystallized for determination of Michaelis constants. Chlorogenic acid and p-coumaric acid were provided by Dr. T. Minamikawa of the Department of Biology, Tokyo Metropolitan University, and Dr. H. Imaseki of this University, respectively. Sodium isoascorbate was given by Dr. S. Tsuruta of Fujisawa Yakuhin Kogyo Co. All other chemicals, unless otherwise stated, were purchased commercially. RESULTS
Isolation of components IIIa and IIIb. Figure 1 shows electrophoretic patterns of polyphenol oxidases extracted from acetone powders of both healthy and cut injured tissue of sweet potato roots. Three kinds of polyphenol oxidase, components IIa, IIb, and IIc, were present in healthy tissue, but in the extract from cut tissue, they were found to contain three more polyphenol oxidases in addition to the above three, which were designated as components IIIa, IIIb, and IV. Three components in healthy root tissue had been investigated as de-
POLYPHENOL
0
20
OXIDASE
IN WOUNDED
60
$0
SWEET
POTATO
303
80
EFFLUENT (tube number) FIG. 4. DEAE-cellulose column chromatogram of polyphenol oxidases. The solid line shows polyphenol oxidase activity in units, and the dotted line, optical density at 280 ml. The abscissa shows effluent (tube numbers). The points labeled with an arrow indicate the fractions which were subjected to polyacrylamide gel electrophoresis.
FIG. 5. Polyacrylamide gel electrophoretic pattern of polyphenol oxidases. IIb, IIIa, and IIIb represent components IIb, IIIa, and IIIb, respectively. A, B, C, D, E, F, G, and H indicate the points labeled in Fig. 4. The numbers 31, 33, 37, 39, 41, 45, 49, and 53 indicate the tube numbers of effluent in Fig. 4.
scribed in t.he previous paper (6, 7). Extracts from healthy tissue which were not injured by cutting contained only a slight amount of component IIIa. The appearance of com-
ponent IIIa might be due to physiological changes or slight damage during storage for about a year. Component IV in cut tissue extract was the only component that mi-
HYODO AND URITANI
20
60
40
EFFLUENT
(tube
SO
number)
FIG. 6. Sephadex G-100 column chromatogram
of polyphenol oxidases. The solid line shows polyphenol oxidase activity in units, and the dotted line, optical density at 280 q. The abscissa shows effluent (tube numbers). The points labeled with an arrow indicate the fractions which were subjected to polyacrylamide gel electrophoresis.
grated to the cathode. During the following isolation procedures of components IIIa and IIIb, a component called IIIc, which migrated far more than IIIb to the anode, was detected, as described below. The original polyphenol oxidase activity in crude extract from acetone powders was almost exclusively recovered (103.8 %) in the 30-55 % saturated ammonium sulfate fraction. One hundred-ml portions of the 30-55 % saturated fraction containing 3.44 gm of protein and 1.7 X lo5 units of polyphenol oxidase were applied to DEAE-cellulose column. The elution pattern is shown in Fig. 2. The components contained in several fractions were confirmed to be components IIa, IIb, IIc, IIIa, and IIlb by the polyacrylamide gel electrophoregram indicated in Fig. 3. That is, three peaks in the chromatographic pattern were essentially composed of components IIa, IIb, and IIIa, respectively. The subsequent tailing area after the appearance of the third peak was found to be a mixture mainly containing components IIb, IIIa, and IIIb. Component
IV was found to be present in the first peak as a minor component. Component IIb, which occupied the second peak, was free from other components, and its specific activity per milligram of protein was 94.5fold greater than that of the crude extract. The component IIIa containing the fraction collected from tubes 75-80 included also IIb and IIIb as minor components. Therefore, the fraction was subjected to Sephadex G-100 column chromatography. Component IIIa, however, could not be separated from the other two components. A further attempt to separate component IIIa from the other two was carried on by DEAE-cellulose column chromatography. As shown in Fig. 4, three peaks equivalent to three components were not obtained, but a single peak with two shoulders appeared. Hence the location of the three components was examined by polyacrylamide gel electrophoresis. As demonstrated in Fig. 5, it was found that only component IIIa was located at the central part of the peak, namely in the effluents from tubes 37-41.
POLYPHENOL
OXIDASE
IN WOUNDED
FIG. 7. Polyacrylamide gel electrophoretic pattern of polyphenol oxidases. IIb, IIIa, IIIb, and 111~ represent components IIb, IIIa, IIIb, and IIIc, respectively. A and B indicate the points labeled in Fig. 6.
