Planta 9 Springer-Verlag 1981
Planta (1981) 152:408414
Redox modulation of a phosphatase from Anacystis nidulans
M. Godeh, J. Udvardy, and G.L. Farkas* Institute of Plant Physiology,BiologicalResearchCenter, HungarianAcademyof Sciences, P.O. Box 521, H-6701 Szeged,Hungary
Abstract. Ascorbic acid (AA) increased the phosphatase activity (pH 6.8) in 10,000 g supernatants from Anacystis nidulans. The enzyme activated by AA was deactivated by dehydroascorbic acid (DHAA). The modulation by AA/DHAA of phosphatase activity in Anacystis appears to be specific; a number of other redox compounds, known to modulate other enzymes, had no effect on the Anacystis phosphatase. A purified phosphatase preparation from Anacystis was also deactivated by DHAA. In contrast, the purified enzyme was not activated by AA, suggesting that a factor mediating the effect of AA was lost during purification. Another factor was found to protect the purified phosphatase against deactivation by DHAA. The enzyme was characterized as a phosphatase with a broad substrate specificity, an apparent molecular weight of 19,000, and a pH optimum of 6.0-7.0. Dialysis of the enzyme preparation against EDTA abolished the phosphatase activity which could be restored by Zn 2§ ions and partially restored by Co 2§ ions. Crude extracts also contained a latent enzyme, the phosphatase activity of which could be detected in the presence of Co 2§ ions only. Zn 2§ ions did not activate this enzymatically inactive protein. The Co 2+_ dependent phosphatase had an apparent mol. wt. of 40,000, a broad substrate specificity, and an alkaline pH-optimum. Infection of Anacystis cultures by cyanophage AS-1 resulted in a decrease in phosphatase activity. The enzyme present in 10,000 g supernatants from infected cells could not be modulated by the AA/DHAA system.
* To whomcorrespondenceshould be sent Abbreviations." AA=ascorbic acid; DEAE=diethylamino ethyl; DHAA=dehydroascorbic acid; EDTA=ethylene-diaminetetraacetate; G6PDH= glucose-6-phosphatedehydrogenase;GSH= reduced glutathione; GSSG=oxidized glutathione; HMP=hexose monophosphate; P~=inorganic phosphorus; pNPP-p-nitrophenylphosphate; pNP=p-nitrophenol; Tris=Tris(hydroxymethyl)aminomethane
0032-0935/81/0152/0408/$01.40
Key words: Anacystis - Cyanobacteria - Cyanophage infection - Oxido-reductive enzyme modulation Phosphatase, acid and alkaline.
Introduction
Redox modulation of enzyme activity is an important aspect of enzyme regulation in chloroplasts (Anderson 1979; Buchanan et al. 1979; Buchanan 1980). Reductive modulation of several cyanobacterial enzymes has also been described (Duggan and Anderson 1975; Pelroy etal. 1976; Schmidt and Christen 1978; Wagner et al. 1978). The oxidative aspects of enzyme modulation in this group of organisms remained, however, largely unexplored. Recently, strong evidence has been obtained for the involvement of oxidative enzyme modulation in the regulation of carbon flow in cyanobacteria. Activation of the oxidative hexose monophosphate (HMP) pathway, associated with an increase in the activity of its first enzyme, glucose-6-P dehydrogenase (G6PDH), has been found to occur in cyanophageinfected Anacystis nidulans (Balogh et al. 1979), apparently due to an oxidative transformation of G6PDH into a hyperactive form (CsOke et al. 1980). Therefore, further work on the oxido-reductive modulation of other cyanobacterial enzymes, in relation to cyanophage-infection, appeared warranted. The present paper deals with the oxido-reductive modulation of the phosphatase ofA. nidulans, because (i) preliminary observations on changes in acid-phosphatase activity in cyanophage-infected Anacystis have already been made (Udvardy et al. 1976), and (ii) the oxido-reductive modulation of acid phosphatases from higher plants has recently been reported (Buchanan et al. 1979).
M. Godeh et al. : Redox modulation of a phosphatase
Materials and methods Strain, growth conditions, and cyanophage infection. Anacystis nidulans (Synechococcus AN PCC 6301) was grown as described previously (Udvardy et al. 1976) in a liquid medium containing the microelements of medium " C " and the major salts of medium " D " of Kratz and Myers (1955). Algal cells were infected by cyanophage AS-I at a multiplicity of infection of 5, as described by Udvardy et aI. (1976).
