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Cell Biology International (2010) 34, 827–835 (Printed in Great Britain)
Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum
Chisa Okada*, Akio Nakamura†, Shigeo Tomioka‡, Kazuhiro Kohama† and Takako S Kaneko§1
*Department of Chemical and Biological Sciences, Graduate School of Science, Japan Womens University, Mejirodai, Bunkyoku, Tokyo 1128681, Japan, †Department of Molecular and Cellular Pharmacology, Graduate School of Medicine, Gunma University, Maebashi, Gunma, Japan, ‡Institute of Molecular and Cellular Bioscience, University of Tokyo, Bunkyo, Tokyo, Japan, and §Department of Chemical and Biological Sciences, Faculty of Science, Japan Womens University, Mejirodai, Bunkyoku, Tokyo 1128681, Japan


A phosphatase was purified through a combination of ion-exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340-fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The Km of the enzyme for PMLC was 10 μM, and the Vmax was 1.17 nkat/mg of protein. Ca2+ (10 μM) inhibited the activity of the enzyme, and Mg2+ (8.5 μM) activated the dephosphorylation of PMLC. Mn2+ (1.6 μM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.


Key words: dephosphorylation, myosin light chain, Physarum plasmodia, sclerotium formation

Abbreviations: CBB, Coomassie Brilliant Blue R-250, MLC, light chain of Physarum myosin II, PMLC, phosphorylated light chain of Physarum myosin II, p-NPP, p-nitrophenyl phosphate

1To whom correspondence should be addressed (email kaneko@fc.jwu.ac.jp).


1. Introduction

A slime mould plasmodium forms a macroplasmodium when grown on agar plates with sufficient nutrients and a microplasmodium when grown in liquid culture. The plasmodium of the true slime mould Physarum polycephalum, which takes the form of a single multinucleated cell, exhibits active shuttle streaming of the cytoplasm and grows rapidly during dark starvation; it also forms a cyst (sclerotium) as a non-growing dormant body (Guttes and Guttes, 1963). It is known that the highly synchronous transformation of microplasmodia into cysts (spherulation) occurs within 36–48 h (Hütterman, 1973), but there have been no corresponding reports on the transformation of macroplasmodia into cysts (also known as sclerotium formation or sclerotization) except for our previous report (Hattori and Kaneko, 1986).

In our previous papers, we reported on a phosphatase [EC 3.1.3.2] in the cytoplasmic soluble fraction of the macroplasmodium of the true slime mould P. polycephalum (Kaneko and Yamaura, 1982; Kaneko and Kato, 1990; Tanaka et al., 2002). Changes in the relative activity of an acid phosphatase have also been reported by Hütterman et al. (1979) during the spherulation of a microplasmodium, which was induced when a plasmodium was transferred into a nutrient-free salt medium and subjected to starvation. The biological function of this enzyme is as yet unknown, however.

Previously, the specific activity of a phosphatase was assayed using p-NPP (p-nitrophenyl phosphate) as a substrate at pH 5.0 during the synchronous formation of sclerotium from plasmodia during dark starvation, induced by starvation using P. polycephalum Ng-1 (Tanaka et al., 2002).

The nearly 2-fold increase in enzymatic activity that we observed after 1 day of starvation, followed by the decrease to a level below the original level after 3.5 days of dark starvation, revealed the precise profiles of phosphatase activity during plasmodial starvation for the first time. The results of electrophoresis of the preparation of a plasmodial phosphatase under native conditions showed two distinct enzyme activity bands, suggesting that the phosphatase preparation consists of at least two isoforms, E1 and E2. The enzyme E1 is strongly suggested to play a leading role in the sclerotium formation of plasmodia during dark starvation (Tanaka et al., 2002). Recently, we found that isoform E1 exhibited a phosphatase activity of dephosphorylation towards PMLC (phosphorylated light chain of Physarum myosin II). In this paper, we describe the identification, purification and characterization of the phosphatase (E1) that exhibits hydrolytic activity towards PMLC and that is involved in sclerotium formation from P. polycephalum plasmodia.

2. Materials and methods

2.1. Culture of Physarum plasmodia

P. polycephalum Ng-1 plasmodia were used. For successive cultures, plasmodia were incubated at 25°C in the dark on 1.5% water agar plates containing a semidefined medium described by Kuroda et al. (1988) and modified according to a method described by K. Kohama, A. Nakamura and Y. Tanaka (unpublished work).

