Planta (Berl.) 113, 115--128 (1973) 9 by Springer-Verlag 1973
A Proteinaceous Inhibitor of Ethylene Biosynthesis by Etiolated Mungbean Hypocotyl Sections Shingo Sakai and Hidemasa Imaseki Research Institute for Biochemical Regulation, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan l~eceived March 12/May 15, 1973
Su,rnmary. A protein which reversibly inhibits auxin-induced ethylene synthesis has been isolated and purified from hypocotyls of etiolated mungbean (Phaseolus aureus Roxb.) seedlings. The molecular weight of the inhibitor was estimated to be 112 000 by gel filtration and polyacrylamide gel-electrophoresis. When treated with sodium dodecylsulfate, the inhibitor gave on polyacrylamide gei-electrophoresis a single band corresponding to a molecular weight of 56 000, indicating that it consisted of two subunits with identical molecular weight. The inhibitor does not degrade nor bind indole-3-acetic acid, and has no peroxidase activity. Introduction
Ethylene is a biologically active substance which greatly influences m a n y growth phenomena in higher plants (Burg, 1962; P r a t t and Goeschl, 1969; Abeles, 1972). Almost all plants produce ethylene, but the rate of production varies depending upon species, age, and kind of organ or tissue. Except for model systems (Lieberman etal., 1965; Yang etal., 1966), ethylene production has only been detected in intact plants, organs or isolated tissues. Disruption of tissues that produce ethylene completely destroys their ethylene-producing activity, and an active ethylenesynthesizing enzyme system has so far never been isolated. Treatment with indole-3-acetic acid (IAA) greatly increases the rate of ethylene production in hypocotyl segments of etiolated mungbean seedlirlgs (Sakai and Imaseki, 1971). Working with mungbean hypocotyl segments, we recently found t h a t crushing one or two segments in the incubation medium severely inhibited ethylene production by other, intact segments in the same medium even though they had been treated with auxin. This result suggests the presence of an inhibitory substance in mungbean hypocotyl tissue which retards ethylene synthesis. We have isolated the inhibitory substance in a pure form and in the present paper, the purification and characterization of the inhibitor are described. The effects of the purified inhibitor on ethylene production and other processes are described elsewhere (Sakai and Imaseki, 1973). 8*
116
S. Sakai and H. Imaseki M a t e r i a l s and M e t h o d s
Plant Material. Etiolated seedlings of mungbean (Phaseolus aureus Roxb.) were grown on an agar-gel bed for 3 days as previously described (Sakai and Imaseki, 1971). For large-scale extraction, hypocotyls were collected and frozen at --15 ~ until a sufficient amount of the material was accumulated. Assay o/ Inhibitory Activity. Ten 6-ram long, subapical hypoeotyl segments excised from 3-days-old seedlings were incubated with 0.5 ml of auxin solution (0.5 mM IAA in 50 mM potassium phosphate buffer, pH 6.8, with 50 ~g/ml of chloramphenicol added) in a 25-ml glass vial sealed with a silicone stopper. The inhibitor was included in the incubation medium. After 6 h incubation at 30 ~ the ethylene content in the gas phase was measured with a gas chromatograph ~JGC-1100; Japan Electron Optics Lab., Tokyo). The amount of inhibitor that caused 50% inhibition'was defined as one unit of activity. All assays were run in triplicate. Isolation and Puri/ication o/the Inhibitor. 3 kg of hypocotyls was homogenized in a blender with 0.1 M phosphate buffer (pH 6.8) containing 0.5% sodium ascorbate and 5 mM 2-mercaptoethanol. The homogenate was filtered through 4 layers of cheese cloth, and the filtrate centrifuged at 9000 • g for 10 rain. (NHd)2SO a was added to the supernatant to give 80% saturation, and the precipitate formed was collected by centrifugation, dissolved in distilled water, and dialyzed against 50 mM phosphate buffer (pH 6.8) for 18 h with several changes of buffer. Any precipitate formed during dialysis was removed by centrifugation and the resultant supernatant was applied to a column of DEAE-cellulose (5.5 • 50 cm) equilibrated with the phosphate buffer. The material that was not absorbed to DEAE-cellulose at pH 6.8 was collected, and then applied to a hydroxyapatite column (5.5 • 39 cm) previously equilibrated with the same phosphate buffer. After the material was charged, the column was thoroughly washed with 0.1 M phosphate buffer (pH 6.8) and the inhibitor eluted with 0.2 M phosphate buffer. The inhibitor was precipitated by 80% saturation of the eluate with (NH~)2SO 4, collected by centrifugation, and dissolved in distilled water. After dialysis against 10 mM Tris-HC1 buffer at pH 8.0, the inhibitor solution was chromatographed on a DEAE-cellulose column (2.5 • 57 cm) with