Enzyme assays. Phosphomonoesterase activity was assayed in both the 10,000 g supernatants of cell sonicates (Udvardy et al. 1976) and in the purified preparations (see below) using two methods. Either the formation of p-nitrophenol (pNP) from p-nitrophenylphosphate (pNPP) or the liberation of inorganic phosphate (P~) from organic phosphorous compounds was measured. Method I: The reaction mixture contained 150~tmol Tris-acetate buffer (pH 6.8), 0.4 txmol pNPP, and a sufficient amount of enzyme (corresponding to 2.108 cells) in a total volume of 1.5 ml. After incubation at 37 ~ C for 30 min, the reaction was stopped by adding 1 ml 0.3 M NaOH after which the reaction mixture was centrifuged at 10,000 g for 10 rain. The pNP released was measured at 400 rim. The complete reaction system minus enzyme served as a blank. When Co 2+ ions were added to the assay system, the reaction was stopped by 0.3 M NaOH containing 20 mM EDTA. Method H: The reaction system contained 100 ~mol of Tris-acetate buffer (pH 6.8), 3 gmol of substrate, and an enzyme aliquot at a total volume of 1 ml. After incubation at 37 ~ C for 30 min, the reaction was terminated by adding 0.5 ml 15% trichloroacetic acid after which the mixture was centrifuged at 10,000 g for i0 rain. Onemilliliter aliquots of the supernatant were assayed for P~ content as described by Lebel etal. (1978). When Co 2+ ions, ascorbic acid (AA), or dehydroascorbic acid (DHAA) were added to the assay system, the P~ liberated was measured according to the method of Chen and Toribara (1956). Phosphatase activity in vivo was measured as described by Ihlenfeldt and Gibson (1975).
Enzyme purification. Cells were harvested and washed as described earlier (Udvardy et al. I976). The washed cells were then frozen. Step 1. The frozen cells were ground to a paste in a mortar with A1203 (1:2; w/w) for 30 rain. This paste was then suspended in a 3-fold volume of 0.05 M Tris-HC1 buffer (pH 7.5). The extract was centrifuged at 20,000 g for 20 min. Fresh A1203 was added to the sediment and the extraction repeated. The two supernatants were combined and centrifuged at 40,000 g for 30 min. The resulting supernatant was used. Step 2. (NH,)zSO4 was added to the supernatant to 30% saturation, stirred for 1 h, and centrifuged at 25,000 g for 30 rain. The sediment was discarded and further (NH4)~SO4 was added to the supernatant to 80% saturation. The centrifugation was repeated and the sediment taken up in 0.02 M Tris-HC1 buffer (pH 7.5). The solution was then dialyzed overnight against 10 1 of the same buffer. Step 3. The dialysate from step 2 was loaded onto a DEAE-cellulose column (2 cm. 30 cm). The column was washed with 0.02 M Tris HC1 buffer (pH 7.5). Proteins were eluted from the column by a NaC1 gradient (0-0.5 M NaC1) at a flow rate of 18 ml h - ~ and the fractions corresponding to major peaks of phosphatase activity combined. The combined fractions were dialyzed against 0.02 M Tris-HC1 buffer (pH 7.5), divided into atiquots, and deep-frozen. Determination of apparent motecular weight. The purified enzymes and reference compounds were chromatographed on Sephadex G100 or G-200 columns (2.4 cm.100 cm) equilibrated with 0.2 M NaC1 in 0.02 M Tris-HC1 buffer (pH 7.5). The marker proteins were identified on the basis of UV absorption at 215 nm (~'-globulin, serum albumin), absorption in the visible range (cytochrome-c), and by enzyme assay (peroxidase), respectively, Phosphatases were
409 detected by the standard assay. It is important to note that more than 90% of phosphatase activity was strongly adsorbed to Sephadex gels and could be eluted only if 0.2 M NaCI was added to the eluting buffer.
DiaIysis. Aliquots of the purified enzymes were dialyzed for 3 days against 3 changes of 1,000 fold volume excess of 50 mM EDTA (pH 5.5). To remove EDTA, the dialysis was followed with three changes of 1,000-fold volume excess of tridistilled water (pH 5.5) for three more days in the cold. Tests for ion requirements were made on this metal-freed enzyme preparation.
Estimation of protein content, Protein content in the crude extracts was determined by the Lowry method (Lowry et al. 1951). During the later stages of purification the protein content was calculated from the absorbance of the solutions at 280 nm.
