- Email: [email protected]
Multiple forms of acid phosphatase from seedling axes of Vigna radiata
Phytochemistry,Vol. 29, No. 9, Pp. 2825-2828,1990. Printed in Great Britain.
MULTIPLE
FORMS
0
OF ACID PHOSPHATASE VIGNA RADIATA PRATIMA DE-KUNDU*
OWL9422/90 S3.OC+O.O0 1990 Pergamon Press plc
FROM SEEDLING
AXES OF
and AMBIKA C. BANERJEE~~
Department of Biochemistry, Calcutta University, 35, Ballygunge Circular Road, Calcutta 700 019, India; TDepartment of Basic Medical Sciences, University of Papua New Guinea, Medical Faculty, P.O. Box 5623, Boroko, Papua New Guinea (Received in revisedform 9 January
Key Word Index--Vigna radiata; Leguminosae;
mung
1990)
bean; seed germination;
axis; acid phosphatase;
multiple
forms.
Abstract-Two major forms of acid phosphatase were isolated and partially purified by alcohol precipitation and ion exchange column chromatography from the axis organs of germinating mung beans (Vigna radiata). Their M,s were 101000 and 118 000 as determined by Sephadex G-200 gel filtration and they both showed strong affinity for pnitrophenyl phosphate with K,s of 0.71 and 0.41 mM, respectively. They were active towards several other phosphate esters, albeit with less affinity, but not phytate. With p-nitrophenyl phosphate as substrate, the two acid phosphatases showed similar pH (pH 5.5) and temperature optima (60”), but their activation energy and stability towards heat and other chemical agents was different.
INTRODUCTION
Acid phosphatases (EC 3.1.3.2) are a group of enzymes that nonspecifically catalyse the hydrolysis of a variety of phosphate esters in an acid environment and they are widely distributed in nature Cl]. In plants, acid phosphatase activity increases with seed germination and seedling growth and is thought to play an important role in phosphate mobilization contributing to the growth of embryonic axes [2-51. Enhancement of acid phosphatase activity in axis has been shown to be due to de novo synthesis of enzyme protein and not due to transfer from germinating cotyledons [6]. However, reports on the characterization of acid phosphatase from axis organs of leguminous seedlings are scanty compared to those from cotyledons and other sources [3-131. We report herein the isolation, partial purification and characterizatiomof the major forms of acid phosphatase from mung bean seedling axes. RESULTS AND DISCUSSION
Acid phosphatase activity present in crude extracts of axes organs of germinating mung beans, Vigna radiata was concentrated by alcohol precipitation, whereby 90% of the total acid phosphatase activity was recovered and two-thirds of proteins were removed, thus leading to a substantial enrichment of enzyme activity. The alcohol precipitated enzyme preparation was subjected to ionexchange chromatography on a DEAE cellulose column. The bulk of contaminating proteins devoid of acid phosphatase activity was removed during the wash. Upon
*Present address: Department of Paediatrics, Kennedy Center, University of Chicago, Chicago, IL 60637, U.S.A. $Author to whom correspondence should be addressed.
