Plant & Cell Physiol. 21 (8): 1475-1482 (1980)
Inorganic pyrophosphatase from. pollen of Typha latifolia Akira Hara, Kousei Kawamoto and Tooru Funaguma
(Received August 27, 1980)
An inorganic pyrophosphatase was purified about 3,800-fold from the pollen of Typha latifolia by chromatography on DEAE-Sephadex A-50, isoelectric focusing and gel filtration through Sephadex G-75. The enzyme had an optimum pH between 8.59.5 and required M g 2+. Since an excess of pyrophosphate over Mg 2+ inhibited the pyrophosphatase reaction, the actual substrate may have been an Mg-pyrophosphate complex. The enzyme degraded inorganic pyrophosphate specifically, showing a Km value of 7.6 X 10-5 M. A possible role of pyrophosphatase was discussed in connection with starch-sucrose conversion. Key words: Inorganic pyrophosphatase - Pollen enzyme - Typhalatifolia.
Inorganic pyrophosphate is produced as a by-product in several biosynthetic reactions, for example, activation of amino acids, polymerization of nucleic acids, and syntheses of coenzymes and sugar nucleotides. Inorganic pyrophosphatase (pyrophosphate phosphohydrolase; EC 3.6.1.1) is likely to promote these biosynthetic reactions by degrading inorganic pyrophosphate into orthophosphate (9). In plants, pyrophosphatases have been investigated in connection with leaf senescence and photosynthesis (8, 10, 13, 14, 17). Simmons and Butler have reported that a large fraction of the total pyrophosphatase activity of the leaf exists in nonaqueous preparations of maize chloroplasts (18). We found an exceedingly high activity of M g2+-dependent alkaline pyrophosphatase in the pollen of Japanese cycad (3, 4), pine, rye, corn and cattails (Typha latifolia), and speculated that this enzyme participates in the processes of starch-sucrose conversion in these species of pollen. Materials and methods
Pollen of Typha latifolia Mature pollen grains were collected in a paddy field at Nissin-cho, Aichi Prefecture, in June 1979, left overnight at room temperature and kept frozen below -20°C until use. Abbreviation: DEAE, diethylaminoethyl. 1475
Downloaded from http://pcp.oxfordjournals.org/ at Mount Royal University on June 6, 2015
Laboratory of Biological Chemistry, Faculty of Agriculture, Meijo University, Tenpaku-ku, Nagoya 468, Japan
1476
A. Hara, K. Kawamoto and T. Funaguma
Enzyme assay Pyrophosphatase was routinely assayed at 37°0 for 15 min in 1 ml of reaction mixture containing 2 mx pyrophosphate, 10 mx MgOI 2, 20 mxr Tris-HOI, pH 9.0, and appropriately diluted enzyme. The reaction was stopped by the addition of 2 ml of 0.15 Mperchloric acid. The orthophosphate liberated was measured according to the method of Furchgott and Gudareff (2). One unit of enzyme is defined as the amount which releases 1 ,umole of orthophosphate per min under the above conditions.
of enzyme
Fifty grams of the pollen grains of Typha latifolia were suspended in 500 ml of 5 mn Tris-HOI, pH 7.0, containing 10 mM MgOI 2, and each 50-ml portion of the suspension was sonicated at 200 watts for 5 min with an Insonator Model M (Kubota). Each 10-ml portion of the sonicated fractions was then disintegrated
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40
60
80
100
120
Tube No. Chromatography of pyrophosphatase on DEAE-Sephadex A-50 column. Experimental conditions are described in Materials and methods. Fractions of 10 ml were collected. -e-, pyrophosphatase activity; -0-, protein; -X-, Cl- concentration. Fig. 1.
Downloaded from http://pcp.oxfordjournals.org/ at Mount Royal University on June 6, 2015
Purification
1477
Inorganic pyrophosphatase of pollen
200
10
10
8
8
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6
t
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0
10
20
30
40
0
Tube No. Fig. 2. Isoelectricfocusing ofpyrophosphatase. Experimental conditions are described in Materials and methods. Fractions of 2.5 g were collected. -e-, pyrophosphatase activity; -0-, protein; •••• , pH.