Results Oxido-reductive modulation o f phosphatase activity. T h e o x i d o - r e d u c t i v e m o d u l a t i o n o f p h o s p h a t a s e act i v i t y w a s f i r s t t e s t e d i n c r u d e , 10,000 g s u p e r n a t a n t s . We modeled our experiments on the paper by Buc h a n a n et al. (1979), r e p o r t i n g r e d o x m o d u l a t i o n o f acid phosphatases from higher plants and on our prev i o u s w o r k o n t h e o x i d o - r e d u c t i v e m o d u l a t i o n o f Anacystis G 6 P D H (Cs6ke et al. 1980). O f t h e w i d e v a r i e t y o f f a c t o r s t e s t e d (e,g., d a r k n e s s / l i g h t , O 2 / a n a e r o b i c conditions, oxidized glutathione/reduced glutathione (GSSG/GSH), dehydroascorbic acid/ascorbic acid (DHAA/AA), NADP/NADPH, etc.), only the
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DEHYDROASCORBIC ACID (mM) ADDED IN ADDITION TO 50mMAA Fig. 1. Activation of phosphatase by ascorbate and deactivation of the enzyme by dehydroascorbate in 10,000 g supernatant from Anacystis nidulans. Aliquots of the supernatant were preincubated for 10 min with 50 mM AA and diluted to a final concentration of 4 mM before enzyme assay. The phosphatase activity of the samples was measured t0 rain after dilution. To other aliqnots of the supernatant treated with 50 mM AA and diluted to a final AA concentration of 4 raM, DHAA was added at various concentrations. The samples were incubated with DHAA for l0 rain and assayed for phosphatase activity. Substrate: pNPP
410
M. Godeh et al. : Redox modulation of a phosphatase
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Fig. 2A, B. DEAE cellulose chromatography of Anacystis proteins in the absence (A) and presence (B) of 0.1 mM ZnC12, respectively. All previous manipulations, before loading the sample on the Zn 2+ containing column (B), including protein extraction and (NH4)2SO~ fractionation (30-80% saturation), were done in the presence of 0.1 mM ZnCI2. Proteins were monitored at 280 nm ( - - ) . Phosphatase activity was measured in the absence (o) and in the presence (0) of 10 mM COC12, respectively. All fractions in variant A were tested for the presence of a factor counteracting the inhibition of the purified Zn z§ phosphatase by dehydroascorbic acid, as described in Table 2 ( zx). Substrate: pNPP
DHAA/AA redox pair affected the enzyme activity to a great extent. In contrast to the acid phosphatases from higher plants known to be activated by oxidants and deactivated by reductants (Buchanan et al. 1979), the Anacystis enzyme was activated by AA and deactivated by DHAA (Fig. 1). Maximum activation by AA was obtained in extracts from cells in the logarithmic phase of growth. Anabaena 7120 was also shown to contain a phosphatase regulated by the AA/DHAA redox system.
The major phosphatase of Anacystis. To test whether or not the purified enzyme retains the properties described above, an Anacystis phosphatase was partially purified. Here we show only the last step of the purification procedure, the DEAE cellulose chromatography (for further details see Materials and Methods). As may be seen in Fig. 2A, part of the acid phosphatase was not bound to the column. A major phosphatase peak was eluted at a NaCI concentration of 0.12 M (average of 5 runs). In most runs, a shoulder on this peak indicated that it may not be homogenous. However, when all manipulations were carried out in the presence of 0.1 mM Zn 2+, in order to prevent the loss of zink possibly present in the enzyme (Applebury et al. 1970), practically all phosphatase activity remained bound to the column and the main eluted peak was symmetrical (bell-shaped) (Fig. 2B). This suggests that Zn 2 § was lost during the manipulations and may play a role in maintaining the " n o r m a l " enzyme form, as described for the E. coli alkaline phosphatase, which can be present in different forms depending on the amount of bound Zn 2§ (Reynolds and Schlessinger 1969). The role of Zn z § in the activity of Anacystis phosphatase was also demonstrated in a more direct way. Dialysis of the pooled phosphatase fractions (Fr. No.