elution with a linear O-O.2 M KC1 gradient acid phosphatase activity was resolved into two peaks, designated as AP-I and AP-II according to their order of elution from the column; AP-I eluted at 0.1 M KC1 and AP-II at 0.13 M KCl. The A2s0 values in the activity regions were very low and no further activity could be eluted from the column by 0.8 M KCl. A typical purification procedure is summarized in Table 1. Because the total activity was resolved into two fractions during purification, the recovery and the purification of each form of acid phosphatase appears to be relatively low. Polyacrylamide gel electrophoresis of AP-I and AP-II showed one major band of acid phosphatase activity with different electrophoretic mobilities. The M,s of AP-I and AP-II were 101000 and 118 000, respectively, as determined by gel filtration through a Sephadex G-200 column. Acid phosphatases from chick pea embryonic axes [7] and Asclepias curassavica latex [9] had much lower M,, viz. 39 000 and 27 000, respectively. AP-I and AP-II exhibited their maximum activity at pH 5.5 and half maximal activities at pH 4.6 and 6.7, when examined in several buffers, viz. Na acetate, Na citrate and Tris-maleate, in the pH range 3.5-7.5. They also showed the same temperature optima at 60”, quite high in comparison to acid phosphatases from cotton seed (37”) [12] and yam (50°) [13]. But at 70”, AP-I and AP-II showed 27 and 60% of their respective maximum activity. The enzymes were stable for several months when stored at - 20”, but at higher temperatures their activities decayed to different extents in both neutral and acidic pH (Fig. I), thus showing the greater heat stability of AP-II. Thermal sensitivity of several plant acid phosphatases have been reported [4, 5, 111; however, the enzyme from Ustilago esculenta showed high thermostability [14]. Both acid phosphatases from mung bean axes possessed broad substrate specificity (Table 2). They exhibited
2826
P. DE-KUNDU and A. C. BANERJEE Table
1. Purification
of acid phosphatase
Total protein (mg)
Step Crude Alcohol ppt. DEAE-cellulose AP-I AP-II
1510 510
Total act. (pkat)
Sp. act. (nkat mg- ’ protein)
24l 18
13.3 35
1.5 6.4
Table
from mung bean seedling
5.0 6.4
2. Substrate
Purification fold 1.0 2.6
661 1000
specificity
axes
50 75
Recovery (%) 100 90 25 32
of acid phosphatases
Relative enzyme activity Substrate
AP-I
AP-II
p-Nitrophenyl phosphate a-Naphthyl phosphate a-Glycerophosphate D-Glucose- 1-phosphate D-Glucose-6-phosphate D-Fructose-1,6-diphosphate Inorganic pyrosphosphate 5’-ATP S-ADP 5’-GMP 5’-GTP 5’-UMP 3’-5’-cyclic AMP Phytate
100 24 29 6 27 20 94 46 20 5 39 5.2 6.4 0
loo 20 15 17 13 18 63 25 16 4.2 21 2.8 5.1 0
(%)
Enzyme activity was assayed in 50 mM Tris-maleate, pH 5.5 as described. The concentration of all substrates used was 2 mM.
100
_/‘ 80 60
.
A II
0
Y)
I.
M
30
I
Time of preincubation (mht Fig. 1. Thermal stability of acid phosphatases. AP-I and AP-II were separately preincubated in 25 mM Tris-HCl, pH 7 (A), or 25 mM Na-acetate, pH 5 (B), at 55” for different time periods and assayed for activity with PNPP as substrate. The activity
remaining was expressed as a percentage of the appropriate zerotime control.
their highest activity towards p-nitrophenyl phosphate (PNPP), and then inorganic pyrophosphate. Nucleoside triphosphates, viz. ATP and GTP were apparently better substrates than nucleoside di- and monophosphates. Highest activity towards PNPP substrate was also reported for peanut acid phosphatase [S]. AP-I and AP-II did not hydrolyse phytate and had very little activity towards nucleoside monophosphates. The effect of various metal ions and chemical agents on the acid phosphatase activities were investigated using PNPP as substrate (Table 3). None of the enzyme forms showed an absolute requirement for a metal ion for its activity. Hg’ + and Ag+ were strong inhibitors for both AP-I and AP-II. Cu2+ and Zn2 ’ were also inhibitory, but the inhibition was greater for AP-I than AP-II, and could be partially relieved by EDTA, however, EDTA itself did not have any effect on mung bean axis acid phosphatase. Ca’ ’ caused some stimulation of both forms. Peanut acid phosphatase is reported to be activated by Ca2+ and Mg2+ [S]. AP-I and AP-II were not inhibited by NaCN, nor were they significantly influenced by B-mercaptoethanal, however, p-chloromercuribenzoate and azide caused some inhibition of AP-I. Arsenate and molybdate inhibited both the enzymes. Tartarate, an inhibitor of animal acid phosphatases, caused significant inhibition of AP-I and AP-II at 5 mM, but not at 1 mM. Detergents, viz. Triton X-100, Tween-80 and Na deoxycholate, stimulated both forms markedly. But. in the presence of 0.1%
Acid phosphatases Table
3. Effects
from mung bean
of some metal ions and chemical activities of acid phosphatases Relative
activity
2827 agents
(% control)
Addition
AP-I
AP-II
None (control) ZnCl,, 1 mM ZnCl,, 1 mM + EDTA, CaCl,, 1 mM
100 37 72 116
100 55 68 120
1 mM
MgCI,, 1 mM CuSO,, 1 mM CuSO,, 1 mM+EDTA, 1 mM FeSO,,
1 mM
Pb(NO,),, 1 mM Co&, 1 mM NiCl,, 1 mM MnCl,, 1 mM HgCl,, 1 mM AgNO,, 1 mM EDTA, 1 mM NaN,, 1 mM NaCN, 1 mM P-MSH, 10 mM PCMB, 1 mM Arsenate, 1 mM Molybdate, 1 mM Tartrate, 1 mM Tartrate, 5 mM Triton X-100, 0.1% Tween-80, 0.1% SDS, 0.1% Sodium deoxycholate, Urea, 8 M
0.5%
on the
95 40 79
117 49 85
95 99 107 104 105 8 19 100 81 101 113 70 54 13 96 49 214 206 13 150 29
110 95 105 94 110 10 30 103 117 120 112 94 45 14 89 48 185 164 72 151 98
Each enzyme was preincubated with or without (control) the indicated metal ion or chemical agent in 50 mM Tris-maleate buffer, pH 5.5 for 10 min at 37”. The enzyme activity was then determined using PNPP as substrate and is expressed as percentage activity of the respective control.