an increasing, linear gradient of NaC1 (0-0.15 M) in the above Tris buffer. Active fractions were combined and the inhibitor was concentrated by precipitation by (NHd)2SO 4 followed by dissolving the precipitate in 3 ml of 50 mM phosphate buffer (pH 6.8). The resulting inhibitor solution was next subjected to gel filtration with Sephadex G-200 using 50 mM phosphate buffer (pH 6.8) containing 0.1 M KC1. The inhibitor fractions were combined, dialyzed against 10 mM Tris-HC1 buffer (pH 8.0), and finally chromatographed on a DE-32 column with an increasing, linear gradient of NaC1 (0-0.1 M) in the above Tris buffer. The highly purified inhibitor was precipitated by dialyzing the combined active fractions against saturated (NH~)2SO a solution. The precipitate was thoroughly dialyzed against 50 mM phosphate buffer (pit 6.8) before use. All procedures were carried out at 4 ~, and all buffers used for dialysis and chromatography contained I mM 2-mercaptoethanol. Estimation o/ Molecular Weight. The molecular weight of the inhibitor was estimated by gel filtration with Sephadex G-200 and by polyacrylamide gelelectrophoresis. A Sephadex G-200 column (2.6 • 90 cm) previously washed with 50 mM phosphate buffer (pH 6.8) containing 0.1 M KC1 was employed, and elntion was conducted with an upward flow with a flow rate of 10 ml h -1. Polyacrylamide gel electrophoresis was carried out with four different concentrations of gel after Hedrick and Smith (1969). Marker proteins employed were purchased from Boehringer, Mannheim, Germany.
Inhibitor of Ethylene Biosynthesis
117
Disc Polyacrylamide Gel-electrophoresis. Disc gel-electrophoresis was carried out by the method of Davis (1964), using a 7.5% gel and a discontinuous buffer system of Tris-glycine (pH 9.4). The gel was stained with Coomassie brilliant blue. For the measurement of inhibitory activity in the gel, unstained gel was consecutively cut into 2-mm-thiek slices. Each slice was placed in a glass vial containing 0.5 ml of auxin medium, then the gel slice was crushed with a glass rod. The gel suspension thus obtained was allowed to stand for 1 h to facilitate extraction of the inhibitor. Then, 10 hypocotyl segments were added to each vial, sealed, and incubated for ethylene production. SDS-Polyacrylamide Gel-electrophoresis. Protein solutions, prepared with 10 mM sodium phosphate buffer (pit 7.0) containing 1% soldium dodecylsulfate (SDS) and 1% 2-mercapteethanol, were incubated at 37 ~ for 4 h and subjected to electrophoresis on 7% acrylamide gel containing 0.1% SDS (Weber and Osborn, 1969). The gels were stained with Coomassie brilliant blue and the molecular weight of the subnnit of the inhibitor was estimated from its mobility. Protein Determination. The amount of protein was measured by the method of Lowry et al. (1951), with bovine serum albumin as standard. IAA-degrading Activity. 50 [zg of the purified inhibitor (41.6 units) was incubated with 2 ~5I [carboxyl-laCJIAA (0.0814 lzCi) in 50 mM phosphate buffer, pH 6.8, with or without an ethanol extract of hypocotyls in a total volume of 0.3 ml. The incubation was carried out at 30 ~ for 1 h in a 5-ml glass vial sealed with a stopper. After the incubation period, the air over the reaction mixture was introduced into an evacuated counting vial which contained 0.2 ml of 20% KOH, and the radioactivity trapped by the K O H was measured. A portion of the reaction mixture was directly subjected to paper chromatography with isopropanolammonium hydroxide-water (8:1:1, v/v) as solvent. A zone of a paper strip corresponding to authentic IAA was cut out and radioactivity of the section was measured with a liquid scintillation spectrometer (Packard Tri-Carb 2311). Equilibrium Dialysis o] the Inhibitor against IAA. An inhibitor solution (0.25 mg protein in 0.5 ml of 50 mM phosphate buffer, p H 6.8) was placed in a dialysis bag and dialyzed against 20 ml of phosphate buffer containing 1.4~M [carboxyl-laC]IAA (0.4 ~Ci) at 4 ~ for 18 h in the dark. The radioactivities of both external and internal solutions were measured. Results
Nature o/the Inhibitor As s h o w n in T a b l e 1, t h e i n h i b i t o r y a c t i v i t y f o u n d in a c r u d e e x t r a c t was h e a t s t a b l e a n d n o n - d i a l y z a b l e , a n d e t h y l e n e p r o d u c t i o n i n d u c e d b y 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d was i n h i b i t e d as well as I A A - i n d u c e d p r o d u c t i o n . I t was also f o u n d t h a t t h e i n h i b i t o r y a c t i v i t y w a s n o t i m p a i r e d b y c h l o r o g e n i c acid, a n d t h a t a b o i l e d c r u d e e x t r a c t r e t a i n i n g inhibitory activity had no peroxidase activity. When a crude extract was s u b j e c t e d to gel f i l t r a t i o n w i t h S e p h a d e x G-200, i n h i b i t o r y a c t i v i t y was d i s t r i b u t e d o v e r n u m e r o u s f r a c t i o n s f r o m a v e r y h i g h - m o l e c u l a r fract i o n to one w i t h a m o l e c u l a r w e i g h t c o r r e s p o n d i n g t o a b o u t 100000 (Fig. 1). H o w e v e r , w h e n t h e s e p a r a t e d f r a c t i o n s w e r e h e a t e d o v e r a boiling water bath and their supernatants obtained after centrifugation assayed, t h e i n h i b i t o r y a c t i v i t y of t h e s l o w - m o v i n g f r a c t i o n s r e m a i n e d in s o l u t i o n