Table 1. Substrate specificity of phosphatase purified from Anacys-
tis nidulans Substrate
Relative enzyme activity (%)a Zn2 +-dependent Co2+-dependent phosphatase latent phosphatase
pNPP Glucose- 1-P Glucose-6-P 6-Phosphogluconate Fructose-6-P Fructose- 1,6-P c~-Glycerophosphate fl-Glycerophosphate Na-pyrophosphate 5'-AMP ADP ATP dAMP dUMP
100 40 2 4 0 16 3 i 16 103 106 101 118 76
100 37 1 0 8 20 2 1 29 0 19 32 7 2
a Enzyme activity was assayed by determining the Pi released
40 55 in Fig. 2A), for three days against 50 mM EDTA, led to a 90-93 % loss of enzyme activity which could be regained up to 70% by incubating the enzyme in the presence of 0.1 mM Zn 2§ Prolonged dialysis was necessary to show this effect. Only a slight inhibition (10%) of enzyme activity was detected immediately after the addition of EDTA to the enzyme. The dialyzed preparation could be reactivated by comparatively high amounts of Co 2§ (110 raM) as well, albeit to a lesser extent than with Zn 2§ (data not shown). The partially purified enzyme preparation (fractions No. 40-55 in Fig. 2A) had a pH optimum of 6.0-7.0, depending on the concentration of pNPP used as the substrate. The apparent K m value for
M. Godeh et al. : Redox modulation of a phosphatase
411 Table 2. Demonstration of the effect of a factor protecting the partially purified Anacystis phosphatase against deactivation with dehydroascorbic acid
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Complete phosphatase assay system~ Complete phosphatase assay system +0.5 mM DHAA Complete phosphatase assay system +0.5 mM DHAA+0.5 ml "protein factor"b
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ASCORBIC OR DEHYDROASCORBICACID
Fig. 3. Effect of different amounts of ascorbic acid (o) and dehydroascorbic acid (o), respectively, on a purified preparation of phosphatase from Anacystis. AA and DHAA were added to the reaction mixture at the start of the assay. Substrate: pNPP
the hydrolysis of p N P P was 2.6 mM. The enzyme exhibited a fairly broad substrate specificity, but had a pronounced preference for the hydrolysis of nucleoside mono-, di- and triphosphates (Table 1, column I). Co 2+, Mn 2+, and Mg 2+ ions (10raM), which affect some other phosphatases (Reid and Wilson 1971; Fernley 1971), had no effect on the activity of the Anacystis phosphatase. The enzyme, purified 250-fold, had an apparent molecular weight of 19,000, as determined by gel filtration. The enzyme activity was inhibited by inorganic phosphate (Ki = 1.65 mM).
Oxido-reductive modulation of the purified enzyme. In contrast to the typical oxido-reductive modulation of phosphatase activity in the crude supernatant, the activity of the purified enzyme decreased upon treatment both with A A and D H A A , although much higher concentrations of A A than D H A A were needed to evoke the same extent of inhibition (Fig. 3). A A preparations of a different origin, subjected to additional purification in our laboratory, as well as isoasorbic acid consistently inhibited the purified enzyme. The inhibitory effect of AA solutions increased upon standing or aeration. Therefore, we concluded that the small amounts of D H A A or of its further breakdown products present in the AA preparations may be responsible for the inhibition of purified phosphatase by AA. Why, then, does A A activate the phosphatase in crude systems? One can assume that in crude extracts there are factors which either remove D H A A or protect in some other way the phosphatase against the deactivating effect of D H A A . Evidence for the existence of the postulated protecting factor is described below.
Pooled fractions No. 41-56 in Fig. 2A were used as a source of partially purified phosphatase b Pooled fractions No. 66-86 in Fig. 2A were used as a source of the factor. These fractions had negligible phosphatase activity a
A factor protects phosphatase against dehydroascorbic acid. After chromatography on a DEAE-cellulose colu m n of algal proteins obtained by (NH4)2SO4-precipitation (Fig. 2A), each fraction was tested for its ability to counteract the phosphatase-deactivating effect of 50 m M AA or 4 m M D H A A . A well defined peak of such a protective activity was found (Fig. 2A), fractions No. 66-86). In the presence of small amounts of the pooled fractions neither AA nor D H A A (the latter only if present in concentrations not higher than 4 raM) inhibited the purified phosphatase. Results of a typical experiment with D H A A are shown in Table 2. The factor, further purified on a Sephadex G-200 column and completely separated from phosphatases (see Fig. 2 and 4), was found to be heat labile. Pending further characterization, on the basis of its chromatographic behavior, UV spectrum, and ninhydrin positivity, we regard the factor to be a protein of an apparent tool. wt. of 130,000, as determined by gel filtration (see Methods).