Downloaded from http://pcp.oxfordjournals.org/ at Mount Royal University on June 6, 2015
with a Teflon glass homogenizer at about 2,000 rpm for 5 min and centrifuged at 8,000 Xg for 10 min. To the combined supernatant (414 ml), 138 ml of chilled acetone was added with stirring. The suspension was centrifuged at 8,000 X g for 10 min and the precipitate was discarded. To the supernatant fluid, 483 ml of chilled acetone was added with stirring. After the mixture had been stirred for 30 min followed by centrifugation as described above, the supernatant fluid was discarded. The precipitate was dissolved in 50 ml of 5 mM Tris-HCI, pH 7.0, containing 10 mn MgCl 2 and dialyzed overnight against the same buffer. The dialysate (53 ml) was applied to a 3 X 32 em column of DEAE-Sephadex A-50 equilibrated with 5 mn Tris-HCI, pH 7.0, containing 10 mM MgCI 2 • After washing with 20 ml of the same buffer, the column was eluted with a linear 0-0.6 M NaCI gradient (total volume of 1,200 ml) made up in the washing buffer (Fig. 1). The active fractions (tubes 45-50) were pooled and dialyzed against 1 mM Tris-HCI buffer, pH 7.0, containing 2 ma MgCI 2 • The dialyzed fraction was applied to an isoelectric focusing apparatus (110 ml, LKB column) at 900 V for 42 hr in the pH range of 3.5-10.0 (Fig. 2). The fractions showing high activity (tubes 15-16) were pooled, dialyzed against 5 mM Tris-Hfll buffer, pH 7.0, containing 10 mM MgCl 2 and concentrated to about 0.5 ml with a collodion bag. The concentrated fraction was passed through
1478
A. Hara, K. Kawamoto and T. Funaguma
0" 3
1
0.2 ~' C
-.-1
0.1
o
8
o,
80
20
Tube No. Fig. 3. Gel filtration of pyrophosphatase on Sephadex G-75. Experimental conditions are described in Materials and methods. Fractions of 3 ml were collected. -e-, pyrophosphatase activity; -0-, protein.
a Sephadex G-75 column (2 X 96 cm) equilibrated with 5 mM Tris-HCl buffer (Fig. 3). The active fractions (tubes 57-62) were collected and used for characterization of the pyrophosphatase. Results
Summary
of the purification ofpyrophosphatase
A pyrophosphatase was obtained from the pollen of Typha latifolia as a single peak during all the procedures including chromatography on DEAE-Sephadex, isoelectric focusing and gel filtration through Sephadex G-75. However, the activity peak did not coincide with the protein peak (Fig. 3), suggesting that the Table 1 Summary ofpurification procedure of pyrophosphatase Step Crude extract a
a
Volume (ml) 414.0
Total protein (mg) 15,401 696.2
Total activity (units)
Specific activity (units/protein)
1,953.8
0.13
1,499.7
2.15
Yield (%) 100 76.8
Acetone fractionation
53.0
DEAE-Sephadex A-50
5~.5
54. 74
829.0
15.2
42.4
Ampholine electrofocusing
21. 0
10.87
519.4
47.8
26.6
Sephadex G-75
17.7
0.549
Fifty grams of pollen grains of Typha latifolia were used.
273. 7
499
14.0
Downloaded from http://pcp.oxfordjournals.org/ at Mount Royal University on June 6, 2015
(1) -+-J
1479
Inorganic pyrophosphatase of pollen
0.04 C
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IVg2+ concentration ,(rnM)
enzyme still contained contaminating proteins. Table 1 shows that the final preparation of the enzyme was purified about 3,800-fold with a yield of 14%.
Effect ofpH onpyrophosphatase actiuity The effect of pH on pyrophosphatase activity was determined with 2 mM pyrophosphate in the presence of 10 mM MgCI 2 • The pyrophosphatase had a pH optimum between 8.5-9.5. Effect of M g 2+ concentration onpyrophosphatase actiuity The effect of MgCl 2 concentration on pyrophosphatase activity was investigated with the pyrophosphate concentration fixed at 2 rmr, Fig. 4 shows that maximum activity of the enzyme was attained with the concentration of MgCl 2 above 3 rmr, whereas the activity was hardly detectable below 1 msr, 0.04
(b)
(a)
(c) o
o 0
0".02
5
10 0
5
10 0
5
10
Pyrophosphate concentration (mM) Fig. 5. Effect of pyrophosphate concentration on pyrophosphatase actioity, presence of 1 rmr (a), 5 mM (b) or 10 mM M g 2+ (c).
Activity was assayed in the
Downloaded from http://pcp.oxfordjournals.org/ at Mount Royal University on June 6, 2015
Fig. 4. Effectof M g 2+ concentration onpyrophosphatase activity. Activity was measured as described in Materials and m.ethods except that the M g 2+ concentration was varied.