2.2. Preparation of plasmodia for dark-starvation experiments

The induction of highly synchronous plasmodial sclerotization was performed as described previously (Hattori and Kaneko, 1986; Tanaka et al., 2002). Every 12 h after the initiation of dark starvation of the plasmodial cultures, 33–34 plates of plasmodia were randomly taken out of the incubator, and the number of plates on which plasmodia had formed sclerotia were counted. Sclerotium formation was determined by two previously described methods (Tanaka et al., 2002).

2.3. Crude enzyme preparation

Enzyme extraction was carried out from plasmodia (∼100 mg fresh weight) and sclerotia (∼50 mg fresh weight) as described previously (Kaneko and Kato, 1990; Tanaka et al., 2002). All steps were carried out at 4°C.

2.4. Purification of phosphatase from Physarum plasmodia

The following buffers were used for phosphatase purification: buffer A, 50 mM Tris/HCl (pH 7.2); buffer B, 50 mM Tris/HCl (pH 8.0). Plasmodia (∼40 g fresh weight) were suspended in approximately 120 ml of buffer A containing 35% (w/v) sucrose and 3 mM EGTA and centrifuged at 1500 g for 10 min. The precipitated plasmodia were then divided into 12 parts. Aliquots of the plasmodia were layered on 40 ml of a solution of 20 ml of buffer A containing 8.6% (w/v) sucrose and 3 mM EGTA and 20 ml of buffer B containing 35% (w/v) sucrose and 3 mM EGTA (Kaneko and Yamaura, 1982) to remove the slime fraction from the plasmodia because the slime fraction contained some phosphatase activity. After centrifugation at 1500 g for 10 min, the slime fraction was detected at the interface between 8.6% (w/v) sucrose and 35% (w/v) sucrose, where the plasmodia had precipitated at the bottom of the tube.

After the slime fraction had been removed, the plasmodia were collected and homogenized in a Teflon–glass homogenizer at 1400 rev./min in approximately 50 ml of buffer A for 5 min at 4°C. Enzyme extraction was carried out as described previously (Kaneko and Kato, 1990; Tanaka et al., 2002). All steps were carried out at 0–4°C. After 1 h of stirring, the homogenate of the plasmodia was centrifuged at 10000 g for 1 h; the supernatant obtained through this process is henceforth called the ‘crude extract fraction’. The crude fraction was adjusted to 40% saturation in (NH4)2SO4 and left to stand for 1 h; it was then collected by means of centrifugation at 20000 g for 1 h, left to stand for 1 h and adjusted to 75% saturation in (NH4)2SO4. The precipitate was collected by means of centrifugation at 20000 g for 1 h and dissolved in 7 ml of buffer A. The solution was dialysed against the same buffer; this dialysate is called the ‘40–75% (NH4)2SO4 saturation fraction’. The fraction was applied to a DEAE-cellulose column (18 mm×500 mm, Sigma–Aldrich) equilibrated with buffer B. The column was washed with buffer B and eluted at a flow rate of 20 ml/h in the same buffer with a linear gradient of 0–0.4 M NaCl (total volume = 400 ml). The phosphatase fraction is henceforth called the ‘DEAE-cellulose fraction’. The DEAE-cellulose fraction was then applied to a Butyl-Toyopearl 650M column (10 mm×150 mm, Tosoh Corporation) equilibrated with buffer B containing (NH4)2SO4 at 30% saturation. The column was washed with buffer B and eluted at a flow rate of 2 ml/h in the same buffer with 20 ml of a mixture of a linear gradient of 30–0 M (NH4)2SO4 followed by 20 ml of a linear gradient of 0–30% ethylene glycol (total volume = 40 ml). The phosphatase fraction obtained was dialysed against buffer B and called the ‘Butyl-Toyopearl 650M fraction’. This fraction was then applied to a DEAE-Toyopearl 650S column (10 mm×150 mm, Tosoh Corporation) equilibrated with buffer B. The column was washed with buffer B and eluted at a flow rate of 2 ml/h in the same buffer with 0–0.2 M NaCl (total volume = 40 ml). The phosphatase fraction is henceforth called the ‘DEAE-Toyopearl 650S fraction’.