With this isolated fraction some properties of component IIla were investigated, as indicated below. Effluents (2880 ml) of the column chromatography from tubes 81-224, composing the tailing part, were collected, and proteins were precipitated by the addition of ammonium sulfate to 80% saturation. The precipitate was dissolved in 0.01 M potassium phosphate buffer, pH 6.8, and the solution was dialyzed against the same buffer. The dialyzate (50 ml) was applied to a Sephadex G-100 column. The column was eluted with 0.01 M potassium phosphate buffer, pH 6.8. As shown in Fig. 6, two peaks appeared. According to polyacrylamide gel electrophoresis, the first peak constituted mainly component IIIb, and the second consisted of a mixture of components IIb, IIIa, IIIb, and
SWEET
POTATO
305
111~ (Fig. 7). In order to purify component IIIb, effluents making up the first peak were collected, concentrated, and rechromatographed on DEAE-cellulose column. Figures 8 and 9, which show the chromatograms and electrophoregrams, illustrate that the last peak constitutes only component IIIb. To elute component IIIb from DEAEcellulose column, sucrose was added to the elution buffer as described in Materials and Nesthods. The addition of sucrose was very effective to prevent tailing and to make a clear-cut elution profile. The specific activity in effluents corresponding to the last peak, which were used for the investigation of properties of component IIIb, corresponded to a 6.7-fold concentration over that in the crude extract. We at’tempted to isolate component 111~ from the mixture obtained as the last peak after Sephadex G-100 column chromatography. It was diflicult, however, to separate component 111~ from component IIIb by DEAE-cellulose column chromatography under varying conditions, and component 111~ has not yet been isolated. Conversion of the components in III group (components IIIa, IIIb, and 111~) to components of the II group (components IIa, IIb, and 11~) and of component IIIb to components IIIa and 111~ were not detected, when either mixture of components IIIa, IIIb, and 111~ or isolated component IIIb was left to stand for one month at 0” as the ammonium sulfate precipitate. The precipitates were then dissolved and chromatographed on a DEAE-cellulose column. Enzymic properties of components IIIa and IIIb and a comparison with those of component IIb. The pH-activity curves of components IIIa and IIIb were differed slightly when caffeic acid was used as the substrate. They also differed slightly from those of components IIb, IIa, and IIc (7). Components IIIa and IIIb were specific for o-diphenols and could oxidize neither monophenols such as p-coumaric acid, p-cresol, m-cresol, o-hydroxybenzoic acid, and p-hydroxyphenylpyruvic acid, nor polyphenols not having vicinal dihydroxy group such as hydroquinone. In the case of component IIlb, tyrosine was also used as a substrate, but it was not oxidized. This was in accord-
306
HYODO
AND
URITANI
40
EFFLUENT
60
80
(tube number)
FIG. 8. DEAE-cellulose column chromatogram of polyphenol oxidases. The solid line shows polyphenol oxidase activity in units, and the dotted line, optical density at 280 rnp. The abscissa shows effluent (tube numbers). The points labeled with an arrow indicate the fractions which were subjected to polyacrylamide gel electrophoresis.
ante with the substrate specificity of components IIa, IIb, and IIc (7). The Michaelis constants (K, values) for several substrates of components IIb, IIIa, and IIIb were determined by the Lineweaver-Burl< equation. The results are summarized in Table I. Components IIb and IIIa possessed the same K, values for chlorogenic acid, caffeic acid, pyrogallol, and pyrocatechol. All of the K, values of Component IIIb for the above four substrates were distinct from those of components IIb and IIIa. A higher affinity to caffeic acid and a lower affinity to chlorogenie acid of component IIIb were indicated. Concerning the maximum velocity, the value of component IIIb for chlorogenic acid was far greater than that for caffeic acid, whereas in the cases of components IIb and IIIa the relation was rather reversed. p-Coumaric acid was found to be a potent competitive inhibitor to the oxidation of caffeic acid by all of three components (Fig. 10). Since the enzyme activities of components IIla and IIIb were inhibited by copper chelating agents such as phenylthiourea, DIECA
(diethyldithiocarbamate), and KCN (Table II), they were regarded as copper enzymes as well as components IIa, IIb, and IIc (7). DISCUSSION
Two polyphenol oxidases (components IIIa and IIIb) produced in cut injured tissue of sweet potato roots were isolated by several steps, although their specific activities per milligram of protein were only slightly raised (4.6- and 6.7-fold than that of the crude extract). Thus it was demonstrated that components IIIa and IIIb were distinct from components IIa, IIb, and IIc as to electrophoretic and chromatographic behaviours and enzymic properties such as pHactivity curve, Michaelis constant, etc. The previous experiments indicated that component III was discriminated from component II in terms of immunological behavior
(3).
All polyphenol oxidases in sweet potato roots, so far as examined, were specific for o-diphenols. They could not oxidize monophenols. It is worthy to note, however, that
POLYPHENOL
OXIDASE
IN WOUNDED TABLE
hlIcH.&ELIS
307
POTATO
I
CONST.~NTS .&ND RELATIVE MAXIMUM VELOCITY VALUES FOR CHLOROGENIC: FOR p-COUM~RIC ACID, PYROC.~TECHOL, hi%~ PYROGALLOL, AND Ki VALUES IIb, IIIa, AND IIIb OF COMPONENTS
Value Km (1x1
Ki(M) v maxa
IIb,
SWEET
Compound
Chlorogenic acid Caffeic acid Pyrocatechol Pyrogallol p-Coumaric acid Chlorogenic acid Caffeic acid Pyrocat,echol Pyrogallol
III.?
2.9 2.9 1.3 1.8 4.8
II&l
x 10-S x 10-3 X 10-Z X 1OV X 1O-4 1.0 1.52 0.55 0.34
2.9 2.9 1.3 1.8 6.8
x 10-a x 10-S X 10-Z X 1O-2 X 1O-4 1.0 1.36 0.75 0.50
TABLE INHIBITORY AGENTS
C~FFEIC
IIIb
4.0 1.7 8.7 2.9 3.3
for chlorogenic a vlmx indicates the relative value when the maximum velocity IIIa, and IlIb represent components IIb, IIIa, and IIIb, respectively.
x IO-3 X 10-a X 1O-3 x 10-Z x 10-d 1.0 0.42 0.06 0.04
acid is taken
as 1.0.