1480
A. Hara, K. Kawamoto and T. Funaguma
Table 2
Effect of divalent cations onpyrophosphatase in the absence orpresence ofM g 2+ Cation concentration at IOmM (Mg2+, 10 mx)
Cation
IOmM (Mg 2+, orna)
Ca 2+
0
0
25
Zn 2+
0
2
38
Mn 2+
0
C 0 2+
0
None
0
1 mM
53 18
2
Activities are expressed in terms of percentage of'the activity in the presence of 10 mx M g 2+.
Effect
ofpyrophosphate concentration on pyrophosphatase activity
Fig. 5 shows that the pyrophosphatase activity was observed when pyrophosphate was present in an amount equimolar to or less than MgCI 2 , whereas the activity was strikingly depressed when more pyrophosphate was present than MgCI 2 .
Determination
of Michaelis constant ofpyrophosphatase
The Michaelis constant was calculated from the Lineweaver-Burk plot.
The
Km value was 7.6 X 10- 5 M. Effect of divalent cations onpyrophosphatase activity Table 2 shows that none of the cations tested could substitute for Mg 2+.
When
Table 3 Substrate specificity of pyrophosphatase Substrate PPi ATP ADP 5'-AMP 2'(3')-AMP UTP
Relative rate of hydrolysis
(%) 100
o o o o 1
UDP 5'-UMP
o
2'(3')-UMP
o o o o o
Glucose-I-phosphate p-Glycerophosphate Bis(p-nitrophenyl)phosphate p-Nitrophenylphosphate
o
Activities were measured by the routine method except that pyrophosphate was replaced by 2 mu of substrate indicated.
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100
Inorganic pyrophosphatase of pollen
1481
each of these cations was added to the reaction mixture in the presence of M g2+, the pyrophosphatase activity was repressed remarkably.
Substrate specificity
ofpyrophosphatase
Hydrolytic activities of the pyrophosphatase against several phosphate compounds were tested in the presence of 10 mn MgOI 2, and the results are summarized in Table 3. The present pyrophosphatase seemed to be highly specific for inorganic pyrophosphate and showed negligible activity for UTP.
Alkaline inorganic pyrophosphatases, widely distributed in microorganisms, animals and plants, require a divalent cation such as Mg2+, Oa 2+, Mn 2+, Zn 2+ and 00 2+ for their activities (1, 3, 5-8, 11, 12, 15, 16, 18-20). The pyrophosphatase from the pollen of Typha latifolia requires M g2+, but not Oa 2+, Mn 2+ or Zn 2+; the latter cations inhibited the enzyme reaction even in the presence of Mg2+ (Table 2). The role of M g2+ in the pyrophosphatase reaction involves the formation of the actual substrate, an Mg-pyrophosphate complex, by its binding to pyrophosphate, which per se acts as a strong competitive inhibitor in its free form (7, 11). We also showed that an excess of pyrophosphate over M g2+ caused inhibition of enzyme in Fig. 5. M g2+ is likely to have an additional role in the pyrophosphatase reaction. We added M g2+ to the enzyme solution during the enzyme purification, because the enzyme was very labile without M g2+ and lost most of its activity, especially during the chromatography on DEAE-Sephadex A-50. This may be explained by the suggestions according to Riddington et al. (16) and Morita et al. (12) that pyrophosphatases bind free M g2+ for the maintenance of stable structures. Pyrophosphatases have a high affinity for pyrophosphate, and our enzyme also showed a low Km value of 7.6 X 10-5 M for it. The substrate specificity of this enzyme seemed to be very strict and no cleavage of phosphate esters except inorganic pyrophosphate was observed as far as tested (Table 3). This result resembled an earlier finding with the pyrophosphatase from the pollen of cycad (3).
We have found M g2+-dependent alkaline pyrophosphatase activity in all pollens from Japanese cycad, rye, corn, pine and Typha latifolia. Thus, we considered what the role of pyrophosphatase in pollen might be. Though carbohydrate levels vary with the plant species, most of their pollen contains starch, sucrose, fructose and glucose. The contents of these carbohydrates change largely during maturation and at the initial stage of germination, there is an increase in sucrose with a concomitant decrease in starch. These facts show that a starchsucrose conversion occurs in pollen. During the process, pyrophosphate is produced by the action ofUDP-glucose pyrophosphorylase in the following manner: Glucose-l-phosphate+ UTP
~
UDP-glucose+PPi.