The fraction was subjected to native PAGE following the method described in Section 2.8 in the ‘Materials and methods section’. When the electrophoretic run was completed, each activity band that corresponded to an enzyme based on Rm was excised from the remaining gel. The enzyme from each activity band was extracted using 100 mM Tris/HCl (pH 7.0), dialysed against 10 mM Tris/HCl (pH 7.0) and concentrated using Millipore Ultrafree CL UFC 4B (Millipore). The enzyme extract from the activity band corresponding to an Rm of 0.6 (E1) is henceforth called ‘the phosphatase’.

2.5. Estimation of native molecular mass of the phosphatase by gel filtration HPLC

The native molecular mass of the phosphatase was estimated by means of gel filtration using a Shodex PROTEIN KW-803 column (Showa Denko) connected to an HPLC system (Lachrom Elite High-Performance Liquid Chromatograph Model L-7000, Hitachi High-Technologies Corporation). The column was equilibrated with 50 mM Tris/HCl (pH 7.0) containing 0.2 M NaCl, then washed with the same buffer and eluted at a flow rate of 1.0 ml/min in the same buffer. The molecular mass was determined using thyroglobulin (670 kDa), gamma-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa) as molecular-mass standards (Bio-Rad Laboratories).

2.6. Assay for phosphatase activity towards p-NPP and PMLC

The phosphatase activity towards p-NPP was assayed using 3.3 mM p-NPP and 67 mM sodium acetate/acetate (pH 5.0) in a final volume of 1.5 ml at 35°C as described previously (Tanaka et al., 2002).

PMLC was prepared using the recombinant MLC (light chain of Physarum myosin II) as described previously (Nakamura et al., 2005). The reaction mixture for the phosphatase assay towards PMLC contained 1.1 μg of enzyme, 0.011 mM PMLC and 17 mM Tris/HCl (pH 7.6) in a final volume of 15 μl at 30°C. A control incubation was performed without the enzyme. The amount of MLC released was determined electrophoretically by means of urea glycerol–PAGE after the enzyme reactions had been terminated by the addition of 15 mg of urea powder according to the method described by Pires et al. (1994) with some previously described modifications (Ozaki et al., 1987). The amount of MLC released was used to determine the amount of Pi (inorganic phosphate) released.

Enzyme activity was measured in katals, with one katal (kat) defined as the amount of activity that converts one mole of substrate per second.

2.7. Substrate specificity of the phosphatase

The substrate specificity of the phosphatase was estimated by incubating the purified enzyme with various substrates at 35°C. Of the enzyme solution (the phosphatase), 0.075 ml was added to 0.175 ml of the reaction mixture containing 3.3 mM of one of the target substrates and 250 mM Tris/HCl (pH 7.6). The amount of phosphorus released during the 20-min incubation period was measured as described previously (Nakamura, 1950).

2.8. Other procedures

Native PAGE was performed in a non-denaturating buffer using 7.5% (w/v) polyacrylamide slab gels at 4°C according to the method described by Davis (1964). Bands representing phosphatase activity were localized using a reaction mixture of Fast Blue B Salt and α-naphthyl phosphate disodium salt as a substrate. The developed gels were stored in 7% acetic acid. SDS/PAGE was carried out in 12% polyacrylamide gels containing 0.1% SDS, as described by Laemmli (1970). The separated protein bands were stained with CBB (Coomassie Brilliant Blue R-250, Sigma–Aldrich) or with silver staining (Merril et al., 1981). The size markers (Amersham Biosciences, Ltd.) were phosphorylase b (97 kDa), BSA (66 kDa), egg ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and α-lactalbumin (14.4 kDa). The protein band was stained with silver staining (Merril et al., 1981).

Densitometric analysis of electrophoretograms of the enzyme preparations were performed according to the method described previously (Tanaka et al., 2002).

Protein concentration was determined by measuring A280 (optical density at 280 nm) with BSA as the standard.

3. Results

3.1. Phosphatase activity towards PMLC during sclerotium formation

Physarum plasmodia, which were bright yellow and migrated rapidly to the oat flakes, were selected for use in the dark starvation experiment for reasons explained previously (Tanaka et al., 2002), namely, because they were expected to undergo synchronous and high-frequency sclerotium formation. In contrast to this expectation, however, no sclerotia were observed 1 day after the start of dark starvation. One and a half days after the start of dark starvation, approximately 97% of plasmodium plates showed dry sclerotia (Figures 1A and 1B).