118
S. Sakai and H. Imaseki
1""r ~
1.6
80
t
;,, ,,, E 1.2 0
/
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~:
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:.:.:.:.:-:.:.:-:-:-:.:.:,:,:.:-:,
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40 Fraction
60 Number
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Fig. 1. Elution profile of crude proteins from mungbean hypocotyls over a Sephadex G-200 column. Precipitate obtained from a buffer extract of 15 g of hypoeotyls b y 80% saturated (NH4)pSO a was applied to t h e column. ---- absorbance at 280 n m ; o inhibitory activities in separated fractions; @ inhibitory activities of supernatants obtained from heat-treated fractions
Table 1. Effect of a crude extract of m u n g b e a n hypocotyl on auxin-induced ethylene production in etiolated m u n g b e a n hypocotyl segments Auxin used
Exp. I
IAA (0.5 mM)
Exp. I I I A A (0.5 mM) 2,4-D (0.5 mM)
Additions
CpH4 produced (nI/10 segments/6 h)
Inhibition (%)
None 200 Crude extract 15 Crude extract, boiled 30 Crude extract, boiled 36 and dialyzed Chlorogenic acid, 0.2 m~[ 198 Crude extract and 32 chlorogenic acid
-93 85 82
None Crude extract None Crude extract
-92 -90
203 17 164 ]6
1 84
Inhibitor of Ethylene Biosynthesis
119
Table 2. Distribution of the inhibitory activity of crude extract into DEAE-passed and -absorbed fractions under different conditions of extraction Tissue
2-MEa
Heat treatment
ground by
fr. wt.
Glass homogenizer Glass homogenizer
60 g 60 g
---
-~
Blender Blender
3 kg 3 kg
-+
---
Total activity (units)
Distribution of activity (%) DEAEpassed
DEAEabsorbed
2890 2 730
43.5 3
56.5 97
91410 107312
47.7 54.6
52.3 45.4
a 2-mercaptoethanol, 5 m3/[ in the extraction medium.
Table 3. Effect of proteolytic enzymes on a partially purified inhibitor Additions
C2H4 produced (nl/10 segments/6 h)
Inhibition
None Inhibitor a Pronase treated inhibitorb Boiled pronase c Trypsin-treated inhibitorb Boiled trypsinc
219 83 219 220 202 220
-62 0 0 8 0
a A fraction passed through DEAE-cellulose column at pH 6.8. b The supernatant obtained after centrifugation of the boiled reaction mixture (partially purified inhibitor, 1 mg protein, and 4 mg of proteolytic enzyme in 4 ml of 50 mM phosphate buffer, pit 6.8). e The supernatant of boiled proteolytic enzyme.
(shaded area i n Fig. 1). While this c o m p o n e n t was n o t absorbed to D E A E cellulose at p I I 6.8, a higher molecular, heat-precipitable c o m p o n e n t was r e t a i n e d in the column. A l t h o u g h t o t a l i n h i b i t o r y a c t i v i t y i n crude e x t r a c t was n o t changed b y a heat t r e a t m e n t , the t r e a t m e n t g r e a t l y diminished the a m o u n t of the form which passed the D E A E . I n contrast, w h e n 5 m M 2 - m e r c a p t o e t h a n o l was included i n the homogenizing m e d i u m , the a m o u n t of the D E A E - p a s s e d form was f o u n d to increase (Table 2). As the l a t t e r c o m p o n e n t seemed to be a n artifact, o n l y the former comp o n e n t was e x a m i n e d further. As shown i n Table 3, i n h i b i t o r y a c t i v i t y i n the p a r t i a l l y purified p r e p a r a t i o n was almost completely lost after 2 h t r e a t m e n t of the p r e p a r a t i o n w i t h pronase ( K a k e n K a g a k u Co., Tokyo, J a p a n ) or t r y p s i n (Miles Labs., E l k h a r t , Ill., U.S.A.) a t 38%
120
S. Sakai and H. Imaseki
200
O m c
v
"o
==
ioo
9
n
cD
N 5o
o
I
2 3 4 5 Incubation Period (hours)
6
Fig. 2. Effect of partially purified inhibitor, a fraction passed through DEAEcellulose at pit 6.8, on auxin-induced ethylene production. 9 control, no inhibitor added; 9 inhibitor added at 3rd h (~); 9 inhibitor added at Oh; [] inhibitor removed at 3rd h (1)
T r e a t m e n t of the inhibitor preparation with N a I O 4 for 18 h at 30 ~ did not destroy the inhibitory activity. The action of the inhibitor is reversible (Fig. 2). Addition of the inhibitor at a n y time after segments are induced to produce ethylene with auxin results in an immediate cessation of ethylene production. Conversely, if the inhibited segments are washed to free t h e m from added inhibitor, the rate of ethylene production increases to t h a t of control segments stimulated with auxin.