The effect of ascorbic acid on the phosphatase is factormediated. Stimulation by A A of the phosphatase activity in purified preparations was not found even in the presence of the factor described above. Thereit was reasonable to suppose that this factor is involved only in the elimination of the inhibitory, effect of the small amounts of D H A A added to the purified preparations or present in the crude extracts. The actual activation of phosphatase by A A is apparently dependent on yet another factor(s) which mediate(s) the effect of AA. In contrast to the purified phosphatase, the phosphatase(s) in the protein fractions obtained by (NH4)zSO4-fractionation (30 80% satura-
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Planta (1981) 152:408414
Redox modulation of a phosphatase from Anacystis nidulans
M. Godeh, J. Udvardy, and G.L. Farkas* Institute of Plant Physiology,BiologicalResearchCenter, HungarianAcademyof Sciences, P.O. Box 521, H-6701 Szeged,Hungary
Abstract. Ascorbic acid (AA) increased the phosphatase activity (pH 6.8) in 10,000 g supernatants from Anacystis nidulans. The enzyme activated by AA was deactivated by dehydroascorbic acid (DHAA). The modulation by AA/DHAA of phosphatase activity in Anacystis appears to be specific; a number of other redox compounds, known to modulate other enzymes, had no effect on the Anacystis phosphatase. A purified phosphatase preparation from Anacystis was also deactivated by DHAA. In contrast, the purified enzyme was not activated by AA, suggesting that a factor mediating the effect of AA was lost during purification. Another factor was found to protect the purified phosphatase against deactivation by DHAA. The enzyme was characterized as a phosphatase with a broad substrate specificity, an apparent molecular weight of 19,000, and a pH optimum of 6.0-7.0. Dialysis of the enzyme preparation against EDTA abolished the phosphatase activity which could be restored by Zn 2§ ions and partially restored by Co 2§ ions. Crude extracts also contained a latent enzyme, the phosphatase activity of which could be detected in the presence of Co 2§ ions only. Zn 2§ ions did not activate this enzymatically inactive protein. The Co 2+_ dependent phosphatase had an apparent mol. wt. of 40,000, a broad substrate specificity, and an alkaline pH-optimum. Infection of Anacystis cultures by cyanophage AS-1 resulted in a decrease in phosphatase activity. The enzyme present in 10,000 g supernatants from infected cells could not be modulated by the AA/DHAA system.
* To whomcorrespondenceshould be sent Abbreviations." AA=ascorbic acid; DEAE=diethylamino ethyl; DHAA=dehydroascorbic acid; EDTA=ethylene-diaminetetraacetate; G6PDH= glucose-6-phosphatedehydrogenase;GSH= reduced glutathione; GSSG=oxidized glutathione; HMP=hexose monophosphate; P~=inorganic phosphorus; pNPP-p-nitrophenylphosphate; pNP=p-nitrophenol; Tris=Tris(hydroxymethyl)aminomethane
0032-0935/81/0152/0408/$01.40
Key words: Anacystis - Cyanobacteria - Cyanophage infection - Oxido-reductive enzyme modulation Phosphatase, acid and alkaline.
Introduction
Redox modulation of enzyme activity is an important aspect of enzyme regulation in chloroplasts (Anderson 1979; Buchanan et al. 1979; Buchanan 1980). Reductive modulation of several cyanobacterial enzymes has also been described (Duggan and Anderson 1975; Pelroy etal. 1976; Schmidt and Christen 1978; Wagner et al. 1978). The oxidative aspects of enzyme modulation in this group of organisms remained, however, largely unexplored. Recently, strong evidence has been obtained for the involvement of oxidative enzyme modulation in the regulation of carbon flow in cyanobacteria. Activation of the oxidative hexose monophosphate (HMP) pathway, associated with an increase in the activity of its first enzyme, glucose-6-P dehydrogenase (G6PDH), has been found to occur in cyanophageinfected Anacystis nidulans (Balogh et al. 1979), apparently due to an oxidative transformation of G6PDH into a hyperactive form (CsOke et al. 1980). Therefore, further work on the oxido-reductive modulation of other cyanobacterial enzymes, in relation to cyanophage-infection, appeared warranted. The present paper deals with the oxido-reductive modulation of the phosphatase ofA. nidulans, because (i) preliminary observations on changes in acid-phosphatase activity in cyanophage-infected Anacystis have already been made (Udvardy et al. 1976), and (ii) the oxido-reductive modulation of acid phosphatases from higher plants has recently been reported (Buchanan et al. 1979).
M. Godeh et al. : Redox modulation of a phosphatase
Materials and methods Strain, growth conditions, and cyanophage infection. Anacystis nidulans (Synechococcus AN PCC 6301) was grown as described previously (Udvardy et al. 1976) in a liquid medium containing the microelements of medium " C " and the major salts of medium " D " of Kratz and Myers (1955). Algal cells were infected by cyanophage AS-I at a multiplicity of infection of 5, as described by Udvardy et aI. (1976).