Based on a previous report, we investigated the phosphatase activity of E1 and E2, the enzymes corresponding to the Rm values of 0.6 and 0.5, respectively, towards the PMLC, according to the method described by Tanaka et al. (2002): briefly, two enzymes were extracted separately from the crude enzyme preparation at pH 7.6 during the synchronous formation of sclerotia from plasmodia during dark starvation. As shown in Figure 1(A), our measurement of each enzyme's activity using p-NPP as a substrate during the first day of dark starvation confirmed that the behaviour of E1 caused an increase in the total enzymatic activity at pH 5.0. As shown in Figure 1(B), the enzymatic activity of E1, as measured at pH 7.6 using PMLC as a substrate, increased up to approximately 4-fold within 0.5 days of the start of dark starvation. Between 0.5 days and 1.5 days after the start of dark starvation, however, it decreased rapidly to zero. As for E2, in contrast, no enzymatic activity was detected during the synchronous formation of sclerotia from plasmodia after the start of dark starvation at pH 7.6 using PMLC as a substrate.

3.2. Purification of phosphatase from Physarum plasmodia

Table 1 shows a summary of the purification parameters. The phosphatase that hydrolyses the phosphoester bond attached to PMLC as a substrate was isolated from P. polycephalum Ng-1 plasmodia that spread to cover the entire agar surface during culture on 1.5% water agar plates containing a semidefined medium. As shown in Table 1, the phosphatase was purified approximately 340-fold through a combination of ion-exchange and hydrophobic chromatography followed first by native PAGE and then by the extraction of the enzyme from the gel, as described in the Materials and methods section. As shown in Table 1, the Butyl-Toyopearl 650M purification step effectively served to remove a significant amount of contaminant proteins from the crude extract. Furthermore, about 80% of contaminant proteins were removed from the Butyl-Toyopearl 650M fraction by the DEAE-Toyopearl 650S purification step (Figure 2A). The DEAE-Toyopearl 650S fraction was assayed for both phosphatase activity towards p-NPP at pH 5.0 and phosphatase activity towards PMLC at pH 7.6.


Table 1 Purification of the phosphatase hydrolysing PMLC from Physarum plasmodia

ND, not detected; Rm value 0.5 corresponds to E2, Rm value of 0.6 corresponds to E1. The enzyme extract from the activity band corresponding to an Rm of 0.6 was what we called ‘the phosphatase’.

Sample Volume (ml) Activity (pkat) Protein (mg) Specific activity (pkat/mg of protein) Purification (-fold) Yield (%)
Crude extract 138 853 718 1.19 1 100
40–75%-satd. (NH4)2SO4 18 765 140 5.46 5 90
DEAE-cellulose 64 608 16.8 36.2 30 71
Butyl-Toyopearl 650M 7.5 540 2.17 249 209 63
DEAE-Toyopearl 650S 4.5 183 0.35 523 439 21
PAGE Rm value 0.5 0.4 ND 0.06 ND ND ND
PAGE Rm value 0.6 0.9 40 0.10 400 336 5



As shown in Figure 2(A), the elution profile of the phosphatase activity towards p-NPP at pH 5.0 and that of the phosphatase activity towards PMLC at pH 7.6 were consistent with each other (Figure 2A). In all column chromatography purification steps, a single chromatographic peak of phosphatase activity towards PMLC was observed (data not shown). Furthermore, native PAGE of the enzyme fraction eluted from DEAE-Toyopearl 650S column chromatography showed two enzymes, E1 and E2, in the activity staining using α-naphthyl phosphate as a substrate (Figure 2B).

3.3. Identification of 50-kDa protein as phosphatase

The enzyme E1 was expected to correspond to the phosphatase exhibiting activity towards PMLC because, of the two enzymes detected in the crude enzyme preparation from plasmodia during dark starvation, only this enzyme exhibited activity towards PMLC, as shown in Figure 1(B). To confirm whether only E1, and not E2, exhibits phosphatase activity towards PMLC, enzyme activity for the two enzymes was measured. As shown in Figure 3, when 1.1 μg of E1 was incubated with 0.011 mM PMLC, the reaction proceeded linearly with time for at least 60 min with no lag phase. In contrast, no activity was detected for E2.