SDS, the activity of AP-I was severely affected whereas much less inhibition was observed for AP-II. Interestingly, such difference was also apparent from the effect of urea. Upon 8 M urea treatment, AP-I lost most of its activity but AP-II remained fully active. The apparent K, of AP-I and AP-II for PNPP were determined from Lineweaver-Burk plots and were 0.71 and 0.41 mM, respectively. They were competitively inhibited by KH,PO, and non-competitively by NaF as revealed by Dixon plots. The apparent Ki values of KH,PO, for AP-I and AP-II were 1.24 and 0.62 mM, respectively, and that of NaF were 0.47 and 0.67 mM, respectively. The Ki of NaF for acid phosphatase from aleurone of rice [3] was 1.29 mM, but that from cultured Ipomoea cells [15] was as low as 8.5 x 10m5 M. The activation energies of AP-I and AP-II were determined from Arrhenius plots constructed from the rate of enzymatic hydrolysis of PNPP (at optimum pH) at various temperatures. The plots were linear in the temperature range 20-60” and the activation energy values of AP-I and AP-II were 6.02 and 7.38 kcal mol- ‘, respect-
ively. These values are lower than those reported for acid phosphatases from Vigna mungo cotyledons (9.4-12.1 kcal mol-‘) [4]. EXPERIMENTAL Materials. Mung bean seeds ( V. radiata) were obtained from the National Seed Corporation, Calcutta, India. PNPP, diazo Fast Garnet GBC and other fine chemicals were from Sigma. Enzyme assay. Acid phosphatase activity was determined by measuring the rate of formation of p-nitrophenol from enzyme catalysed hydrolysis of PNPP substrate. Reaction mixts (1 ml) containing 50 mM Tris-maleate buffer (pH 5.5), 2 mM PNPP and a suitable aliquot of the enzyme prepn were incubated at 37 for 15 min. The reaction was stopped by adding 4 ml 0.1 M NaOH and the A of the liberated p-nitrophenol measured at 410 nm. When other phosphate esters were used as substrate, the liberated inorganic phosphate was measured according to the method of ref. [16]. Enzyme purification. Mung beans were surface sterilized with 0.1% Hg&, washed thoroughly with H,O and then allowed to
2828
P. DE-KUNDU and A. C. BANERJEE
germinate in the dark at 28”. Axes organs (ca 100 g) were harvested from 3-day germinated seeds, thoroughly washed with ice-cold H,O and homogenized in 200 ml 10 mM Tris-HCI buffer, pH 7.2 (buffer A) using a precooled mortar and pestle. Unless otherwise stated, all operations were carried out at O-4”. The homogenate was strained through two layers of cheesecloth and spun at 12,000 9 for 30 min. The supernatant obtained was mixed with 2.5 vol of chilled EtOH (-20”) with continuous shaking. The resulting mixt. was kept at -2O’, for 30min and then centrifuged at 10000 9 for 15 min. The pellet thus obtained was dissolved in a minimum vol of buffer A and dialysed against 100 vol of the same buffer for 4-5 hr with one change. The dialysate was centrifuged to remove any undissolved particles and the clear supernatant slowly applied to a DE-52 column (1.5 x 14.5 cm) pre-equilibrated with buffer A. The column was eluted first with buffer A and then with 200 ml of a linear O-200 mM KC1 gradient in buffer A. Column frs (2.5 ml) were assayed for acid phosphatase activity and scanned at 280 nm for protein. Frs under each acid phosphatase peak were pooled, or dialysed against buffer A for 6 hr and stored at -20’ lyophilized. Protein estimation. Protein was measured according to the method of ref. [17] using BSA as std. Polyacrylamide gef ekctrophoresis was carried out at 4” following the method of ref. 1181 using a 7.5% gel containing 0.15% Triton X-100 at pH 4.6. Gels were stained for acid phosphatase activity by incubation with 0.1% Na a-naphthyl acid phosphate and 0.1% diazo Fast Garnet GBC in 50 mM NaOAc buffer pH 5.0 for 15-20 min at 37” [19]. Zones containing enzyme activity appeared as coloured bands. Developed gels were stored in 7.5% HOAc and 30% EtOH. Geljibation of purified enzyme proteins was carried out on a Sephadex G-200 column pre-equilibrated with 0.1 M KC1 in buffer A and calibrated with standard marker proteins for determination of M, according to ref. 1201. Acknowledyements-Financial and other help from the University of Calcutta and the University of Papua New Guinea are gratefully acknowledged.