Since phosphorylase, UDP-glucose pyrophosphorylase and sucrose synthase, which are concerned with sucrose synthesis from starch, are found in the pollen of Typha latifolia, we presume that the present Mg2+-dependent alkaline pyrophos-
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Discussion
1482
A. Hara, K. Kawamoto and T. Funaguma
phatase hydrolyzes pyrophosphate which is released in the formation of UDPglucose. References
leaf development and senescence.
Plant Physiol. 63: 318-323 (1979).
(14) Rauser, W. E.: Inorganic pyrophosphatases in leaves during plant development and senescence. Can. J. Bot. 49: 311-316 (1971). (15) Riddington, J. W. and L. G. Butler: Yeast inorganic pyrophosphatase I. Binding of pyrophosphatase, metal ion, and metal ion-pyrophosphate complexes. J. Biol. Chern. 247: 73037307 (1972). (16) Riddington, J. W., W. Y. Yang and L. G. Butler: Yeast inorganic pyrophosphatase IV. Purification, quaternary structure, and evidence for strongly bound M g 2+. Arch. Biochern. Biophys. 153: 714-725 (1972). (17) Schwenn, J. D., R. McC. Lilley and D. A. Walker: Inorganic pyrophosphatase and photosynthesis by isolated chloroplasts I. Characterization of chloroplast pyrophosphatase and its relation to the response to exogeneous pyrophosphate. Biochim. Biophys. Acta 325: 586-595 (1973). (18) Simmons, S. and L. G. Butler: Alkaline inorganic pyrophosphatase of maize leaves. ibid. 172: 150-157 (1969). (19) Tominaga, N. and T. Mori: Purification and characterization of inorganic pyrophosphatase from Thiobacillus thiooxidans. J. Biochern. 81: 477-483 (1977). (20) Tono, H. and A. Konberg: Biochemical studies of bacterial sporulation III. Inorganic pyrophosphatase of vegetative cells and spores of Bacillus subtilis. J. Biol. Chern. 242: 2375-2382 (1967).
Downloaded from http://pcp.oxfordjournals.org/ at Mount Royal University on June 6, 2015
( 1) Bloch-Frankenthal, L.: The role of magnesium in the hydrolysis of sodium pyrophosphate by inorganic pyrophosphatase. Biochem. J. 57: 87-92 (1954). ( 2) Furchgott, R. F. and T. de Gudareff: The determination of inorganic phosphate and creatine phosphate in tissue extracts. J. Biol. Chern. 223: 377-388 (1956). ( 3) Hara, A. and T. Funaguma: M g 2+ dependent inorganic pyrophosphatase of Japanese cycad pollen. Jap. J. Palynol. 21: 1-7 (1978). ( 4) Hara, A., K. Matsugano and A. Kobayashi: Protease associated with the cell surface of cycad pollens. ibid. 15: 41-44 (1975). ( 5) Irie, M., A. Yabuta, K. Kimura, Y. Shindo and K. Tomita: Distribution and properties of alkaline pyrophosphatase of rat liver. J. Biochem. 67: 47-58 (1970). ( 6) ] osse,]. : Constitutive inorganic pyrophosphatase of Escherichia coli I. Purification and catalytic properties. J. Biol. Chern. 241: 1938-1947 (1966). ( 7) ]osse,].: Constitutive inorganic pyrophosphatase of Escherichia coli II. Nature and binding of active substrate and the role of magnesium. ibid. 241: 1948-1957 (1966). ( 8) Kar, M. and D. Mishra: Inorganic pyrophosphatase activity during rice leaf senescence. Can. J. Bot. 53: 503-511 (1975). ( .9) Kornberg, A.: In Horizons in Biochemistry, Edited by M. Kasha and B. Pullman. Academic Press, New York. p. 251-264. 1962. (10) McC. Lilley, R., j, D. Schwenn and D. A. Walker: Inorganic pyrophosphatase and photosynthesis by isolated chloroplasts II. The controlling influence of orthophosphate. Biochirn. Biophys. Acta 325: 596-604 (1973). (11) Moe, o. A. and L. G. Butler: .Yeast inorganic pyrophosphatase II. Kinetics of M g 2+ activation. J. Biol. Chern. 247: 7308-7314 (1972). (12) Morita,]. and T. Yasui: Purification and some properties ofa neutral muscle pyrophosphatase. J. Biochern. 83: 719-726 (1978). (13) Patra, H. K. and D. Mishra: Pyrophosphatase, peroxidase, and phenoloxidase activities during
Inorganic pyrophosphatase from. pollen of Typha latifolia Akira Hara, Kousei Kawamoto and Tooru Funaguma
(Received August 27, 1980)
An inorganic pyrophosphatase was purified about 3,800-fold from the pollen of Typha latifolia by chromatography on DEAE-Sephadex A-50, isoelectric focusing and gel filtration through Sephadex G-75. The enzyme had an optimum pH between 8.59.5 and required M g 2+. Since an excess of pyrophosphate over Mg 2+ inhibited the pyrophosphatase reaction, the actual substrate may have been an Mg-pyrophosphate complex. The enzyme degraded inorganic pyrophosphate specifically, showing a Km value of 7.6 X 10-5 M. A possible role of pyrophosphatase was discussed in connection with starch-sucrose conversion. Key words: Inorganic pyrophosphatase - Pollen enzyme - Typhalatifolia.