Puri/ication o/the Inhibitor DEAE-cellulose c h r o m a t o g r a p h y of a crude extract at p i t 6.8 efficiently separated the inhibitor from other inhibitory substances. F u r t h e r purification of inhibitor b y successive column c h r o m a t o g r a p h y with hydroxylapatite, DEAE-cellulose at p H 8.0, Sephadex G-200 and DE-32 finally gave an inhibitor preparation which contained a single b a n d on polyacrylamide gel-elcctrophoresis (Fig. 3). I n h i b i t o r y activity on the gel
Inhibitor of Ethylene Biosynthesis i
i
,
i
i
gel top
121
i
i
dye front
4
4
II
r (-)
I
(+)
60
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20
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i
i ~
0
I
r
I
I
I
i
2
3
4
5
6
Gel Length
(cm)
Fig. 3. Polyacrylamide gel electrophoresis of the purified inhibitor
Table 4. Purification of a n inhibitor of ethylene production from mungbean hypocotyl Purification step
Crude extract
Volume Protein
Activity
(ml)
(mg)
(units)
Specific Degree activity of purl(units/mg fication protein) (• )
Yield
Thermostability
(%)
7170
12900
92525 a
7.2
1
80% (NH4)~SO 4
920
9856
91414 a
9.3
1.3
100 98.8
DEAE-cellulose (pH 6.8)
1400
2 292
43613 a
19.3
2.7
47.1
185
+
Hydroxyapatite
1500
32862
177.7
24.7
35.5
--
DEAE-cellulose (pH 8.0)
298
74.5
15684
210.5
29.2
20.0
--
Sephadex G 200
106
36.5
10495
287.5
39.3
11.3
--
86
8.5
7363
866.2
120.3
8.0
--
D E 32
a For activity measurement of those fractions, 0.2 M ehlorogenic acid was added to the incubation medium to prevent oxidative reactions b y peroxidase.
122
S. Sakai and H. Imaseki: Inhibitor of Ethylene Biosynthesis
--~100
a_ 8o o)
~ 6o ~40
I
0
I
I
I
I 2 3 4 Inhibitor added (.ug protein)
i
5
Fig. 4. Concentration-activity relationship of the purified inhibitor
coincided with the stained band. With 7.5% acrylamide gel and Trisglycine buffer, p H 8.9, the inhibitor has an l%m value (mobility relative to the tracking dye) of 0.23. A summary of the purification procedure is presented in Table 4. At the final step, ca. 120fold purification was achieved with on overall yield of 8 %. At each step of the purification, the thermostability of the inhibitory activity was examined by heating the preparation over a boiling water bath for 5 min. The thermostability was lost when the inhibitor had been absorbed to an ion exchanger. It did not matter where, as in the procedure described here, we used hydroxyapatite before the second DEAE-cellulose chromatography was run before the hydroxyapatite chromatography. Fig. 4 shows a concentration-activity relationship; one unit of the inhibitory activity corresponded to 1.2 ~g of the inhibitor.
Molecular Weight o[ the Inhibitor The elution pattern of the purified inhibitor from a Sephadex G-200 column showed a symmetrical, single peak for inhibitory activity coincident with the absorbance peak at 280 nm (Fig. 5). The ratio of the elution volume of the inhibitor to the elution volume of blue dextran (Ve/Vo) was 1.525. From the calibration curve made with standard proteins, a molecular weight of about 112000 was estimated for the inhibitor (Fig. 6). The molcular weight was also determined by the method of Hedrick and Smith (1968). Fig. 7a shows the effect of different gel concentration on the mobility of the inhibitor protein. The slope of the linear line obtained was 7.38, giving a molecular weight of 110000 (Fig. 7b).