The native molecular mass of the phosphatase exhibiting activity towards PMLC was estimated at approximately 50 kDa through analytical gel filtration on a Shodex Protein KW-803 column (Figure 4A). SDS/PAGE after each purification step is shown in Figure 4(B). The one major band was detected through CBB staining between 45 and 66 kDa molecular markers. Through this six-step protocol, enzyme activity was enriched ∼340-fold compared with the specific activity of the crude extract as described in section 2.4 above and shown in Table 1. Final yield after the six-step purification process was 5% of the crude extract (Table 1). The subunit molecular mass was estimated to be 50 kDa according to SDS/PAGE (Figure 4B). All these results indicate that the native enzyme is a monomer in its native state.

3.4. Substrate specificity

The phosphatase was tested for hydrolytic activity at pH 7.6 against various phosphorylated substrates (Table 2). No hydrolytic activity of the enzyme was detected towards nucleotides such as ATP, GTP, TTP and CTP. AMP and ADP were not hydrolysed by the phosphatase (data not shown). It had no hydrolytic activity towards phosphothreonine and no hydrolytic activity against protein tyrosine PTPS [phosphatase substrate monophosphate: Tyr-Arg-Asp-Ile-Tyr(PO3H2)-Glu-Tyr-Asp-Tyr-Arg-Lys], but high hydrolytic activity towards phosphoserine. Other phosphorylated compounds such as p-NPP, inorganic pyrophosphate, bis (p-nitrophenyl) phosphate and glucose 1-phosphate were not hydrolysed by the phosphatase. Among the phosphorylated compounds tested, in fact, hydrolytic activity was observed only against PMLC. (The concentration of PMLC used was at about the Km value of 0.011 mM.)


Table 2 Substrate specificity of the Physarum phosphatase

ND, not detected.

Substrate Specific activity (nkat/mg of protein)
PMLC 0.41
TTP ND
GTP ND
ATP ND
CTP ND
p-NPP ND
Phosphoserine 12.63
Phosphothreonine ND
PTPS ND
PP(i) ND
BNPP ND
IMP ND
G1P ND



3.5. Kinetic properties

The phosphatase showed a relatively narrow pH-dependent PMLC dephosphorylation activity profile with maximal activity at pH 7.6. The pH values for half maximal activity were pH 7.05 and 8.05 (data not shown).

The effect of temperature on the stability of PMLC dephosphorylation activity was investigated. As shown in Figure 5, above 40°C the activity decreased sharply and was completely lost at approximately 75°C. Activation energy was estimated to be 38 kJ/mol (Figure 5, inset). The Q10 value of the phosphatase was estimated at around 2.3.

3.6. Substrate saturation kinetics

The rate of the enzyme reaction was measured in the presence of PMLC at various concentrations (Figure 6). The double-reciprocal plots of reaction velocity and substrate concentration reveal that the Km of the enzyme for PMLC is 10 μM, and the Vmax is 1.17 nkat/mg of protein (Figure 6, inset).

3.7. Effects of Ca2+, Mg2+, Mn2+, EGTA and NaF on phosphatase activity

These results are summarized in Table 3. The bivalent cations Ca2+ and Mg2+ have opposite effects on the activity of the enzyme towards PMLC. Ca2+ (10 μM) inhibits the activity of the enzyme by approximately 80%, whereas EGTA (0.1 mM) inhibits it by only 14%. Mg2+ (8.5 mM), in contrast, enhances the dephosphorylation of PMLC by approximately 170%. In the presence of Mg2+ (8.5 mM), the inhibitory effect of Ca2+ (10 μM) was restored. Mn2+ (1.6 mM) highly enhanced the phosphatase activity towards PMLC, increasing it by approximately 280%. In the presence of 5 mM NaF, a common phosphatase inhibitor, the enzyme activity was not inhibited, though 50 mM NaF inhibited enzyme activity by approximately 50%.


Table 3 Effects of EGTA, Mn2+, Mg2+, Ca2+ and NaF on the Physarum phosphatase

Addition Concentration (mM) Specific activity (nkat/mg of protein)
Control 0.41
EGTA 0.1 0.35
MnCl2 1.6 1.14
MgCl2 8.5 0.69
CaCl2 0.01 0.07
MgCl2CaCl2 8.50.01 0.42
NaF 5 0.40
NaF 50 0.22



4. Discussion

A highly synchronous induction of plasmodial sclerotization was performed experimentally. In this investigation, we determined that the rapid increase in the phosphatase activity of E1 towards PMLC at pH 7.6 preceded sclerotium formation by Physarum plasmodia. A native PAGE of the DEAE-Toyopearl 650S fraction (Figure 2A) revealed two enzymes, E1 and E2, through enzyme activity staining (Figure 2B); the two bands were thought likely to correspond to the enzymes detected in the crude extract from plasmodia (data not shown) within half a day after the start of dark starvation (Figures 1A and 1B). Other experiments revealed that E1 is the phosphatase that hydrolyses a phosphoester bond attached to PMLC (Figure 3).