Inorganic pyrophosphate is produced as a by-product in several biosynthetic reactions, for example, activation of amino acids, polymerization of nucleic acids, and syntheses of coenzymes and sugar nucleotides. Inorganic pyrophosphatase (pyrophosphate phosphohydrolase; EC 3.6.1.1) is likely to promote these biosynthetic reactions by degrading inorganic pyrophosphate into orthophosphate (9). In plants, pyrophosphatases have been investigated in connection with leaf senescence and photosynthesis (8, 10, 13, 14, 17). Simmons and Butler have reported that a large fraction of the total pyrophosphatase activity of the leaf exists in nonaqueous preparations of maize chloroplasts (18). We found an exceedingly high activity of M g2+-dependent alkaline pyrophosphatase in the pollen of Japanese cycad (3, 4), pine, rye, corn and cattails (Typha latifolia), and speculated that this enzyme participates in the processes of starch-sucrose conversion in these species of pollen. Materials and methods
Pollen of Typha latifolia Mature pollen grains were collected in a paddy field at Nissin-cho, Aichi Prefecture, in June 1979, left overnight at room temperature and kept frozen below -20°C until use. Abbreviation: DEAE, diethylaminoethyl. 1475
Downloaded from http://pcp.oxfordjournals.org/ at Mount Royal University on June 6, 2015
Laboratory of Biological Chemistry, Faculty of Agriculture, Meijo University, Tenpaku-ku, Nagoya 468, Japan
1476
A. Hara, K. Kawamoto and T. Funaguma
Enzyme assay Pyrophosphatase was routinely assayed at 37°0 for 15 min in 1 ml of reaction mixture containing 2 mx pyrophosphate, 10 mx MgOI 2, 20 mxr Tris-HOI, pH 9.0, and appropriately diluted enzyme. The reaction was stopped by the addition of 2 ml of 0.15 Mperchloric acid. The orthophosphate liberated was measured according to the method of Furchgott and Gudareff (2). One unit of enzyme is defined as the amount which releases 1 ,umole of orthophosphate per min under the above conditions.
of enzyme
Fifty grams of the pollen grains of Typha latifolia were suspended in 500 ml of 5 mn Tris-HOI, pH 7.0, containing 10 mM MgOI 2, and each 50-ml portion of the suspension was sonicated at 200 watts for 5 min with an Insonator Model M (Kubota). Each 10-ml portion of the sonicated fractions was then disintegrated
30 - - - - - - - - - - - - - - - - - - - - - - , 6
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4
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20
40
60
80
100
120
Tube No. Chromatography of pyrophosphatase on DEAE-Sephadex A-50 column. Experimental conditions are described in Materials and methods. Fractions of 10 ml were collected. -e-, pyrophosphatase activity; -0-, protein; -X-, Cl- concentration. Fig. 1.
Downloaded from http://pcp.oxfordjournals.org/ at Mount Royal University on June 6, 2015
Purification
1477
Inorganic pyrophosphatase of pollen
200
10
10
8
8
r-i
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en
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6
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Pol
0
10
20
30
40
0
Tube No. Fig. 2. Isoelectricfocusing ofpyrophosphatase. Experimental conditions are described in Materials and methods. Fractions of 2.5 g were collected. -e-, pyrophosphatase activity; -0-, protein; •••• , pH.