II
EFFECT OF COPPER CHEL.~TING ON THE ENZYME ACTIVITY OF ETCH COMFONENT
Inhibitor
Phenylthiourea Diethyldithiocarbamate Potassium cyanide
FIG. 9. Polyacrylamide gel electrophoretic pattern of polyphenol oxidases. IIa, IIb, and IIIb represent components IIa, IIb, and IIIb, respect,ively. A, B, and C indicate the points labeled in Fig. 8.
ACID, ACID
Concentration CM)
5 1 5 1 5 1
x X x x x X
10-J lo-3 lo-4 lo-3 1*4 lcr3
Inhibition
(%)
IIIa
IIIb
80.6 86.1 22.2 19.4 83.3 97.2
62.7 82.3 46.4 57.2 42.9 53.7
p-coumaric acid, which has been assumed to be a precursor of caffeic acid or chlorogenic acid (9), was a potent competitive inhibitor to the enzymes. The affinity of p-coumaric acid to enzymes was stronger t,han that of substrates. It might be postulated that pcoumaric acid could be a substance which regulates the activity of polyphenol oxidases in intact cells by binding to the enzymes. Addition of sucrose to the elution buffer stimulated the elution of component IIIb through the DEAE-cellulose column. Sucrose may prevent absorption of polyphenol oxidases to polyhydroxy groups of DEAEcellulose. This suggests an assumption that polyphenol oxidases might be kept inactive in intact cells by binding with polyhydroxycontaining substances in cellular membrane. The previous papers reported that poly-
308
HYODO AND URITANI
lllb
0
,‘I. I/S
FIG. 10. Competitive
inhibition
4
3
2
(X103
of the activity
5
6
7
I/M)
of each component by p-coumaric acid.
IIb, IIIa, and IIIb represent components, IIb, IIIa, and IIIb, respectively. 0, without inhibitor; A, with 6.75 X 1W M p-coumaric acid.
phenol oxidase activity in the cut injured tissue increased in a sigmoidal fashion over a loo-hour period (4), and that the rise in the enzyme activity was presumably due to de nno2/osynthesis of enzyme protein (5). It has been widely known in the plant kingdom that polyphenol oxidase activity increases in response to infection or cutting. A role of
this enzyme in diseased tissue has been considered to be associated with a defense action of the host (l,lO). It will be of interest and importance to make clear a role or a meaning of polyphenol oxidases inducibly formed in the infected or cut tissue and to elucidate a triggering mechanism which causes synthesis of the enzyme protein.
POLYPHENOL
OXIDASE
IN WOUNDED
SCKNOWLEDGMENTS The authors are grateful to Professor M. A. Stahmann of the Department of Biochemistry, University of Wisconsin, for his valuable advice in preparing this manuscript. REFERENCES 1. FARKAS, G. L., AND KIR~~LY, Z., Phytopathol. 2. 44, 105 (1962). 2. GRITANI, I., AND MURAMATSU, K., J. Agr. Chem. Sot. Japan 26,289 (1952). 3. HYODO, H., AND URITANI, I., J. Biochem. 67, 161 (1965). 4. HYODO, H., AND URITANI, I., Plant Cell Physiol. 7, 137 (1966).
SWEET
POTATO
309
5. HYODO, H., AND URITANI, I., Agr. Biol. Chem. 30, 1083 (1966). 6. HYODO, H., AND BANDO, S., Bgr. Biol. Chem. 2S, 763 (1965). 7. HYODO, H., AND URITANI, I., J. Biochem. 68, 388 (1965). 8. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. BioZ. Chem. 193, 2G5 (1951). 9. BROWN, S. A,, Ann. Rev. Plant Physiol. 17, 223 (1966). 10. RUBIN, B. A., AND ARTSIKHOVSKAYA, YE. I’., “Biochemistry and Physiology of Plant Immunity.” Pergamon Press, New York (1963)
OF
Properties
BIOCHEMISTRY
AND
BIOPHYSICS
of Polyphenol
Oxidases after
HIROSHI Laboratory
of Metabolic
122, 299-309 (1967)
Produced
in Sweet
H‘T-ODO
February
Tissue
Wounding’ Ai’TD
IKUZO
URITAXI
Regulation, Institute for Biochemical Regulation, Nagoya University, Nagoya, Japan Received
Potato
21, 1967; accepted
May
Faculty
of Bgriculture,
18, 1967
Two polyphenol oxidases, component IIIa and IIIb, produced in the sweet potato roots tissue in response to infection or cutting, have been isolated from cut injured t.issue and separated from other polyphenol oxidase components. Their enaymic properties were investigated and compared with those of component IIb in healthy root t,issue. Components IIIa and IIIb were shown to be distinct from either component IIb or other kinds of polyphenol oxidase in sweet potato roots by elect.rophoretic, chromatographic, and enzymological behaviors.