The phosphatase was purified from Physarum plasmodia in four steps, specifically through two ion-exchange column chromatographies, each using a different type of carrier, hydrophobic column chromatography and native PAGE. As shown in Table 1, the phosphatase exhibiting activity towards PMLC was purified 340-fold to a final phosphatase specific activity of 400 pkat/mg of protein with a recovery of 5%. Although the DEAE-cellulose fraction was retained after Con A Sepharose column chromatography, the elution profile of the enzyme was too broad to obtain an enzyme preparation for further purification (data not shown). However, the Con A affinity of the enzyme suggests that the enzyme is a glycoprotein.

We were able to determine that the phosphatase we sought was not among one of the following animal smooth muscle phosphatases, because it did not share a native molecular mass and subunit molecular masses with any of them (Onishi et al., 1982; Werth et al., 1982; McClure and Korn, 1983; Yoshida and Yagi, 1988; Alessi et al., 1992; Pato and Kerc, 1990; Tulloch and Pato, 1991; Mitsui et al., 1992; Shirazi et al., 1994). The phosphatase from Physarum plasmodium, in contrast with all of these, is composed of a single subunit with a molecular mass of 50 kDa (Figures 4A and B). Its molecular mass and subunit structure are different from those of the animal smooth muscle phosphatases described above.

In this study, the Physarum plasmodial phosphatase hydrolysing PMLC at pH 7.6 was purified; it has a hydrolytic activity towards p-NPP (Figure 2A) and α-naphthyl phosphate at pH 5.0 (Figure 2B). Although the enzyme was observed to exhibit a high level of activity towards p-NPP at pH 5.0, as shown in Figure 1(A), it exhibited no hydrolytic activity towards p-NPP at pH 7.6 (Table 2). The hydrolysis of p-NPP is not catalysed by the myosin light chain phosphatase from the soluble fraction of chicken gizzard smooth muscle (Onishi et al., 1982), by the phosphatase purified from the Acanthamoeba soluble fraction (McClure and Korn, 1983) or by the Physarum soluble phosphatase under investigation in this study. The phosphatase is thus likely to be characterized by activity that hydrolyses a phosphoester bond attached to PMLC and by a lack of activity towards p-NPP at pH 7.6, an artificial substrate that is useful for assaying the activity of some other phosphatases. The hydrolysis reactions of p-NPP, pyrophosphate and glucose 1-phosphate were not catalysed by the phosphatase, though these phosphorylated compounds serve in other situations as good substrates for determining whether a phosphatase has a broad enzyme specificity.

Several phosphorylated compounds were tested at pH 7.6, and PMLC was found to be the substrate hydrolysed by the enzyme. Of the two phosphoamino acids and one phosphoamino peptide, only phosphoserine was hydrolysed by the Physarum phosphatase. It is likely that the enzyme demonstrates a high preference for phosphoserine because the enzyme hydrolysed a phosphoester bond attached to PMLC, which was prepared as a substrate by phosphorylating 18 kDa of the recombinant phosphorylatable light chain of Physarum myosin II at Ser18 using Physarum kinase as described previously (Nakamura et al., 2005). Based on these results, we suggest that the phosphatase could be classified as a serine/threonine protein phosphatase.

The optimal pH for more than 70% of phosphatases with high catalytic activities towards myosin light chain is 7.5 (Pato and Adelstein, 1983a, 1983b; Pato and Kerc, 1985; Tulloch and Pato, 1991); the optimal pH for the enzyme in this study is comparable with this value.

The activation energy for the phosphatase investigated in this study is consistent with those required for various other enzymatic reactions, most of which fall into the range of 21–63 kJ/mol. The Q10 values of animal smooth muscle myosin phosphatases range from 2.7 to 5.3 (Mitsui et al., 1994). The Q10 value of the phosphatase from Physarum plasmodia was estimated to be about 2.3, a value that is relatively low compared with those of the animal smooth muscle myosin phosphatases (Mitsui et al., 1994).

