Eur. J. Biochem. 208,747-752 (1992) 0FEBS 1992

Characterization of N"-phosphoarginine hydrolase from rat liver Masako KUBA, Hitoshi OHMORI and Akira KUMON Department of Biochemistry, Saga Medical School, Japan (Received June 9, 1992) - EJB 92 0801

N"-Phosphoarginine hydrolase from rat liver hydrolyzed N"-phosphoarginine into arginine and inorganic phosphate, whereas it did not release inorganic phosphate from 19 other phosphorylated compounds containing a N-P bond, an 0 - P bond or a C-P bond. In addition, it was not able to transfer the phosphoryl moiety from NO-phosphoarginine to ADP. These results indicated that this enzyme was distinct from both phosphoamidase and arginine kinase. Its properties were as follows: thiol compounds were essential for its activity; it was stimulated by 1.5-2-fold in the presence of 0.001 YoLubrol, Tween 20, poly(oxyethy1ene) 9-lauryl ether and Nonidet P-40, while 0.004% sodium lauryl sulfate inhibited the activity completely; concentrations of sodium molybdate and sodium vanadate necessary for 50% inhibition were 7 pM and 12 pM, respectively; some proteins stimulated the activity, while lysophosphatidic acid, lysophosphatidylinositol, and phosphatidic acid suppressed the activity even in the presence of poly(oxyethy1ene) 9-lauryl ether.

Nu-Phosphoarginine was first isolated from fresh-water crab muscle [l]. It is synthesized in vivo by arginine kinase and serves as the immediate buffer of myoplasmic ATP [2]. Arginine kinase is distributed in protostomians, while it is replaced with creatine kinase in chordates. Consequently, the natural occurrence of N"-phosphoarginine and the enzymes participating in its metabolism are not expected to occur in mammalian tissues. Contrary to our expectations, one phosphoamide hydrolase that released arginine and inorganic phosphate from N"phosphoarginine was detected in rat liver cytosol. In this study, some properties of this N"-phosphoarginine hydrolase (Nu-phosphoarginine phosphatase; PAPase) are described and compared with those of various phosphatases; it did not hydrolyze any compound other than Nu-phosphoarginine. Since phosphoamidase hydrolyzes the N-P bond of phosphocreatine [3, 41 and the 0 - P bond of phosphotyrosine [3], inorganic pyrophosphate [4] and glucose 6-phosphate [5], this PAPase is a novel phosphoamide hydrolase different from the phosphoamidase reported previously.

synthesized according to methods of Sheridan et al. [6], Zetterqvist and Engstrom [7]and Hultquist et al. [8], respectively. Each synthetic compound was free of inorganic phosphate when analysed by high-voltage paper electrophoresis

PI. Detergents and lipids

Chaps was a product of Dojindo Laboratories. Poly(oxyethylene) 9-lauryl ether (polidocanol), Tween 20, Lubrol and Nonidet P-40 were purchased from Sigma Chemical Company. Okadaic acid, digitonin, sodium lauryl sulfate, Triton X-100 and sodium cholate were obtained from Wako Pure Chemical Industry. Phosphatidylcholine from egg yolk, phosphatidylethanolamine from bovine brain, phosphatidylinositol from pig liver, phosphatidic acid from egg lecithin, lysophosphatidylcholine from egg, lysophosphatidylethanolamine and lysophosphatidylinositol from pig liver and lysophosphatidic acid from egg lecithin were products of Serdary Research Laboratories Inc. The various phospholipids were sonicated just prior to use.

MATERIALS AND METHODS

Additional chemicals

Materials

Tetramisole, polylysine, herring protamine, calf thymus histones 11s and IIIS, bovine serum albumin, bovine brain S100 protein and bovine brain calmodulin were products of Sigma Chemical Company. Other chemicals were of analytical grade.

Phosphorylated compounds

N"-phosphoarginine, phosphocreatine, methylenediphosphonic acid, imidodiphosphate and adenylyl imidodiphosphate were purchased from Sigma Chemical Company. Phosphoramidate, 6-phospholysine and 3-phosphohistidine were Correspondence to A. Kumon, Department of Biochemistry, Saga Medical School, Nabeshima 5-1-1, Saga-shi, Saga, Japan 849 Abbreviations. PAPase, NO-phosphoarginine phosphatase; polidocanol, poly(oxyethy1ene) 9-lauryl ether. Enzymes. Phosphoamidase (EC 3.9.1.1); arginine kinase (EC 2.7.3.3).