Enzyme assays. Phosphomonoesterase activity was assayed in both the 10,000 g supernatants of cell sonicates (Udvardy et al. 1976) and in the purified preparations (see below) using two methods. Either the formation of p-nitrophenol (pNP) from p-nitrophenylphosphate (pNPP) or the liberation of inorganic phosphate (P~) from organic phosphorous compounds was measured. Method I: The reaction mixture contained 150~tmol Tris-acetate buffer (pH 6.8), 0.4 txmol pNPP, and a sufficient amount of enzyme (corresponding to 2.108 cells) in a total volume of 1.5 ml. After incubation at 37 ~ C for 30 min, the reaction was stopped by adding 1 ml 0.3 M NaOH after which the reaction mixture was centrifuged at 10,000 g for 10 rain. The pNP released was measured at 400 rim. The complete reaction system minus enzyme served as a blank. When Co 2+ ions were added to the assay system, the reaction was stopped by 0.3 M NaOH containing 20 mM EDTA. Method H: The reaction system contained 100 ~mol of Tris-acetate buffer (pH 6.8), 3 gmol of substrate, and an enzyme aliquot at a total volume of 1 ml. After incubation at 37 ~ C for 30 min, the reaction was terminated by adding 0.5 ml 15% trichloroacetic acid after which the mixture was centrifuged at 10,000 g for i0 rain. Onemilliliter aliquots of the supernatant were assayed for P~ content as described by Lebel etal. (1978). When Co 2+ ions, ascorbic acid (AA), or dehydroascorbic acid (DHAA) were added to the assay system, the P~ liberated was measured according to the method of Chen and Toribara (1956). Phosphatase activity in vivo was measured as described by Ihlenfeldt and Gibson (1975).
Enzyme purification. Cells were harvested and washed as described earlier (Udvardy et al. I976). The washed cells were then frozen. Step 1. The frozen cells were ground to a paste in a mortar with A1203 (1:2; w/w) for 30 rain. This paste was then suspended in a 3-fold volume of 0.05 M Tris-HC1 buffer (pH 7.5). The extract was centrifuged at 20,000 g for 20 min. Fresh A1203 was added to the sediment and the extraction repeated. The two supernatants were combined and centrifuged at 40,000 g for 30 min. The resulting supernatant was used. Step 2. (NH,)zSO4 was added to the supernatant to 30% saturation, stirred for 1 h, and centrifuged at 25,000 g for 30 rain. The sediment was discarded and further (NH4)~SO4 was added to the supernatant to 80% saturation. The centrifugation was repeated and the sediment taken up in 0.02 M Tris-HC1 buffer (pH 7.5). The solution was then dialyzed overnight against 10 1 of the same buffer. Step 3. The dialysate from step 2 was loaded onto a DEAE-cellulose column (2 cm. 30 cm). The column was washed with 0.02 M Tris HC1 buffer (pH 7.5). Proteins were eluted from the column by a NaC1 gradient (0-0.5 M NaC1) at a flow rate of 18 ml h - ~ and the fractions corresponding to major peaks of phosphatase activity combined. The combined fractions were dialyzed against 0.02 M Tris-HC1 buffer (pH 7.5), divided into atiquots, and deep-frozen. Determination of apparent motecular weight. The purified enzymes and reference compounds were chromatographed on Sephadex G100 or G-200 columns (2.4 cm.100 cm) equilibrated with 0.2 M NaC1 in 0.02 M Tris-HC1 buffer (pH 7.5). The marker proteins were identified on the basis of UV absorption at 215 nm (~'-globulin, serum albumin), absorption in the visible range (cytochrome-c), and by enzyme assay (peroxidase), respectively, Phosphatases were
409 detected by the standard assay. It is important to note that more than 90% of phosphatase activity was strongly adsorbed to Sephadex gels and could be eluted only if 0.2 M NaCI was added to the eluting buffer.
DiaIysis. Aliquots of the purified enzymes were dialyzed for 3 days against 3 changes of 1,000 fold volume excess of 50 mM EDTA (pH 5.5). To remove EDTA, the dialysis was followed with three changes of 1,000-fold volume excess of tridistilled water (pH 5.5) for three more days in the cold. Tests for ion requirements were made on this metal-freed enzyme preparation.
Estimation of protein content, Protein content in the crude extracts was determined by the Lowry method (Lowry et al. 1951). During the later stages of purification the protein content was calculated from the absorbance of the solutions at 280 nm.