REFERENCES
2.
3. 4.
6.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Dixon, M. and Webb, E. C. (1979) Eniymes, p. 255. Longman, London. Bewley, J. D. and Black, M. (1985) Seeds Physiology of Development and Germination, p. 298. Plenum Press, New York. Yamagata, H., Tanaka, K. and Kasai, Z. (1980) Plant Cell Physiol. 21, 1449. Tamura, T., Minamikawa, T. and Koshiba, T. (1982) J. Expt. Botany 33, 1332. Basha, S. M. (1984) Can. J. Botany 62, 385. Okuda. S., Kaneko, .I., Ogawa, T., Yamaguchi, T., Izaki, K. and Takahashi, H. (1987) Agric. Biol. Chem. 51, 109. Angosto, T., Gonzalez. F. and Matilla. A. (1988) Physiol. Plant. 74, 715. Park, H. C. and Van Etten. R. L. (1986) Phytochemistry 25, 351. Giordani, R., Nari, J., Noat; G. and Sauve, P. (1986) Plant Sci. 43, 207. Murray, D. R. and Collier, M. D. (1977) Aust. J. Plant Physiol. 4, 843. Ullah, A. H. J. and Gibson, D. M. (1988) Arch. Biochem. Biophys. 260, 514. Bhargava, R. and Sachar, R. C. (1987) Phytochemistry 26, 1293. Kamenan, A. and Diopoh, J. (1982) Plant Sci. Letters 24,173. Funagama, T., Kawamura. Y. and Hara, A. (1982) Agric. Biol. Chem. 46, 2117. Zink, M. W. and Velicky, I. A. (1982) Plant Cell Tiss. Organ Cult. 1, 265. Chen, P. S., Toribara, T. Y. (1956) Anal. Chem. 28, 1756. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Bio[. Chem. 193, 265. Reisfeld, R. A., Lewis, V. J. and Williams, D. E. (1962) Nature 195, 281. Barka, T. (1961) J. Histochem. Cytochem. 9, 542. Andrews, P. (1964) Biochem. J. 91, 222.
MULTIPLE
FORMS
0
OF ACID PHOSPHATASE VIGNA RADIATA PRATIMA DE-KUNDU*
OWL9422/90 S3.OC+O.O0 1990 Pergamon Press plc
FROM SEEDLING
AXES OF
and AMBIKA C. BANERJEE~~
Department of Biochemistry, Calcutta University, 35, Ballygunge Circular Road, Calcutta 700 019, India; TDepartment of Basic Medical Sciences, University of Papua New Guinea, Medical Faculty, P.O. Box 5623, Boroko, Papua New Guinea (Received in revisedform 9 January
Key Word Index--Vigna radiata; Leguminosae;
mung
1990)
bean; seed germination;
axis; acid phosphatase;
multiple
forms.