Numerous observations on the increase in polyphenol oxidase activity in plant tissues following infection or cut injury (wounding) have been reported, as reviewed by Farkas and Kirsly (1). When sweet potato roots were infected by Ceratocystis jimbriata or injured by cutting, accumulation of polyphenolic compounds and enhancement of polyphenol oxidase activity occurred in the injured tissues (2-4). Besides polyphenol oxidases detect’ed in the healthy root tissue, other kinds of polyphenol oxidase were found in response to either infection or cutting by means of column chromatography, electrophoresis, and antigen-antibody reaction. The former was designated as component II and the latter as components I and III (3). The increase in pol;yphenol oxidase activity was investigated m slices excised from sweet potato roots, namely in cut injured Gssue. After a lag phase during the first 30 hours, a rapid increase in enzyme activity occurred in a sigmoidal fashion over the next TO hours. The increment of polyphenols cccurred over a shorter period of time and was evident before an increase in polyphenol 1 This paper constitutes part 58 of the phytopathological chemistry of sweet potato with black rot.
oxidase activity could be detected (4). The enhancement of polyphenol oxidase activity in cut injured tissue was inferred to be derived from de novo synthesis of the enzyme protein, since by administration of antibiotics such as actinomycin D, puromycin, and blasticidin S, the increase in the enzyme activity was markedly suppressed (5). Component II was found to be a mixture of three subcomponents (components IIa, IIb, and 11~) which were separated by DEAEcellulose column chromatography. Among them, the major component, component IIb, was further purified to the extent that the specific activity increased about 500-fold over that’ of the crude extract, and the ultracentrifugal pattern of the enzyme showed a single peak. Its copper content was determined to be 0.27% by t’he neutron activation analysis (6, 7). All of the three components were specific for o-diphenolic compounds, and their enzyme activities were inhibited by copper chelating agents. This paper deals with the isolation and the properties of some subcomponents const,ituting component III, such as components IIIa and IIIb (3), which are formed in cut injured tissue. Comparative enzymic studies of components IIIa and IIIb with component IIb are also reported.
300
HYODO MATERIALS
AND
AND
METHODS
Norin No. 1, a variety of sweet potato, was used for the experiments. The roots were harvested in 1965 at the Kariya farm, Aichi, and were stored at l&11” until used. They were scrubbed in water, immersed in 0.1 ‘% sodium hypochlorite solution for 30 minutes to sterilize the outer parts, and rinsed in running tap water. The roots from which the cortices has been removed were crosssectionally cut into slices 2 mm thick by a ham slicer. The slices were incubated in the dark at 2&30” under high humidity for 3 days. Preparation of acetone powder. Six hundred-gm portions of the incubated slices (cut injured tissue) were chopped and homogenized in a Homomixer (Nihonseiki Co. Ltd.) with 2.4 liters of cold acetone containing 0.1% ascorbic acid at -30” for 2 minutes. The homogenate was filtered on a Buchner funnel. The residue was washed with 600 ml of cold acetone and 600 ml of cold ethyl et,her, and then dried to powder in a desiccator over P205 in 2racuo. Finally 3.2 kg of acetone powder was obtained from 9.0 kg of the incubated slices by repitition of the same process 15 times. The powder was stored in a deep freezer at -20” until used for enzyme extraction. Enzyme extraction from acetone powder. Unless otherwise noted, all subsequent operations were performed in a cold room at 2-4”. The above dried powder (2.8 kg) was suspended in 22 liters of 0.02 M potassium phosphate buffer, pH 7.0, containing 1% sodium isoascorbate. The suspension was stirred for 30 minutes and then squeezed through chesse cloth. The filtrate was left standing overnight to precipitate starch. The resultant yellowish brown supernatant fluid (20 liters) was subjected to ammonium sulfate fractionation. Ammonium sulfate fractionation. To the crude extract (20 liters) 3580 gm of ammonium sulfate was added, with constant stirring. During the addition of ammonium sulfate, the pH of the solution was adjusted to between 6 and 7 with 5 N NHaOH. The resultant suspension, which was 25yo saturated with respect to ammonium sulfate, was mixed with standard Super-Cel (Johns-Manville) and filtered on a Buchner funnel with the aid of a suction pump. To the filtrate were added 7160 gm of ammonium sulfate to make 75% saturation, and the suspension was again mixed with SuperCel and filtered on a Buchner funnel. The residue was suspended in 0.02 M potassium phosphate buffer, pH 7.0, containing 1% sodium isoascorbate and ammonium sulfate of 200% saturation. The suspension was stirred for 1 hour and filtered on a Buchner funnel to remove undissolved materials. To the filtrate (4.4 liters) was added 1575 gm of ammonium sulfate (about 70% saturation). The
URITANI resultant suspension was centrifuged at 809Og for 10 minutes. The precipitate was dissolved in 400 ml of 0.02 M potassium phosphate buffer, pH 7.0, containing 1% sodium isoascorbate. This solution was dialyzed 4 times against 5-liter portions of the same buffer for 40 hours. To the dialyzate (520 ml) was added 107 gm of ammonium sulfate (30% saturation), and the suspension was centrifuged 8000g for 10 minutes. The precipitate was dissolved in 100 ml of 0.02 M Tris-HCl buffer, pH 7.6, and the solution was dialyzed against the same buffer (30y0 saturated fraction). To the supernatant solution was added 90 gm of ammonium sulfate, and the suspension was centrifuged at 8OOOgfor 10 minutes. The precipitate was dissolved in 100 ml of 0.02 M Tris-HCl buffer, pH 7.6, and the solution was dialyzed against the same buffer (3&55$& saturated fraction). To the supernatant fluid was added a further 54 gm of ammonium sulfate, and centrifugation, dissolving, and dialysis were performed as described above (5570 ‘% saturated fraction). The analytical results on polyphenol oxidase activity in the above three
FIG. 1. Polyacrylamide gel electrophoretic pattern of polyphenol oxidases. IIa, IIb, IIc, IIIa, IIIb, and IV represent component IIa, IIb, IIc, IIIa, IIIb and IV, respectively. H indicates an extract from acetone powders of healthy tissue of sweet potato roots; C, an extract from acetone powders of cut injured tissue of sweet potato roots.