Results Oxido-reductive modulation o f phosphatase activity. T h e o x i d o - r e d u c t i v e m o d u l a t i o n o f p h o s p h a t a s e act i v i t y w a s f i r s t t e s t e d i n c r u d e , 10,000 g s u p e r n a t a n t s . We modeled our experiments on the paper by Buc h a n a n et al. (1979), r e p o r t i n g r e d o x m o d u l a t i o n o f acid phosphatases from higher plants and on our prev i o u s w o r k o n t h e o x i d o - r e d u c t i v e m o d u l a t i o n o f Anacystis G 6 P D H (Cs6ke et al. 1980). O f t h e w i d e v a r i e t y o f f a c t o r s t e s t e d (e,g., d a r k n e s s / l i g h t , O 2 / a n a e r o b i c conditions, oxidized glutathione/reduced glutathione (GSSG/GSH), dehydroascorbic acid/ascorbic acid (DHAA/AA), NADP/NADPH, etc.), only the
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DEHYDROASCORBIC ACID (mM) ADDED IN ADDITION TO 50mMAA Fig. 1. Activation of phosphatase by ascorbate and deactivation of the enzyme by dehydroascorbate in 10,000 g supernatant from Anacystis nidulans. Aliquots of the supernatant were preincubated for 10 min with 50 mM AA and diluted to a final concentration of 4 mM before enzyme assay. The phosphatase activity of the samples was measured t0 rain after dilution. To other aliqnots of the supernatant treated with 50 mM AA and diluted to a final AA concentration of 4 raM, DHAA was added at various concentrations. The samples were incubated with DHAA for l0 rain and assayed for phosphatase activity. Substrate: pNPP
410
M. Godeh et al. : Redox modulation of a phosphatase
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Fig. 2A, B. DEAE cellulose chromatography of Anacystis proteins in the absence (A) and presence (B) of 0.1 mM ZnC12, respectively. All previous manipulations, before loading the sample on the Zn 2+ containing column (B), including protein extraction and (NH4)2SO~ fractionation (30-80% saturation), were done in the presence of 0.1 mM ZnCI2. Proteins were monitored at 280 nm ( - - ) . Phosphatase activity was measured in the absence (o) and in the presence (0) of 10 mM COC12, respectively. All fractions in variant A were tested for the presence of a factor counteracting the inhibition of the purified Zn z§ phosphatase by dehydroascorbic acid, as described in Table 2 ( zx). Substrate: pNPP
DHAA/AA redox pair affected the enzyme activity to a great extent. In contrast to the acid phosphatases from higher plants known to be activated by oxidants and deactivated by reductants (Buchanan et al. 1979), the Anacystis enzyme was activated by AA and deactivated by DHAA (Fig. 1). Maximum activation by AA was obtained in extracts from cells in the logarithmic phase of growth. Anabaena 7120 was also shown to contain a phosphatase regulated by the AA/DHAA redox system.
The major phosphatase of Anacystis. To test whether or not the purified enzyme retains the properties described above, an Anacystis phosphatase was partially purified. Here we show only the last step of the purification procedure, the DEAE cellulose chromatography (for further details see Materials and Methods). As may be seen in Fig. 2A, part of the acid phosphatase was not bound to the column. A major phosphatase peak was eluted at a NaCI concentration of 0.12 M (average of 5 runs). In most runs, a shoulder on this peak indicated that it may not be homogenous. However, when all manipulations were carried out in the presence of 0.1 mM Zn 2+, in order to prevent the loss of zink possibly present in the enzyme (Applebury et al. 1970), practically all phosphatase activity remained bound to the column and the main eluted peak was symmetrical (bell-shaped) (Fig. 2B). This suggests that Zn 2 § was lost during the manipulations and may play a role in maintaining the " n o r m a l " enzyme form, as described for the E. coli alkaline phosphatase, which can be present in different forms depending on the amount of bound Zn 2§ (Reynolds and Schlessinger 1969). The role of Zn z § in the activity of Anacystis phosphatase was also demonstrated in a more direct way. Dialysis of the pooled phosphatase fractions (Fr. No.