Abstract-Two major forms of acid phosphatase were isolated and partially purified by alcohol precipitation and ion exchange column chromatography from the axis organs of germinating mung beans (Vigna radiata). Their M,s were 101000 and 118 000 as determined by Sephadex G-200 gel filtration and they both showed strong affinity for pnitrophenyl phosphate with K,s of 0.71 and 0.41 mM, respectively. They were active towards several other phosphate esters, albeit with less affinity, but not phytate. With p-nitrophenyl phosphate as substrate, the two acid phosphatases showed similar pH (pH 5.5) and temperature optima (60”), but their activation energy and stability towards heat and other chemical agents was different.
INTRODUCTION
Acid phosphatases (EC 3.1.3.2) are a group of enzymes that nonspecifically catalyse the hydrolysis of a variety of phosphate esters in an acid environment and they are widely distributed in nature Cl]. In plants, acid phosphatase activity increases with seed germination and seedling growth and is thought to play an important role in phosphate mobilization contributing to the growth of embryonic axes [2-51. Enhancement of acid phosphatase activity in axis has been shown to be due to de novo synthesis of enzyme protein and not due to transfer from germinating cotyledons [6]. However, reports on the characterization of acid phosphatase from axis organs of leguminous seedlings are scanty compared to those from cotyledons and other sources [3-131. We report herein the isolation, partial purification and characterizatiomof the major forms of acid phosphatase from mung bean seedling axes. RESULTS AND DISCUSSION
Acid phosphatase activity present in crude extracts of axes organs of germinating mung beans, Vigna radiata was concentrated by alcohol precipitation, whereby 90% of the total acid phosphatase activity was recovered and two-thirds of proteins were removed, thus leading to a substantial enrichment of enzyme activity. The alcohol precipitated enzyme preparation was subjected to ionexchange chromatography on a DEAE cellulose column. The bulk of contaminating proteins devoid of acid phosphatase activity was removed during the wash. Upon
*Present address: Department of Paediatrics, Kennedy Center, University of Chicago, Chicago, IL 60637, U.S.A. $Author to whom correspondence should be addressed.
elution with a linear O-O.2 M KC1 gradient acid phosphatase activity was resolved into two peaks, designated as AP-I and AP-II according to their order of elution from the column; AP-I eluted at 0.1 M KC1 and AP-II at 0.13 M KCl. The A2s0 values in the activity regions were very low and no further activity could be eluted from the column by 0.8 M KCl. A typical purification procedure is summarized in Table 1. Because the total activity was resolved into two fractions during purification, the recovery and the purification of each form of acid phosphatase appears to be relatively low. Polyacrylamide gel electrophoresis of AP-I and AP-II showed one major band of acid phosphatase activity with different electrophoretic mobilities. The M,s of AP-I and AP-II were 101000 and 118 000, respectively, as determined by gel filtration through a Sephadex G-200 column. Acid phosphatases from chick pea embryonic axes [7] and Asclepias curassavica latex [9] had much lower M,, viz. 39 000 and 27 000, respectively. AP-I and AP-II exhibited their maximum activity at pH 5.5 and half maximal activities at pH 4.6 and 6.7, when examined in several buffers, viz. Na acetate, Na citrate and Tris-maleate, in the pH range 3.5-7.5. They also showed the same temperature optima at 60”, quite high in comparison to acid phosphatases from cotton seed (37”) [12] and yam (50°) [13]. But at 70”, AP-I and AP-II showed 27 and 60% of their respective maximum activity. The enzymes were stable for several months when stored at - 20”, but at higher temperatures their activities decayed to different extents in both neutral and acidic pH (Fig. I), thus showing the greater heat stability of AP-II. Thermal sensitivity of several plant acid phosphatases have been reported [4, 5, 111; however, the enzyme from Ustilago esculenta showed high thermostability [14]. Both acid phosphatases from mung bean axes possessed broad substrate specificity (Table 2). They exhibited
2826
P. DE-KUNDU and A. C. BANERJEE Table
1. Purification
of acid phosphatase
Total protein (mg)
Step Crude Alcohol ppt. DEAE-cellulose AP-I AP-II
1510 510
Total act. (pkat)
Sp. act. (nkat mg- ’ protein)
24l 18
13.3 35
1.5 6.4
Table
from mung bean seedling
5.0 6.4
2. Substrate
Purification fold 1.0 2.6
661 1000
specificity
axes
50 75
Recovery (%) 100 90 25 32
of acid phosphatases
Relative enzyme activity Substrate
AP-I
AP-II
p-Nitrophenyl phosphate a-Naphthyl phosphate a-Glycerophosphate D-Glucose- 1-phosphate D-Glucose-6-phosphate D-Fructose-1,6-diphosphate Inorganic pyrosphosphate 5’-ATP S-ADP 5’-GMP 5’-GTP 5’-UMP 3’-5’-cyclic AMP Phytate
100 24 29 6 27 20 94 46 20 5 39 5.2 6.4 0
loo 20 15 17 13 18 63 25 16 4.2 21 2.8 5.1 0
(%)
Enzyme activity was assayed in 50 mM Tris-maleate, pH 5.5 as described. The concentration of all substrates used was 2 mM.