Downloaded from http://pcp.oxfordjournals.org/ at Mount Royal University on June 6, 2015
with a Teflon glass homogenizer at about 2,000 rpm for 5 min and centrifuged at 8,000 Xg for 10 min. To the combined supernatant (414 ml), 138 ml of chilled acetone was added with stirring. The suspension was centrifuged at 8,000 X g for 10 min and the precipitate was discarded. To the supernatant fluid, 483 ml of chilled acetone was added with stirring. After the mixture had been stirred for 30 min followed by centrifugation as described above, the supernatant fluid was discarded. The precipitate was dissolved in 50 ml of 5 mM Tris-HCI, pH 7.0, containing 10 mn MgCl 2 and dialyzed overnight against the same buffer. The dialysate (53 ml) was applied to a 3 X 32 em column of DEAE-Sephadex A-50 equilibrated with 5 mn Tris-HCI, pH 7.0, containing 10 mM MgCI 2 • After washing with 20 ml of the same buffer, the column was eluted with a linear 0-0.6 M NaCI gradient (total volume of 1,200 ml) made up in the washing buffer (Fig. 1). The active fractions (tubes 45-50) were pooled and dialyzed against 1 mM Tris-HCI buffer, pH 7.0, containing 2 ma MgCI 2 • The dialyzed fraction was applied to an isoelectric focusing apparatus (110 ml, LKB column) at 900 V for 42 hr in the pH range of 3.5-10.0 (Fig. 2). The fractions showing high activity (tubes 15-16) were pooled, dialyzed against 5 mM Tris-Hfll buffer, pH 7.0, containing 10 mM MgCl 2 and concentrated to about 0.5 ml with a collodion bag. The concentrated fraction was passed through
1478
A. Hara, K. Kawamoto and T. Funaguma
0" 3
1
0.2 ~' C
-.-1
0.1
o
8
o,
80
20
Tube No. Fig. 3. Gel filtration of pyrophosphatase on Sephadex G-75. Experimental conditions are described in Materials and methods. Fractions of 3 ml were collected. -e-, pyrophosphatase activity; -0-, protein.
a Sephadex G-75 column (2 X 96 cm) equilibrated with 5 mM Tris-HCl buffer (Fig. 3). The active fractions (tubes 57-62) were collected and used for characterization of the pyrophosphatase. Results
Summary
of the purification ofpyrophosphatase
A pyrophosphatase was obtained from the pollen of Typha latifolia as a single peak during all the procedures including chromatography on DEAE-Sephadex, isoelectric focusing and gel filtration through Sephadex G-75. However, the activity peak did not coincide with the protein peak (Fig. 3), suggesting that the Table 1 Summary ofpurification procedure of pyrophosphatase Step Crude extract a
a
Volume (ml) 414.0
Total protein (mg) 15,401 696.2
Total activity (units)
Specific activity (units/protein)
1,953.8
0.13
1,499.7
2.15
Yield (%) 100 76.8
Acetone fractionation
53.0
DEAE-Sephadex A-50
5~.5
54. 74
829.0
15.2
42.4
Ampholine electrofocusing
21. 0
10.87
519.4
47.8
26.6
Sephadex G-75
17.7
0.549
Fifty grams of pollen grains of Typha latifolia were used.
273. 7
499
14.0
Downloaded from http://pcp.oxfordjournals.org/ at Mount Royal University on June 6, 2015
(1) -+-J
1479
Inorganic pyrophosphatase of pollen
0.04 C
.r-i
e 'r-f... o e
o
~
. . . . . 0.02 "0
Q)
+J
co
~
Q)
.c
-r-f
r-f
.r-i ~
o
5
10
IVg2+ concentration ,(rnM)
enzyme still contained contaminating proteins. Table 1 shows that the final preparation of the enzyme was purified about 3,800-fold with a yield of 14%.
Effect ofpH onpyrophosphatase actiuity The effect of pH on pyrophosphatase activity was determined with 2 mM pyrophosphate in the presence of 10 mM MgCI 2 • The pyrophosphatase had a pH optimum between 8.5-9.5. Effect of M g 2+ concentration onpyrophosphatase actiuity The effect of MgCl 2 concentration on pyrophosphatase activity was investigated with the pyrophosphate concentration fixed at 2 rmr, Fig. 4 shows that maximum activity of the enzyme was attained with the concentration of MgCl 2 above 3 rmr, whereas the activity was hardly detectable below 1 msr, 0.04
(b)
(a)
(c) o
o 0
0".02
5
10 0
5
10 0
5
10
Pyrophosphate concentration (mM) Fig. 5. Effect of pyrophosphate concentration on pyrophosphatase actioity, presence of 1 rmr (a), 5 mM (b) or 10 mM M g 2+ (c).