Table 1. Substrate specificity of phosphatase purified from Anacys-
tis nidulans Substrate
Relative enzyme activity (%)a Zn2 +-dependent Co2+-dependent phosphatase latent phosphatase
pNPP Glucose- 1-P Glucose-6-P 6-Phosphogluconate Fructose-6-P Fructose- 1,6-P c~-Glycerophosphate fl-Glycerophosphate Na-pyrophosphate 5'-AMP ADP ATP dAMP dUMP
100 40 2 4 0 16 3 i 16 103 106 101 118 76
100 37 1 0 8 20 2 1 29 0 19 32 7 2
a Enzyme activity was assayed by determining the Pi released
40 55 in Fig. 2A), for three days against 50 mM EDTA, led to a 90-93 % loss of enzyme activity which could be regained up to 70% by incubating the enzyme in the presence of 0.1 mM Zn 2§ Prolonged dialysis was necessary to show this effect. Only a slight inhibition (10%) of enzyme activity was detected immediately after the addition of EDTA to the enzyme. The dialyzed preparation could be reactivated by comparatively high amounts of Co 2§ (110 raM) as well, albeit to a lesser extent than with Zn 2§ (data not shown). The partially purified enzyme preparation (fractions No. 40-55 in Fig. 2A) had a pH optimum of 6.0-7.0, depending on the concentration of pNPP used as the substrate. The apparent K m value for
M. Godeh et al. : Redox modulation of a phosphatase
411 Table 2. Demonstration of the effect of a factor protecting the partially purified Anacystis phosphatase against deactivation with dehydroascorbic acid
100
so
1
/1
10
30
Composition of the system
Rate of reaction : nkat 1 1
%
Complete phosphatase assay system~ Complete phosphatase assay system +0.5 mM DHAA Complete phosphatase assay system +0.5 mM DHAA+0.5 ml "protein factor"b
50 10
100 20
58
116
50 mM
ASCORBIC OR DEHYDROASCORBICACID
Fig. 3. Effect of different amounts of ascorbic acid (o) and dehydroascorbic acid (o), respectively, on a purified preparation of phosphatase from Anacystis. AA and DHAA were added to the reaction mixture at the start of the assay. Substrate: pNPP
the hydrolysis of p N P P was 2.6 mM. The enzyme exhibited a fairly broad substrate specificity, but had a pronounced preference for the hydrolysis of nucleoside mono-, di- and triphosphates (Table 1, column I). Co 2+, Mn 2+, and Mg 2+ ions (10raM), which affect some other phosphatases (Reid and Wilson 1971; Fernley 1971), had no effect on the activity of the Anacystis phosphatase. The enzyme, purified 250-fold, had an apparent molecular weight of 19,000, as determined by gel filtration. The enzyme activity was inhibited by inorganic phosphate (Ki = 1.65 mM).
Oxido-reductive modulation of the purified enzyme. In contrast to the typical oxido-reductive modulation of phosphatase activity in the crude supernatant, the activity of the purified enzyme decreased upon treatment both with A A and D H A A , although much higher concentrations of A A than D H A A were needed to evoke the same extent of inhibition (Fig. 3). A A preparations of a different origin, subjected to additional purification in our laboratory, as well as isoasorbic acid consistently inhibited the purified enzyme. The inhibitory effect of AA solutions increased upon standing or aeration. Therefore, we concluded that the small amounts of D H A A or of its further breakdown products present in the AA preparations may be responsible for the inhibition of purified phosphatase by AA. Why, then, does A A activate the phosphatase in crude systems? One can assume that in crude extracts there are factors which either remove D H A A or protect in some other way the phosphatase against the deactivating effect of D H A A . Evidence for the existence of the postulated protecting factor is described below.
Pooled fractions No. 41-56 in Fig. 2A were used as a source of partially purified phosphatase b Pooled fractions No. 66-86 in Fig. 2A were used as a source of the factor. These fractions had negligible phosphatase activity a
A factor protects phosphatase against dehydroascorbic acid. After chromatography on a DEAE-cellulose colu m n of algal proteins obtained by (NH4)2SO4-precipitation (Fig. 2A), each fraction was tested for its ability to counteract the phosphatase-deactivating effect of 50 m M AA or 4 m M D H A A . A well defined peak of such a protective activity was found (Fig. 2A), fractions No. 66-86). In the presence of small amounts of the pooled fractions neither AA nor D H A A (the latter only if present in concentrations not higher than 4 raM) inhibited the purified phosphatase. Results of a typical experiment with D H A A are shown in Table 2. The factor, further purified on a Sephadex G-200 column and completely separated from phosphatases (see Fig. 2 and 4), was found to be heat labile. Pending further characterization, on the basis of its chromatographic behavior, UV spectrum, and ninhydrin positivity, we regard the factor to be a protein of an apparent tool. wt. of 130,000, as determined by gel filtration (see Methods).
The effect of ascorbic acid on the phosphatase is factormediated. Stimulation by A A of the phosphatase activity in purified preparations was not found even in the presence of the factor described above. Thereit was reasonable to suppose that this factor is involved only in the elimination of the inhibitory, effect of the small amounts of D H A A added to the purified preparations or present in the crude extracts. The actual activation of phosphatase by A A is apparently dependent on yet another factor(s) which mediate(s) the effect of AA. In contrast to the purified phosphatase, the phosphatase(s) in the protein fractions obtained by (NH4)zSO4-fractionation (30 80% satura-
412
M. Godeh et al. : Redox modulation of a phosphatase 04 0.3
;oow
= -
o c
cEo02
80~
'~ 0.1
4o~
6o:
5
20