POLYPHENOL
OXIDASE
IN WOUNDED
IO0 EFFLUENT
SWEET
POTATO
150 (tubs number)
FIG. 2. DEAE-cellulose column chromatogram of polyphenol oxidases. The solid line shows polyphenol oxidase activity in units, and the dotted line, optical density at 280 mp. The abscissa shows effluent (tube numbers). The points labeled with an arrow indicate the fractions which were subjected to polyacrylamide gel electrophoresis.
fractions showed that the activity was exclusively recovered in 3&55y0 saturated fraction. DEAE-cellulose column chromatography. Diethylaminoethyl (DEAE)-cellulose (0.90 meq/gm, Brown Chem. Co.) in a chromatographic column (6 X 37 cm) was pre-equilibrated with 0.02 M TrisHCl buffer, pH 7.6. One hundred ml of a solution of the fraction precipitated by 3&55% ammonium sulfate saturation was applied to the top of the column. The column was t’hen eluted at 4 ml/ minute with an exponential gradient of sodium chloride by allowing 1.6 liters of 0.02 M Tris-HCl buffer, pH 7.6, containing 1 M sodium chloride to flow with, constant stirring, into 3.1 liters of 0.02 M Tris-HCl buffer, pH 7.6. Fractions, each containing 22 ml, were collected automatically by a G.M.E. (Gilson Medical Electronics) fraction collector. As shown in the chromatographic pattern (Fig. 2), polyphenol oxidases were separated to several fractions containing components IIa, IIb, IIc, IIIa, and IIIb, which were named previously (3, 7). This was ascertained by polyacrylamide gel electrophoresis (Fig. 3). The confirmation will be shown in det,ail in Results. Components IIa, IIb, and IIc in healthy tissue were investigated in the previous experiments (7). Separat,ion of components IIIa and ITIb was carried by the
following procedures, during which another component, component IIIc, was detected in the component IIIb fraction. Polyacrylamide gel electrophoresis. “Cyanogum 41” (Weyerhaeuser Co., Hartford, Connecticut), consisting of a mixture of acrylamide monomer and NJ’-methylenebisacrylamide, was used for gel formation. Five gm of Cyanogum 41 was disbuffer, pH 9.3, solved in 95 ml of 0.02 M Tris-HCl and the solution was filtered to remove a trace of insoluble matter. To the solution were added two catalysts, 1.0 ml of 10% dimethylaminopropionitryle (DMAPN) and 1.0 ml of 10% ammonium persulfate. All these solutions were freshly prepared and mixed just before use. The flask which contained the mixture was evacuated by an aspirator to avoid formation of air bubbles, and the solution was poured into a plastic mould (13 X 30 X 0.15 cm). The solution was covered with a plastic sheet and allowed to stand for 1 hour until gelled. Eight plastic pieces (1 X 0.1 X 0.05 cm) had been set in a row on the plastic cover to make slots for sample insertion. After gel formation was complete, samples were inserted into the slots. The polyacrylamide slab was connected by filter papers to the electrode vessels which contained 0.02 M Tris-HCl buffer, pH 9.3. Electrophoresis was
302
HYODO
AND
FIG. 3. Polyacrylamide gel electrophoretic pattern of polyphenol oxidases. IIa, IIb, IIc, IIIa, IIIb, and IV represent components IIa, IIb, IIc, IIIa, IIIb, and IV, respectively. A, B, C, D, E, F, G, and H indicate the points labeled in Fig. 2. carried out routinely at 16 V/cm and 0.4 mA/cm for 12 hours. The positions of polyphenol oxidases in polyacryalmide gel after electrophoresis were revealed by staining the gel with a solution made up of equal volumes of 0.9% caffeic acid and 0.1% p-phenylenediamine (5). Sephadex G-100 column chromatography. Sephadex G-106 (Pharmacia, Sweden) in a chromatographic column was pre-equilibrated with 0.02 M Tris-HCl buffer, pH 7.6, in the case of component IIIa fraction, or 0.01 M potassium phosphate buffer, pH 6.8, in the case of component IIIb fraction and the fractions obtained by DEAE-cellulose column chromatography. The column was eluted with the same buffer. Effluents were collected automatically by a G.M.E. fraction collector. DEAE-cellulose column chromatography for isolation of components IIIa and IIIb. To a DEAEcellulose column (1.8 X 24 cm) previously equilibrated with 0.02 M Tris-HCl buffer, pH 7.6, was applied 1.8 ml of the solution containing the component IIIa obtained with Sephadex G-100 column chromatography. The column was eluted with an exponential gradient, of sodium chloride from 0 to
URITANI 0.5 M. In the case of component IIIb, 4 ml of the solution obtained with Sephadex G-100 column chromatography was applied to DEAE-cellulose column (1.8 X 24 cm) previously equilibrated with 0.05 M Tris-HCI buffer, pH 7.6, containing 12.57, sucrose. The column was developed by the same buffer with an exponential gradient of sodium chloride from 0 to 1.0 -II. Assay of polyphenol oxidase activity. An oxygen electrode was used for determining polyphenol oxidase activity. The ingredients of the mixture in the reaction cell were as follows: 3.5 ml of 0.1 M potassium phosphate buffer (pH 6.0), 0.1 ml of the enzyme solution, and 0.1 ml of substrate solution in a total volume 3.7 ml. The procedures used in this assay and the unit of the enzyme activity are described in more detail elsewhere (7). Caffeic acid (5 X 1W M) adjusted to pH 6.0 was used as the substrate for routine assay system. In the experiments to measure Michaelis constants or to determine the substrate specificity, the following compounds were used: chlorogenic acid, pyrocatechol, pyrogallol, caffeic acid, dihydroxyphenylalanine, hydroquinone, p-coumaric acid, p-cresol, m-cresol, o-hydroxybenzoic acid, and phydroxyphenylpyruvic acid. Estimation of protein content. The protein content was determined by the method of Lowry et al. (8). The amount of protein was calculated on the basis of a standard curve prepared by using bovine serum albumin (fraction V, Armour & Co.).