J
L
I00
0.1
E c o co o,1
E ~o
"6
~o.o5
50
A Proteinaceous Inhibitor of Ethylene Biosynthesis by Etiolated Mungbean Hypocotyl Sections Shingo Sakai and Hidemasa Imaseki Research Institute for Biochemical Regulation, Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan l~eceived March 12/May 15, 1973
Su,rnmary. A protein which reversibly inhibits auxin-induced ethylene synthesis has been isolated and purified from hypocotyls of etiolated mungbean (Phaseolus aureus Roxb.) seedlings. The molecular weight of the inhibitor was estimated to be 112 000 by gel filtration and polyacrylamide gel-electrophoresis. When treated with sodium dodecylsulfate, the inhibitor gave on polyacrylamide gei-electrophoresis a single band corresponding to a molecular weight of 56 000, indicating that it consisted of two subunits with identical molecular weight. The inhibitor does not degrade nor bind indole-3-acetic acid, and has no peroxidase activity. Introduction
Ethylene is a biologically active substance which greatly influences m a n y growth phenomena in higher plants (Burg, 1962; P r a t t and Goeschl, 1969; Abeles, 1972). Almost all plants produce ethylene, but the rate of production varies depending upon species, age, and kind of organ or tissue. Except for model systems (Lieberman etal., 1965; Yang etal., 1966), ethylene production has only been detected in intact plants, organs or isolated tissues. Disruption of tissues that produce ethylene completely destroys their ethylene-producing activity, and an active ethylenesynthesizing enzyme system has so far never been isolated. Treatment with indole-3-acetic acid (IAA) greatly increases the rate of ethylene production in hypocotyl segments of etiolated mungbean seedlirlgs (Sakai and Imaseki, 1971). Working with mungbean hypocotyl segments, we recently found t h a t crushing one or two segments in the incubation medium severely inhibited ethylene production by other, intact segments in the same medium even though they had been treated with auxin. This result suggests the presence of an inhibitory substance in mungbean hypocotyl tissue which retards ethylene synthesis. We have isolated the inhibitory substance in a pure form and in the present paper, the purification and characterization of the inhibitor are described. The effects of the purified inhibitor on ethylene production and other processes are described elsewhere (Sakai and Imaseki, 1973). 8*
116
S. Sakai and H. Imaseki M a t e r i a l s and M e t h o d s
Plant Material. Etiolated seedlings of mungbean (Phaseolus aureus Roxb.) were grown on an agar-gel bed for 3 days as previously described (Sakai and Imaseki, 1971). For large-scale extraction, hypocotyls were collected and frozen at --15 ~ until a sufficient amount of the material was accumulated. Assay o/ Inhibitory Activity. Ten 6-ram long, subapical hypoeotyl segments excised from 3-days-old seedlings were incubated with 0.5 ml of auxin solution (0.5 mM IAA in 50 mM potassium phosphate buffer, pH 6.8, with 50 ~g/ml of chloramphenicol added) in a 25-ml glass vial sealed with a silicone stopper. The inhibitor was included in the incubation medium. After 6 h incubation at 30 ~ the ethylene content in the gas phase was measured with a gas chromatograph ~JGC-1100; Japan Electron Optics Lab., Tokyo). The amount of inhibitor that caused 50% inhibition'was defined as one unit of activity. All assays were run in triplicate. Isolation and Puri/ication o/the Inhibitor. 3 kg of hypocotyls was homogenized in a blender with 0.1 M phosphate buffer (pH 6.8) containing 0.5% sodium ascorbate and 5 mM 2-mercaptoethanol. The homogenate was filtered through 4 layers of cheese cloth, and the filtrate centrifuged at 9000 • g for 10 rain. (NHd)2SO a was added to the supernatant to give 80% saturation, and the precipitate formed was collected by centrifugation, dissolved in distilled water, and dialyzed against 50 mM phosphate buffer (pH 6.8) for 18 h with several changes of buffer. Any precipitate formed during dialysis was removed by centrifugation and the resultant supernatant was applied to a column of DEAE-cellulose (5.5 • 50 cm) equilibrated with the phosphate buffer. The material that was not absorbed to DEAE-cellulose at pH 6.8 was collected, and then applied to a hydroxyapatite column (5.5 • 39 cm) previously equilibrated with the same phosphate buffer. After the material was charged, the column was thoroughly washed with 0.1 M phosphate buffer (pH 6.8) and the inhibitor eluted with 0.2 M phosphate buffer. The inhibitor was precipitated by 80% saturation of the eluate with (NH~)2SO 4, collected by centrifugation, and dissolved in distilled water. After dialysis against 10 mM Tris-HC1 buffer at pH 8.0, the inhibitor solution was chromatographed on a DEAE-cellulose column (2.5 • 57 cm) with an increasing, linear gradient of NaC1 (0-0.15 M) in the above Tris buffer. Active fractions were combined and the inhibitor was concentrated by precipitation by (NHd)2SO 4 followed by dissolving the precipitate in 3 ml of 50 mM phosphate buffer (pH 6.8). The resulting inhibitor solution was next subjected to gel filtration with Sephadex G-200 using 50 mM phosphate buffer (pH 6.8) containing 0.1 M KC1. The inhibitor fractions were combined, dialyzed against 10 mM Tris-HC1 buffer (pH 8.0), and finally chromatographed on a DE-32 column with an increasing, linear gradient of NaC1 (0-0.1 M) in the above Tris buffer. The highly purified inhibitor was precipitated by dialyzing the combined active fractions against saturated (NH~)2SO a solution. The precipitate was thoroughly dialyzed against 50 mM phosphate buffer (pit 6.8) before use. All procedures were carried out at 4 ~, and all buffers used for dialysis and chromatography contained I mM 2-mercaptoethanol. Estimation o/ Molecular Weight. The molecular weight of the inhibitor was estimated by gel filtration with Sephadex G-200 and by polyacrylamide gelelectrophoresis. A Sephadex G-200 column (2.6 • 90 cm) previously washed with 50 mM phosphate buffer (pH 6.8) containing 0.1 M KC1 was employed, and elntion was conducted with an upward flow with a flow rate of 10 ml h -1. Polyacrylamide gel electrophoresis was carried out with four different concentrations of gel after Hedrick and Smith (1969). Marker proteins employed were purchased from Boehringer, Mannheim, Germany.