The Km values of phosphatases with high catalytic activity towards myosin light chain can fall in the range of approximately 1.5 μM to 10 μM (Pato and Adelstein, 1983a, 1983b; Pato and Kerc, 1985; Yoshida and Yagi, 1988; Tulloch and Pato, 1991; Mitsui et al., 1992). The Km of our enzyme was consistent with this Km range. Although the Vmax values of the phosphatases with high catalytic activities towards myosin light chain tend to have a relatively wide range of 33–883 nkat/mg of protein, in this study the Vmax ( = 1.17 nkat /mg of protein) of our enzyme was less than 1/30 the minimum Vmax previously reported for smooth muscle phosphatases (Pato and Adelstein, 1983a, 1983b; Pato and Kerc, 1985; Yoshida and Yagi, 1988; Tulloch and Pato, 1991; Mitsui et al., 1992).

The phosphatase under investigation is not Mg2+-dependent, as the myosin phosphatase MAPPI is (Mitsui et al., 1992), though two phosphatases with high catalytic activities towards the myosin light chains of TG SMP-II and RU SMP-II have been reported to be Mg2+-dependent (Pato and Adelstein, 1983b; Pato and Kerc, 1985). The results obtained in this study, however, show that the bivalent cations Ca2+ and Mg2+ have opposing effects on the activity of the phosphatase towards the myosin light chain and that the inhibitory effect of Ca2+ is cancelled out by the enhancing effect of Mg2+; this is an enzymatic property of the phosphatase. Ca2+ and Mg2+ tend to inhibit the activities of TG SMP-I and RU SMP-I towards the myosin light chain (Pato and Adelstein, 1983a; Pato and Kerc, 1990). A strong stimulatory effect of Mn2+ on the dephosphorylation of PMLC by the enzyme was observed in this study, as well as on the activities of TG SMP-III and RU SMP-II (Pato and Kerc,1990; Tulloch and Pato, 1991). The addition of NaF inhibited the activity of TG SMP-II by 50%, reducing it to 1.0 mM (Pato and Adelstein, 1983b). An inhibitory effect on myosin phosphatases from chicken gizzard smooth muscle, also of approximately 50%, was therefore observed at NaF concentrations less than 8 mM (Yoshida and Yagi, 1988) as well as at 12 mM (MAPPI) (Mitsui et al., 1992), although the inhibitory effect of NaF on the Physarum plasmodium phosphatase was not observed until the NaF concentration reached approximately 50 mM.

Furthermore, the activity of the phosphatase towards PMLC was inhibited by okadaic acid (ID50 was 10 nM) (data not shown). This concentration is more similar to the concentrations of okadaic acid that inhibit chicken smooth myosin phosphatase (IC50: 40 nM) (Alessi et al., 1990) and MAPPI (IC50: 70 nM) (Mitsui et al., 1992). It has no Ca2+ or Mg2+ dependency. These results suggest that the phosphatase gathered from Physarum plasmodia can be classified into the serine/threonine protein phosphatase category of PP1-like phosphatases (Cohen, 1989).

Our findings show that the Physarum plasmodium enzyme is likely to be a novel phosphatase exhibiting hydrolytic activity towards PMLC.

Author contribution

The major contribution to the study was carried out by Chisa Okada under the guidance of Takako Kaneko. Akio Nakamura and Kazuhiro Kohama contributed to the study by advising Chisa Okada how to assay for phosphatase activity towards phosphorylated myosin light chain and providing the substrate and Physarum polycepophalum Ng-1 plasmodia. The contribution to the study of Shigeo Tomioka was advising Chisa Okada how to purify further ‘the DEAE-cellulose fraction’ of the phosphatase to obtain the purified enzyme.

Acknowledgements

We would like to thank Dr Hisae Maki and Miss Satoka Ishii of Japan Women's University for providing technical assistance and help with the preparation of this paper.

Funding

This research was supported by Japan Women' University and Department of Molecular and Cellular Bioscience, Graduate School of Medicine, Gunma University.

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Received 31 October 2009/4 January 2010; accepted 13 April 2010

Published as Cell Biology International Immediate Publication 13 April 2010, doi:10.1042/CBI20090340


© The Author(s) Journal compilation © 2010 Portland Press Limited


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)