100
_/‘ 80 60
.
A II
0
Y)
I.
M
30
I
Time of preincubation (mht Fig. 1. Thermal stability of acid phosphatases. AP-I and AP-II were separately preincubated in 25 mM Tris-HCl, pH 7 (A), or 25 mM Na-acetate, pH 5 (B), at 55” for different time periods and assayed for activity with PNPP as substrate. The activity
remaining was expressed as a percentage of the appropriate zerotime control.
their highest activity towards p-nitrophenyl phosphate (PNPP), and then inorganic pyrophosphate. Nucleoside triphosphates, viz. ATP and GTP were apparently better substrates than nucleoside di- and monophosphates. Highest activity towards PNPP substrate was also reported for peanut acid phosphatase [S]. AP-I and AP-II did not hydrolyse phytate and had very little activity towards nucleoside monophosphates. The effect of various metal ions and chemical agents on the acid phosphatase activities were investigated using PNPP as substrate (Table 3). None of the enzyme forms showed an absolute requirement for a metal ion for its activity. Hg’ + and Ag+ were strong inhibitors for both AP-I and AP-II. Cu2+ and Zn2 ’ were also inhibitory, but the inhibition was greater for AP-I than AP-II, and could be partially relieved by EDTA, however, EDTA itself did not have any effect on mung bean axis acid phosphatase. Ca’ ’ caused some stimulation of both forms. Peanut acid phosphatase is reported to be activated by Ca2+ and Mg2+ [S]. AP-I and AP-II were not inhibited by NaCN, nor were they significantly influenced by B-mercaptoethanal, however, p-chloromercuribenzoate and azide caused some inhibition of AP-I. Arsenate and molybdate inhibited both the enzymes. Tartarate, an inhibitor of animal acid phosphatases, caused significant inhibition of AP-I and AP-II at 5 mM, but not at 1 mM. Detergents, viz. Triton X-100, Tween-80 and Na deoxycholate, stimulated both forms markedly. But. in the presence of 0.1%
Acid phosphatases Table
3. Effects
from mung bean
of some metal ions and chemical activities of acid phosphatases Relative
activity
2827 agents
(% control)
Addition
AP-I
AP-II
None (control) ZnCl,, 1 mM ZnCl,, 1 mM + EDTA, CaCl,, 1 mM
100 37 72 116
100 55 68 120
1 mM
MgCI,, 1 mM CuSO,, 1 mM CuSO,, 1 mM+EDTA, 1 mM FeSO,,
1 mM
Pb(NO,),, 1 mM Co&, 1 mM NiCl,, 1 mM MnCl,, 1 mM HgCl,, 1 mM AgNO,, 1 mM EDTA, 1 mM NaN,, 1 mM NaCN, 1 mM P-MSH, 10 mM PCMB, 1 mM Arsenate, 1 mM Molybdate, 1 mM Tartrate, 1 mM Tartrate, 5 mM Triton X-100, 0.1% Tween-80, 0.1% SDS, 0.1% Sodium deoxycholate, Urea, 8 M
0.5%
on the
95 40 79
117 49 85
95 99 107 104 105 8 19 100 81 101 113 70 54 13 96 49 214 206 13 150 29
110 95 105 94 110 10 30 103 117 120 112 94 45 14 89 48 185 164 72 151 98
Each enzyme was preincubated with or without (control) the indicated metal ion or chemical agent in 50 mM Tris-maleate buffer, pH 5.5 for 10 min at 37”. The enzyme activity was then determined using PNPP as substrate and is expressed as percentage activity of the respective control.