Inhibitor of Ethylene Biosynthesis
117
Disc Polyacrylamide Gel-electrophoresis. Disc gel-electrophoresis was carried out by the method of Davis (1964), using a 7.5% gel and a discontinuous buffer system of Tris-glycine (pH 9.4). The gel was stained with Coomassie brilliant blue. For the measurement of inhibitory activity in the gel, unstained gel was consecutively cut into 2-mm-thiek slices. Each slice was placed in a glass vial containing 0.5 ml of auxin medium, then the gel slice was crushed with a glass rod. The gel suspension thus obtained was allowed to stand for 1 h to facilitate extraction of the inhibitor. Then, 10 hypocotyl segments were added to each vial, sealed, and incubated for ethylene production. SDS-Polyacrylamide Gel-electrophoresis. Protein solutions, prepared with 10 mM sodium phosphate buffer (pit 7.0) containing 1% soldium dodecylsulfate (SDS) and 1% 2-mercapteethanol, were incubated at 37 ~ for 4 h and subjected to electrophoresis on 7% acrylamide gel containing 0.1% SDS (Weber and Osborn, 1969). The gels were stained with Coomassie brilliant blue and the molecular weight of the subnnit of the inhibitor was estimated from its mobility. Protein Determination. The amount of protein was measured by the method of Lowry et al. (1951), with bovine serum albumin as standard. IAA-degrading Activity. 50 [zg of the purified inhibitor (41.6 units) was incubated with 2 ~5I [carboxyl-laCJIAA (0.0814 lzCi) in 50 mM phosphate buffer, pH 6.8, with or without an ethanol extract of hypocotyls in a total volume of 0.3 ml. The incubation was carried out at 30 ~ for 1 h in a 5-ml glass vial sealed with a stopper. After the incubation period, the air over the reaction mixture was introduced into an evacuated counting vial which contained 0.2 ml of 20% KOH, and the radioactivity trapped by the K O H was measured. A portion of the reaction mixture was directly subjected to paper chromatography with isopropanolammonium hydroxide-water (8:1:1, v/v) as solvent. A zone of a paper strip corresponding to authentic IAA was cut out and radioactivity of the section was measured with a liquid scintillation spectrometer (Packard Tri-Carb 2311). Equilibrium Dialysis o] the Inhibitor against IAA. An inhibitor solution (0.25 mg protein in 0.5 ml of 50 mM phosphate buffer, p H 6.8) was placed in a dialysis bag and dialyzed against 20 ml of phosphate buffer containing 1.4~M [carboxyl-laC]IAA (0.4 ~Ci) at 4 ~ for 18 h in the dark. The radioactivities of both external and internal solutions were measured. Results
Nature o/the Inhibitor As s h o w n in T a b l e 1, t h e i n h i b i t o r y a c t i v i t y f o u n d in a c r u d e e x t r a c t was h e a t s t a b l e a n d n o n - d i a l y z a b l e , a n d e t h y l e n e p r o d u c t i o n i n d u c e d b y 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d was i n h i b i t e d as well as I A A - i n d u c e d p r o d u c t i o n . I t was also f o u n d t h a t t h e i n h i b i t o r y a c t i v i t y w a s n o t i m p a i r e d b y c h l o r o g e n i c acid, a n d t h a t a b o i l e d c r u d e e x t r a c t r e t a i n i n g inhibitory activity had no peroxidase activity. When a crude extract was s u b j e c t e d to gel f i l t r a t i o n w i t h S e p h a d e x G-200, i n h i b i t o r y a c t i v i t y was d i s t r i b u t e d o v e r n u m e r o u s f r a c t i o n s f r o m a v e r y h i g h - m o l e c u l a r fract i o n to one w i t h a m o l e c u l a r w e i g h t c o r r e s p o n d i n g t o a b o u t 100000 (Fig. 1). H o w e v e r , w h e n t h e s e p a r a t e d f r a c t i o n s w e r e h e a t e d o v e r a boiling water bath and their supernatants obtained after centrifugation assayed, t h e i n h i b i t o r y a c t i v i t y of t h e s l o w - m o v i n g f r a c t i o n s r e m a i n e d in s o l u t i o n
118
S. Sakai and H. Imaseki
1""r ~
1.6
80
t
;,, ,,, E 1.2 0
/
:.:.:.:.:.:.:.:.:. '::':':::+:':
a 0.8
L o
:':-2":':-:':':-:';
i \
0.4
s ---~
,I
/
/t
,
6 0 ~_
',
~0
~
"-~-
~,...v...v...,.,,
~
:.:.:.:.:,:-:.:.:,:,
I