Chemicals. Caffeic acid and pyrocatechol were recrystallized for determination of Michaelis constants. Chlorogenic acid and p-coumaric acid were provided by Dr. T. Minamikawa of the Department of Biology, Tokyo Metropolitan University, and Dr. H. Imaseki of this University, respectively. Sodium isoascorbate was given by Dr. S. Tsuruta of Fujisawa Yakuhin Kogyo Co. All other chemicals, unless otherwise stated, were purchased commercially. RESULTS
Isolation of components IIIa and IIIb. Figure 1 shows electrophoretic patterns of polyphenol oxidases extracted from acetone powders of both healthy and cut injured tissue of sweet potato roots. Three kinds of polyphenol oxidase, components IIa, IIb, and IIc, were present in healthy tissue, but in the extract from cut tissue, they were found to contain three more polyphenol oxidases in addition to the above three, which were designated as components IIIa, IIIb, and IV. Three components in healthy root tissue had been investigated as de-
POLYPHENOL
0
20
OXIDASE
IN WOUNDED
60
$0
SWEET
POTATO
303
80
EFFLUENT (tube number) FIG. 4. DEAE-cellulose column chromatogram of polyphenol oxidases. The solid line shows polyphenol oxidase activity in units, and the dotted line, optical density at 280 ml. The abscissa shows effluent (tube numbers). The points labeled with an arrow indicate the fractions which were subjected to polyacrylamide gel electrophoresis.
FIG. 5. Polyacrylamide gel electrophoretic pattern of polyphenol oxidases. IIb, IIIa, and IIIb represent components IIb, IIIa, and IIIb, respectively. A, B, C, D, E, F, G, and H indicate the points labeled in Fig. 4. The numbers 31, 33, 37, 39, 41, 45, 49, and 53 indicate the tube numbers of effluent in Fig. 4.
scribed in t.he previous paper (6, 7). Extracts from healthy tissue which were not injured by cutting contained only a slight amount of component IIIa. The appearance of com-
ponent IIIa might be due to physiological changes or slight damage during storage for about a year. Component IV in cut tissue extract was the only component that mi-
HYODO AND URITANI
20
60
40
EFFLUENT
(tube
SO
number)
FIG. 6. Sephadex G-100 column chromatogram
of polyphenol oxidases. The solid line shows polyphenol oxidase activity in units, and the dotted line, optical density at 280 q. The abscissa shows effluent (tube numbers). The points labeled with an arrow indicate the fractions which were subjected to polyacrylamide gel electrophoresis.
grated to the cathode. During the following isolation procedures of components IIIa and IIIb, a component called IIIc, which migrated far more than IIIb to the anode, was detected, as described below. The original polyphenol oxidase activity in crude extract from acetone powders was almost exclusively recovered (103.8 %) in the 30-55 % saturated ammonium sulfate fraction. One hundred-ml portions of the 30-55 % saturated fraction containing 3.44 gm of protein and 1.7 X lo5 units of polyphenol oxidase were applied to DEAE-cellulose column. The elution pattern is shown in Fig. 2. The components contained in several fractions were confirmed to be components IIa, IIb, IIc, IIIa, and IIlb by the polyacrylamide gel electrophoregram indicated in Fig. 3. That is, three peaks in the chromatographic pattern were essentially composed of components IIa, IIb, and IIIa, respectively. The subsequent tailing area after the appearance of the third peak was found to be a mixture mainly containing components IIb, IIIa, and IIIb. Component
IV was found to be present in the first peak as a minor component. Component IIb, which occupied the second peak, was free from other components, and its specific activity per milligram of protein was 94.5fold greater than that of the crude extract. The component IIIa containing the fraction collected from tubes 75-80 included also IIb and IIIb as minor components. Therefore, the fraction was subjected to Sephadex G-100 column chromatography. Component IIIa, however, could not be separated from the other two components. A further attempt to separate component IIIa from the other two was carried on by DEAE-cellulose column chromatography. As shown in Fig. 4, three peaks equivalent to three components were not obtained, but a single peak with two shoulders appeared. Hence the location of the three components was examined by polyacrylamide gel electrophoresis. As demonstrated in Fig. 5, it was found that only component IIIa was located at the central part of the peak, namely in the effluents from tubes 37-41.