Activity was assayed in the
Downloaded from http://pcp.oxfordjournals.org/ at Mount Royal University on June 6, 2015
Fig. 4. Effectof M g 2+ concentration onpyrophosphatase activity. Activity was measured as described in Materials and m.ethods except that the M g 2+ concentration was varied.
1480
A. Hara, K. Kawamoto and T. Funaguma
Table 2
Effect of divalent cations onpyrophosphatase in the absence orpresence ofM g 2+ Cation concentration at IOmM (Mg2+, 10 mx)
Cation
IOmM (Mg 2+, orna)
Ca 2+
0
0
25
Zn 2+
0
2
38
Mn 2+
0
C 0 2+
0
None
0
1 mM
53 18
2
Activities are expressed in terms of percentage of'the activity in the presence of 10 mx M g 2+.
Effect
ofpyrophosphate concentration on pyrophosphatase activity
Fig. 5 shows that the pyrophosphatase activity was observed when pyrophosphate was present in an amount equimolar to or less than MgCI 2 , whereas the activity was strikingly depressed when more pyrophosphate was present than MgCI 2 .
Determination
of Michaelis constant ofpyrophosphatase
The Michaelis constant was calculated from the Lineweaver-Burk plot.
The
Km value was 7.6 X 10- 5 M. Effect of divalent cations onpyrophosphatase activity Table 2 shows that none of the cations tested could substitute for Mg 2+.
When
Table 3 Substrate specificity of pyrophosphatase Substrate PPi ATP ADP 5'-AMP 2'(3')-AMP UTP
Relative rate of hydrolysis
(%) 100
o o o o 1
UDP 5'-UMP
o
2'(3')-UMP
o o o o o
Glucose-I-phosphate p-Glycerophosphate Bis(p-nitrophenyl)phosphate p-Nitrophenylphosphate
o
Activities were measured by the routine method except that pyrophosphate was replaced by 2 mu of substrate indicated.
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100
Inorganic pyrophosphatase of pollen
1481
each of these cations was added to the reaction mixture in the presence of M g2+, the pyrophosphatase activity was repressed remarkably.
Substrate specificity
ofpyrophosphatase
Hydrolytic activities of the pyrophosphatase against several phosphate compounds were tested in the presence of 10 mn MgOI 2, and the results are summarized in Table 3. The present pyrophosphatase seemed to be highly specific for inorganic pyrophosphate and showed negligible activity for UTP.
Alkaline inorganic pyrophosphatases, widely distributed in microorganisms, animals and plants, require a divalent cation such as Mg2+, Oa 2+, Mn 2+, Zn 2+ and 00 2+ for their activities (1, 3, 5-8, 11, 12, 15, 16, 18-20). The pyrophosphatase from the pollen of Typha latifolia requires M g2+, but not Oa 2+, Mn 2+ or Zn 2+; the latter cations inhibited the enzyme reaction even in the presence of Mg2+ (Table 2). The role of M g2+ in the pyrophosphatase reaction involves the formation of the actual substrate, an Mg-pyrophosphate complex, by its binding to pyrophosphate, which per se acts as a strong competitive inhibitor in its free form (7, 11). We also showed that an excess of pyrophosphate over M g2+ caused inhibition of enzyme in Fig. 5. M g2+ is likely to have an additional role in the pyrophosphatase reaction. We added M g2+ to the enzyme solution during the enzyme purification, because the enzyme was very labile without M g2+ and lost most of its activity, especially during the chromatography on DEAE-Sephadex A-50. This may be explained by the suggestions according to Riddington et al. (16) and Morita et al. (12) that pyrophosphatases bind free M g2+ for the maintenance of stable structures. Pyrophosphatases have a high affinity for pyrophosphate, and our enzyme also showed a low Km value of 7.6 X 10-5 M for it. The substrate specificity of this enzyme seemed to be very strict and no cleavage of phosphate esters except inorganic pyrophosphate was observed as far as tested (Table 3). This result resembled an earlier finding with the pyrophosphatase from the pollen of cycad (3).
We have found M g2+-dependent alkaline pyrophosphatase activity in all pollens from Japanese cycad, rye, corn, pine and Typha latifolia. Thus, we considered what the role of pyrophosphatase in pollen might be. Though carbohydrate levels vary with the plant species, most of their pollen contains starch, sucrose, fructose and glucose. The contents of these carbohydrates change largely during maturation and at the initial stage of germination, there is an increase in sucrose with a concomitant decrease in starch. These facts show that a starchsucrose conversion occurs in pollen. During the process, pyrophosphate is produced by the action ofUDP-glucose pyrophosphorylase in the following manner: Glucose-l-phosphate+ UTP
~
UDP-glucose+PPi.