!:i:!:!:i:i:i:i:i:i:i ...:i:i:!:!:i:i:i:!:i:i :::::::::::::::::::::::::::::::
~:
~ ~
2 0 __=
:.:.:.:.:-:.:.:-:-:-:.:.:,:,:.:-:,
0
20
40 Fraction
60 Number
80
IO0
Fig. 1. Elution profile of crude proteins from mungbean hypocotyls over a Sephadex G-200 column. Precipitate obtained from a buffer extract of 15 g of hypoeotyls b y 80% saturated (NH4)pSO a was applied to t h e column. ---- absorbance at 280 n m ; o inhibitory activities in separated fractions; @ inhibitory activities of supernatants obtained from heat-treated fractions
Table 1. Effect of a crude extract of m u n g b e a n hypocotyl on auxin-induced ethylene production in etiolated m u n g b e a n hypocotyl segments Auxin used
Exp. I
IAA (0.5 mM)
Exp. I I I A A (0.5 mM) 2,4-D (0.5 mM)
Additions
CpH4 produced (nI/10 segments/6 h)
Inhibition (%)
None 200 Crude extract 15 Crude extract, boiled 30 Crude extract, boiled 36 and dialyzed Chlorogenic acid, 0.2 m~[ 198 Crude extract and 32 chlorogenic acid
-93 85 82
None Crude extract None Crude extract
-92 -90
203 17 164 ]6
1 84
Inhibitor of Ethylene Biosynthesis
119
Table 2. Distribution of the inhibitory activity of crude extract into DEAE-passed and -absorbed fractions under different conditions of extraction Tissue
2-MEa
Heat treatment
ground by
fr. wt.
Glass homogenizer Glass homogenizer
60 g 60 g
---
-~
Blender Blender
3 kg 3 kg
-+
---
Total activity (units)
Distribution of activity (%) DEAEpassed
DEAEabsorbed
2890 2 730
43.5 3
56.5 97
91410 107312
47.7 54.6
52.3 45.4
a 2-mercaptoethanol, 5 m3/[ in the extraction medium.
Table 3. Effect of proteolytic enzymes on a partially purified inhibitor Additions
C2H4 produced (nl/10 segments/6 h)
Inhibition
None Inhibitor a Pronase treated inhibitorb Boiled pronase c Trypsin-treated inhibitorb Boiled trypsinc
219 83 219 220 202 220
-62 0 0 8 0
a A fraction passed through DEAE-cellulose column at pH 6.8. b The supernatant obtained after centrifugation of the boiled reaction mixture (partially purified inhibitor, 1 mg protein, and 4 mg of proteolytic enzyme in 4 ml of 50 mM phosphate buffer, pit 6.8). e The supernatant of boiled proteolytic enzyme.
(shaded area i n Fig. 1). While this c o m p o n e n t was n o t absorbed to D E A E cellulose at p I I 6.8, a higher molecular, heat-precipitable c o m p o n e n t was r e t a i n e d in the column. A l t h o u g h t o t a l i n h i b i t o r y a c t i v i t y i n crude e x t r a c t was n o t changed b y a heat t r e a t m e n t , the t r e a t m e n t g r e a t l y diminished the a m o u n t of the form which passed the D E A E . I n contrast, w h e n 5 m M 2 - m e r c a p t o e t h a n o l was included i n the homogenizing m e d i u m , the a m o u n t of the D E A E - p a s s e d form was f o u n d to increase (Table 2). As the l a t t e r c o m p o n e n t seemed to be a n artifact, o n l y the former comp o n e n t was e x a m i n e d further. As shown i n Table 3, i n h i b i t o r y a c t i v i t y i n the p a r t i a l l y purified p r e p a r a t i o n was almost completely lost after 2 h t r e a t m e n t of the p r e p a r a t i o n w i t h pronase ( K a k e n K a g a k u Co., Tokyo, J a p a n ) or t r y p s i n (Miles Labs., E l k h a r t , Ill., U.S.A.) a t 38%
120
S. Sakai and H. Imaseki
200
O m c
v
"o
==
ioo
9
n
cD
N 5o
o
I
2 3 4 5 Incubation Period (hours)
6
Fig. 2. Effect of partially purified inhibitor, a fraction passed through DEAEcellulose at pit 6.8, on auxin-induced ethylene production. 9 control, no inhibitor added; 9 inhibitor added at 3rd h (~); 9 inhibitor added at Oh; [] inhibitor removed at 3rd h (1)
T r e a t m e n t of the inhibitor preparation with N a I O 4 for 18 h at 30 ~ did not destroy the inhibitory activity. The action of the inhibitor is reversible (Fig. 2). Addition of the inhibitor at a n y time after segments are induced to produce ethylene with auxin results in an immediate cessation of ethylene production. Conversely, if the inhibited segments are washed to free t h e m from added inhibitor, the rate of ethylene production increases to t h a t of control segments stimulated with auxin.