SDS, the activity of AP-I was severely affected whereas much less inhibition was observed for AP-II. Interestingly, such difference was also apparent from the effect of urea. Upon 8 M urea treatment, AP-I lost most of its activity but AP-II remained fully active. The apparent K, of AP-I and AP-II for PNPP were determined from Lineweaver-Burk plots and were 0.71 and 0.41 mM, respectively. They were competitively inhibited by KH,PO, and non-competitively by NaF as revealed by Dixon plots. The apparent Ki values of KH,PO, for AP-I and AP-II were 1.24 and 0.62 mM, respectively, and that of NaF were 0.47 and 0.67 mM, respectively. The Ki of NaF for acid phosphatase from aleurone of rice [3] was 1.29 mM, but that from cultured Ipomoea cells [15] was as low as 8.5 x 10m5 M. The activation energies of AP-I and AP-II were determined from Arrhenius plots constructed from the rate of enzymatic hydrolysis of PNPP (at optimum pH) at various temperatures. The plots were linear in the temperature range 20-60” and the activation energy values of AP-I and AP-II were 6.02 and 7.38 kcal mol- ‘, respect-
ively. These values are lower than those reported for acid phosphatases from Vigna mungo cotyledons (9.4-12.1 kcal mol-‘) [4]. EXPERIMENTAL Materials. Mung bean seeds ( V. radiata) were obtained from the National Seed Corporation, Calcutta, India. PNPP, diazo Fast Garnet GBC and other fine chemicals were from Sigma. Enzyme assay. Acid phosphatase activity was determined by measuring the rate of formation of p-nitrophenol from enzyme catalysed hydrolysis of PNPP substrate. Reaction mixts (1 ml) containing 50 mM Tris-maleate buffer (pH 5.5), 2 mM PNPP and a suitable aliquot of the enzyme prepn were incubated at 37 for 15 min. The reaction was stopped by adding 4 ml 0.1 M NaOH and the A of the liberated p-nitrophenol measured at 410 nm. When other phosphate esters were used as substrate, the liberated inorganic phosphate was measured according to the method of ref. [16]. Enzyme purification. Mung beans were surface sterilized with 0.1% Hg&, washed thoroughly with H,O and then allowed to
2828
P. DE-KUNDU and A. C. BANERJEE
germinate in the dark at 28”. Axes organs (ca 100 g) were harvested from 3-day germinated seeds, thoroughly washed with ice-cold H,O and homogenized in 200 ml 10 mM Tris-HCI buffer, pH 7.2 (buffer A) using a precooled mortar and pestle. Unless otherwise stated, all operations were carried out at O-4”. The homogenate was strained through two layers of cheesecloth and spun at 12,000 9 for 30 min. The supernatant obtained was mixed with 2.5 vol of chilled EtOH (-20”) with continuous shaking. The resulting mixt. was kept at -2O’, for 30min and then centrifuged at 10000 9 for 15 min. The pellet thus obtained was dissolved in a minimum vol of buffer A and dialysed against 100 vol of the same buffer for 4-5 hr with one change. The dialysate was centrifuged to remove any undissolved particles and the clear supernatant slowly applied to a DE-52 column (1.5 x 14.5 cm) pre-equilibrated with buffer A. The column was eluted first with buffer A and then with 200 ml of a linear O-200 mM KC1 gradient in buffer A. Column frs (2.5 ml) were assayed for acid phosphatase activity and scanned at 280 nm for protein. Frs under each acid phosphatase peak were pooled, or dialysed against buffer A for 6 hr and stored at -20’ lyophilized. Protein estimation. Protein was measured according to the method of ref. [17] using BSA as std. Polyacrylamide gef ekctrophoresis was carried out at 4” following the method of ref. 1181 using a 7.5% gel containing 0.15% Triton X-100 at pH 4.6. Gels were stained for acid phosphatase activity by incubation with 0.1% Na a-naphthyl acid phosphate and 0.1% diazo Fast Garnet GBC in 50 mM NaOAc buffer pH 5.0 for 15-20 min at 37” [19]. Zones containing enzyme activity appeared as coloured bands. Developed gels were stored in 7.5% HOAc and 30% EtOH. Geljibation of purified enzyme proteins was carried out on a Sephadex G-200 column pre-equilibrated with 0.1 M KC1 in buffer A and calibrated with standard marker proteins for determination of M, according to ref. 1201. Acknowledyements-Financial and other help from the University of Calcutta and the University of Papua New Guinea are gratefully acknowledged.
REFERENCES
2.
3. 4.
6.
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
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