POLYPHENOL
OXIDASE
IN WOUNDED
FIG. 7. Polyacrylamide gel electrophoretic pattern of polyphenol oxidases. IIb, IIIa, IIIb, and 111~ represent components IIb, IIIa, IIIb, and IIIc, respectively. A and B indicate the points labeled in Fig. 6.
With this isolated fraction some properties of component IIla were investigated, as indicated below. Effluents (2880 ml) of the column chromatography from tubes 81-224, composing the tailing part, were collected, and proteins were precipitated by the addition of ammonium sulfate to 80% saturation. The precipitate was dissolved in 0.01 M potassium phosphate buffer, pH 6.8, and the solution was dialyzed against the same buffer. The dialyzate (50 ml) was applied to a Sephadex G-100 column. The column was eluted with 0.01 M potassium phosphate buffer, pH 6.8. As shown in Fig. 6, two peaks appeared. According to polyacrylamide gel electrophoresis, the first peak constituted mainly component IIIb, and the second consisted of a mixture of components IIb, IIIa, IIIb, and
SWEET
POTATO
305
111~ (Fig. 7). In order to purify component IIIb, effluents making up the first peak were collected, concentrated, and rechromatographed on DEAE-cellulose column. Figures 8 and 9, which show the chromatograms and electrophoregrams, illustrate that the last peak constitutes only component IIIb. To elute component IIIb from DEAEcellulose column, sucrose was added to the elution buffer as described in Materials and Nesthods. The addition of sucrose was very effective to prevent tailing and to make a clear-cut elution profile. The specific activity in effluents corresponding to the last peak, which were used for the investigation of properties of component IIIb, corresponded to a 6.7-fold concentration over that in the crude extract. We at’tempted to isolate component 111~ from the mixture obtained as the last peak after Sephadex G-100 column chromatography. It was diflicult, however, to separate component 111~ from component IIIb by DEAE-cellulose column chromatography under varying conditions, and component 111~ has not yet been isolated. Conversion of the components in III group (components IIIa, IIIb, and 111~) to components of the II group (components IIa, IIb, and 11~) and of component IIIb to components IIIa and 111~ were not detected, when either mixture of components IIIa, IIIb, and 111~ or isolated component IIIb was left to stand for one month at 0” as the ammonium sulfate precipitate. The precipitates were then dissolved and chromatographed on a DEAE-cellulose column. Enzymic properties of components IIIa and IIIb and a comparison with those of component IIb. The pH-activity curves of components IIIa and IIIb were differed slightly when caffeic acid was used as the substrate. They also differed slightly from those of components IIb, IIa, and IIc (7). Components IIIa and IIIb were specific for o-diphenols and could oxidize neither monophenols such as p-coumaric acid, p-cresol, m-cresol, o-hydroxybenzoic acid, and p-hydroxyphenylpyruvic acid, nor polyphenols not having vicinal dihydroxy group such as hydroquinone. In the case of component IIlb, tyrosine was also used as a substrate, but it was not oxidized. This was in accord-
306
HYODO
AND
URITANI
40
EFFLUENT
60
80
(tube number)
FIG. 8. DEAE-cellulose column chromatogram of polyphenol oxidases. The solid line shows polyphenol oxidase activity in units, and the dotted line, optical density at 280 rnp. The abscissa shows effluent (tube numbers). The points labeled with an arrow indicate the fractions which were subjected to polyacrylamide gel electrophoresis.
ante with the substrate specificity of components IIa, IIb, and IIc (7). The Michaelis constants (K, values) for several substrates of components IIb, IIIa, and IIIb were determined by the Lineweaver-Burl< equation. The results are summarized in Table I. Components IIb and IIIa possessed the same K, values for chlorogenic acid, caffeic acid, pyrogallol, and pyrocatechol. All of the K, values of Component IIIb for the above four substrates were distinct from those of components IIb and IIIa. A higher affinity to caffeic acid and a lower affinity to chlorogenie acid of component IIIb were indicated. Concerning the maximum velocity, the value of component IIIb for chlorogenic acid was far greater than that for caffeic acid, whereas in the cases of components IIb and IIIa the relation was rather reversed. p-Coumaric acid was found to be a potent competitive inhibitor to the oxidation of caffeic acid by all of three components (Fig. 10). Since the enzyme activities of components IIla and IIIb were inhibited by copper chelating agents such as phenylthiourea, DIECA
(diethyldithiocarbamate), and KCN (Table II), they were regarded as copper enzymes as well as components IIa, IIb, and IIc (7). DISCUSSION
Two polyphenol oxidases (components IIIa and IIIb) produced in cut injured tissue of sweet potato roots were isolated by several steps, although their specific activities per milligram of protein were only slightly raised (4.6- and 6.7-fold than that of the crude extract). Thus it was demonstrated that components IIIa and IIIb were distinct from components IIa, IIb, and IIc as to electrophoretic and chromatographic behaviours and enzymic properties such as pHactivity curve, Michaelis constant, etc. The previous experiments indicated that component III was discriminated from component II in terms of immunological behavior
(3).