Puri/ication o/the Inhibitor DEAE-cellulose c h r o m a t o g r a p h y of a crude extract at p i t 6.8 efficiently separated the inhibitor from other inhibitory substances. F u r t h e r purification of inhibitor b y successive column c h r o m a t o g r a p h y with hydroxylapatite, DEAE-cellulose at p H 8.0, Sephadex G-200 and DE-32 finally gave an inhibitor preparation which contained a single b a n d on polyacrylamide gel-elcctrophoresis (Fig. 3). I n h i b i t o r y activity on the gel
Inhibitor of Ethylene Biosynthesis i
i
,
i
i
gel top
121
i
i
dye front
4
4
II
r (-)
I
(+)
60
._o =
o CL
40 LIJ
20
P, c
i
i ~
0
I
r
I
I
I
i
2
3
4
5
6
Gel Length
(cm)
Fig. 3. Polyacrylamide gel electrophoresis of the purified inhibitor
Table 4. Purification of a n inhibitor of ethylene production from mungbean hypocotyl Purification step
Crude extract
Volume Protein
Activity
(ml)
(mg)
(units)
Specific Degree activity of purl(units/mg fication protein) (• )
Yield
Thermostability
(%)
7170
12900
92525 a
7.2
1
80% (NH4)~SO 4
920
9856
91414 a
9.3
1.3
100 98.8
DEAE-cellulose (pH 6.8)
1400
2 292
43613 a
19.3
2.7
47.1
185
+
Hydroxyapatite
1500
32862
177.7
24.7
35.5
--
DEAE-cellulose (pH 8.0)
298
74.5
15684
210.5
29.2
20.0
--
Sephadex G 200
106
36.5
10495
287.5
39.3
11.3
--
86
8.5
7363
866.2
120.3
8.0
--
D E 32
a For activity measurement of those fractions, 0.2 M ehlorogenic acid was added to the incubation medium to prevent oxidative reactions b y peroxidase.
122
S. Sakai and H. Imaseki: Inhibitor of Ethylene Biosynthesis
--~100
a_ 8o o)
~ 6o ~40
I
0
I
I
I
I 2 3 4 Inhibitor added (.ug protein)
i
5
Fig. 4. Concentration-activity relationship of the purified inhibitor
coincided with the stained band. With 7.5% acrylamide gel and Trisglycine buffer, p H 8.9, the inhibitor has an l%m value (mobility relative to the tracking dye) of 0.23. A summary of the purification procedure is presented in Table 4. At the final step, ca. 120fold purification was achieved with on overall yield of 8 %. At each step of the purification, the thermostability of the inhibitory activity was examined by heating the preparation over a boiling water bath for 5 min. The thermostability was lost when the inhibitor had been absorbed to an ion exchanger. It did not matter where, as in the procedure described here, we used hydroxyapatite before the second DEAE-cellulose chromatography was run before the hydroxyapatite chromatography. Fig. 4 shows a concentration-activity relationship; one unit of the inhibitory activity corresponded to 1.2 ~g of the inhibitor.
Molecular Weight o[ the Inhibitor The elution pattern of the purified inhibitor from a Sephadex G-200 column showed a symmetrical, single peak for inhibitory activity coincident with the absorbance peak at 280 nm (Fig. 5). The ratio of the elution volume of the inhibitor to the elution volume of blue dextran (Ve/Vo) was 1.525. From the calibration curve made with standard proteins, a molecular weight of about 112000 was estimated for the inhibitor (Fig. 6). The molcular weight was also determined by the method of Hedrick and Smith (1968). Fig. 7a shows the effect of different gel concentration on the mobility of the inhibitor protein. The slope of the linear line obtained was 7.38, giving a molecular weight of 110000 (Fig. 7b).
J
L
I00
0.1
E c o co o,1
E ~o
"6
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50