Since phosphorylase, UDP-glucose pyrophosphorylase and sucrose synthase, which are concerned with sucrose synthesis from starch, are found in the pollen of Typha latifolia, we presume that the present Mg2+-dependent alkaline pyrophos-
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Discussion
1482
A. Hara, K. Kawamoto and T. Funaguma
phatase hydrolyzes pyrophosphate which is released in the formation of UDPglucose. References
leaf development and senescence.
Plant Physiol. 63: 318-323 (1979).
(14) Rauser, W. E.: Inorganic pyrophosphatases in leaves during plant development and senescence. Can. J. Bot. 49: 311-316 (1971). (15) Riddington, J. W. and L. G. Butler: Yeast inorganic pyrophosphatase I. Binding of pyrophosphatase, metal ion, and metal ion-pyrophosphate complexes. J. Biol. Chern. 247: 73037307 (1972). (16) Riddington, J. W., W. Y. Yang and L. G. Butler: Yeast inorganic pyrophosphatase IV. Purification, quaternary structure, and evidence for strongly bound M g 2+. Arch. Biochern. Biophys. 153: 714-725 (1972). (17) Schwenn, J. D., R. McC. Lilley and D. A. Walker: Inorganic pyrophosphatase and photosynthesis by isolated chloroplasts I. Characterization of chloroplast pyrophosphatase and its relation to the response to exogeneous pyrophosphate. Biochim. Biophys. Acta 325: 586-595 (1973). (18) Simmons, S. and L. G. Butler: Alkaline inorganic pyrophosphatase of maize leaves. ibid. 172: 150-157 (1969). (19) Tominaga, N. and T. Mori: Purification and characterization of inorganic pyrophosphatase from Thiobacillus thiooxidans. J. Biochern. 81: 477-483 (1977). (20) Tono, H. and A. Konberg: Biochemical studies of bacterial sporulation III. Inorganic pyrophosphatase of vegetative cells and spores of Bacillus subtilis. J. Biol. Chern. 242: 2375-2382 (1967).
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( 1) Bloch-Frankenthal, L.: The role of magnesium in the hydrolysis of sodium pyrophosphate by inorganic pyrophosphatase. Biochem. J. 57: 87-92 (1954). ( 2) Furchgott, R. F. and T. de Gudareff: The determination of inorganic phosphate and creatine phosphate in tissue extracts. J. Biol. Chern. 223: 377-388 (1956). ( 3) Hara, A. and T. Funaguma: M g 2+ dependent inorganic pyrophosphatase of Japanese cycad pollen. Jap. J. Palynol. 21: 1-7 (1978). ( 4) Hara, A., K. Matsugano and A. Kobayashi: Protease associated with the cell surface of cycad pollens. ibid. 15: 41-44 (1975). ( 5) Irie, M., A. Yabuta, K. Kimura, Y. Shindo and K. Tomita: Distribution and properties of alkaline pyrophosphatase of rat liver. J. Biochem. 67: 47-58 (1970). ( 6) ] osse,]. : Constitutive inorganic pyrophosphatase of Escherichia coli I. Purification and catalytic properties. J. Biol. Chern. 241: 1938-1947 (1966). ( 7) ]osse,].: Constitutive inorganic pyrophosphatase of Escherichia coli II. Nature and binding of active substrate and the role of magnesium. ibid. 241: 1948-1957 (1966). ( 8) Kar, M. and D. Mishra: Inorganic pyrophosphatase activity during rice leaf senescence. Can. J. Bot. 53: 503-511 (1975). ( .9) Kornberg, A.: In Horizons in Biochemistry, Edited by M. Kasha and B. Pullman. Academic Press, New York. p. 251-264. 1962. (10) McC. Lilley, R., j, D. Schwenn and D. A. Walker: Inorganic pyrophosphatase and photosynthesis by isolated chloroplasts II. The controlling influence of orthophosphate. Biochirn. Biophys. Acta 325: 596-604 (1973). (11) Moe, o. A. and L. G. Butler: .Yeast inorganic pyrophosphatase II. Kinetics of M g 2+ activation. J. Biol. Chern. 247: 7308-7314 (1972). (12) Morita,]. and T. Yasui: Purification and some properties ofa neutral muscle pyrophosphatase. J. Biochern. 83: 719-726 (1978). (13) Patra, H. K. and D. Mishra: Pyrophosphatase, peroxidase, and phenoloxidase activities during