All polyphenol oxidases in sweet potato roots, so far as examined, were specific for o-diphenols. They could not oxidize monophenols. It is worthy to note, however, that
POLYPHENOL
OXIDASE
IN WOUNDED TABLE
hlIcH.&ELIS
307
POTATO
I
CONST.~NTS .&ND RELATIVE MAXIMUM VELOCITY VALUES FOR CHLOROGENIC: FOR p-COUM~RIC ACID, PYROC.~TECHOL, hi%~ PYROGALLOL, AND Ki VALUES IIb, IIIa, AND IIIb OF COMPONENTS
Value Km (1x1
Ki(M) v maxa
IIb,
SWEET
Compound
Chlorogenic acid Caffeic acid Pyrocatechol Pyrogallol p-Coumaric acid Chlorogenic acid Caffeic acid Pyrocat,echol Pyrogallol
III.?
2.9 2.9 1.3 1.8 4.8
II&l
x 10-S x 10-3 X 10-Z X 1OV X 1O-4 1.0 1.52 0.55 0.34
2.9 2.9 1.3 1.8 6.8
x 10-a x 10-S X 10-Z X 1O-2 X 1O-4 1.0 1.36 0.75 0.50
TABLE INHIBITORY AGENTS
C~FFEIC
IIIb
4.0 1.7 8.7 2.9 3.3
for chlorogenic a vlmx indicates the relative value when the maximum velocity IIIa, and IlIb represent components IIb, IIIa, and IIIb, respectively.
x IO-3 X 10-a X 1O-3 x 10-Z x 10-d 1.0 0.42 0.06 0.04
acid is taken
as 1.0.
II
EFFECT OF COPPER CHEL.~TING ON THE ENZYME ACTIVITY OF ETCH COMFONENT
Inhibitor
Phenylthiourea Diethyldithiocarbamate Potassium cyanide
FIG. 9. Polyacrylamide gel electrophoretic pattern of polyphenol oxidases. IIa, IIb, and IIIb represent components IIa, IIb, and IIIb, respect,ively. A, B, and C indicate the points labeled in Fig. 8.
ACID, ACID
Concentration CM)
5 1 5 1 5 1
x X x x x X
10-J lo-3 lo-4 lo-3 1*4 lcr3
Inhibition
(%)
IIIa
IIIb
80.6 86.1 22.2 19.4 83.3 97.2
62.7 82.3 46.4 57.2 42.9 53.7
p-coumaric acid, which has been assumed to be a precursor of caffeic acid or chlorogenic acid (9), was a potent competitive inhibitor to the enzymes. The affinity of p-coumaric acid to enzymes was stronger t,han that of substrates. It might be postulated that pcoumaric acid could be a substance which regulates the activity of polyphenol oxidases in intact cells by binding to the enzymes. Addition of sucrose to the elution buffer stimulated the elution of component IIIb through the DEAE-cellulose column. Sucrose may prevent absorption of polyphenol oxidases to polyhydroxy groups of DEAEcellulose. This suggests an assumption that polyphenol oxidases might be kept inactive in intact cells by binding with polyhydroxycontaining substances in cellular membrane. The previous papers reported that poly-
308
HYODO AND URITANI
lllb
0
,‘I. I/S
FIG. 10. Competitive
inhibition
4
3
2
(X103
of the activity
5
6
7
I/M)
of each component by p-coumaric acid.
IIb, IIIa, and IIIb represent components, IIb, IIIa, and IIIb, respectively. 0, without inhibitor; A, with 6.75 X 1W M p-coumaric acid.
phenol oxidase activity in the cut injured tissue increased in a sigmoidal fashion over a loo-hour period (4), and that the rise in the enzyme activity was presumably due to de nno2/osynthesis of enzyme protein (5). It has been widely known in the plant kingdom that polyphenol oxidase activity increases in response to infection or cutting. A role of
this enzyme in diseased tissue has been considered to be associated with a defense action of the host (l,lO). It will be of interest and importance to make clear a role or a meaning of polyphenol oxidases inducibly formed in the infected or cut tissue and to elucidate a triggering mechanism which causes synthesis of the enzyme protein.
POLYPHENOL
OXIDASE
IN WOUNDED
SCKNOWLEDGMENTS The authors are grateful to Professor M. A. Stahmann of the Department of Biochemistry, University of Wisconsin, for his valuable advice in preparing this manuscript. REFERENCES 1. FARKAS, G. L., AND KIR~~LY, Z., Phytopathol. 2. 44, 105 (1962). 2. GRITANI, I., AND MURAMATSU, K., J. Agr. Chem. Sot. Japan 26,289 (1952). 3. HYODO, H., AND URITANI, I., J. Biochem. 67, 161 (1965). 4. HYODO, H., AND URITANI, I., Plant Cell Physiol. 7, 137 (1966).
SWEET
POTATO
309
5. HYODO, H., AND URITANI, I., Agr. Biol. Chem. 30, 1083 (1966). 6. HYODO, H., AND BANDO, S., Bgr. Biol. Chem. 2S, 763 (1965). 7. HYODO, H., AND URITANI, I., J. Biochem. 68, 388 (1965). 8. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. BioZ. Chem. 193, 2G5 (1951). 9. BROWN, S. A,, Ann. Rev. Plant Physiol. 17, 223 (1966). 10. RUBIN, B. A., AND ARTSIKHOVSKAYA, YE. I’., “Biochemistry and Physiology of Plant Immunity.” Pergamon Press, New York (1963)