Preparation of PAPase from rat liver Rat liver (150 g) was homogenized in a Waring blender with 450 ml buffer A (50 mM Tris/HCl, pH 7.5, 10 mM 2mercaptoethanol), containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 1 mM benzamidine hydrochloride. The homogenate was centrifuged at 15000 x g for

748 40 min and the supernatant was centrifuged again at I00000 x g for 60 min. The latter supernatant was fractionated with ammonium sulfate in the range 35-60%, and the precipitate was dissolved in a minimal volume of buffer A. The dissolved solution was applied to a Sepharose 4B column ( 5 cm x 95 cm) equilibrated with buffer A and the column was eluted with the same buffer. Fractions exhibiting PAPase activity were combined and precipitated by 80% ammonium sulfate. The precipitate was dissolved in 25 ml buffer A and dialyzed against buffer A. The dialysate was applied to a DEAE cellulose column (2.7 cm x 40 cm) equilibrated previously with buffer A and the column was washed with the same buffer. Fractions of 10 ml were collected during washing of the column. PAPase was eluted in the retarded portions (fractions 19- 26) of flow-through fractions. These fractions were combined and dialyzed against buffer B (50 mM Mes, pH 6.5, 10 mM 2-mercaptoethanol). The dialysate was applied to a CM cellulose column (1.6 cm x 30 cm) equilibrated with buffer B and the column was chromatographed with a 600-ml linear 0 - 0.2-M NaCl gradient. PAPase was eluted at 0.10 M NaC1. Fractions of PAPase activity were combined and dialyzed against buffer A. PAPase was purified 900-fold compared to the supernatant of crude homogenate, and 1 pg purified PAPase released 0.1 nmol inorganic phosphate every min from N"-phosphoarginine under standard assay conditions.

4

6

8

10

12

PH

Fig. 1. Effect of pH on PAPase activity. Assays were carried out in reaction mixtures containing 3.2 pg PAPase, and 50 mM acetate/ NaOH, pH 4.0-5.5 (A), 50 mM Tris/HCl, pH 5.5-9.5 ( O ) , or 50 mM glycine/NaOH, pH 9.5 - 11 (0).

However, the spot of N"-phosphoarginine was detectable with Rosenberg's reagent, when the second reagent was sprayed after heating the plate previously treated with the first HC1 solution.

PAPase assay

Determination of protein amounts

A standard reaction mixture (100 pl) consisted of 50 mM Tris/HCl, pH 7.0, 1 mM N"-phosphoarginine, 10 mM 2mercaptoethanol, 0.01YO polidocanol and purified PAPase (3-10 pg). The mixture was incubated at 30°C for 3060 min and the inorganic phosphate released was assayed by successive additions of 1.O ml malachite green reagent and 50 pl 34% citric acid [9, 101. Citric acid solution was added to the reaction mixture 10 s after the addition of malachite green reagent. The absorbance of blue color, due to an inorganic phosphate/molybdate complex, was measured at 630 nm.

Protein concentration was determined by the method of Bradford [131, employing the Bio-Rad prepared reagent and bovine y-globulin standard.

Thin-layer chromatography and electrophoresis 5 mM N"-phosphoarginine or 5 mM arginine was incubated for 60 rnin in the absence or presence of 5 mM ADP or 5 mM ATP in a 100-pl reaction mixture containing 50 mM Tris/HCl, pH 7.0, 0.01YOpolidocanol, 43 pg PAPase, 10 mM MgClz and 10 mM 2-mercaptoethanol. The reaction was terminated by the addition of 1 p110 mM sodium vanadate, and an aliquot ( 5 pl) of the reaction mixture was applied to a sheet of PEI-cellulose F (Kodak-5725) or two thin layer plates of cellulose (Kodak-13265). The PEI-cellulose plate was chromatographed with 1 M LiCl and nucleotide spots were visualized with radiation at 254 nm [ll]. The two thin-layer sheets of cellulose were electrophoresed with 0.86 M pyridine/ acetate, pH 7.5. After drying, these were stained with ninhydrin reagent or Rosenberg's reagent for detection of arginine and N"-phosphoarginine, or detection of inorganic phosphate [12], respectively. When spots of 6-phospholysine and 3-phosphohistidine were developed with Rosenberg's reagent, these compounds were hydrolyzed by the first solution of HCl and the spots were stained by the second Rosenberg's reagent. However, NO-phosphoarginine was more stable to the first spray solution of HC1 than 6-phospholysine and 3phosphohistidine, and the spot of N"-phosphoarginine was not stained at room temperature by Rosenberg's solution.

RESULTS Compounds and pH required for full activity of PAPase

ThioI compounds PAPase activity was lost by dialysis of the enzyme preparation against 50 mM Tris/HCl, pH 7.0, and was restored by the addition of a thiol compound to the reaction mixture. Concentrations for half-maximal activity were 1 mM for both glutathione and dithiothreitol and 5 mM for cysteine and 2mercaptoethanol. Detergents

0.001% Polidocanol, Lubrol, Tween 20, Triton X-100 and Nonidet P-40 stimulated the PAPase activity by 1.5 -2-fold, and activation of the enzyme to the same extent was observed with additions of 0.004% Chaps and 0.004% digitonin. 0.004% sodium lauryl sulfate, an anionic detergent, inhibited PAPase completely. However, another anionic detergent, sodium cholate did not inhibit PAPase, even at 0.004%, and PAPase was stimulated twofold with 0.1 % sodium cholate.

PH The optimal pH was observed in the range 6.5 - 7.0, and pH below 5.5 and above 9.5 were not appropriate for the activity (Fig. 1). Substrate specificities Release of inorganic phosphate was measured under the standard assay conditions, using 20 phosphorylated com-

749

+ - P-His J P-Lys 7P-Arg

. Arg

-0 -0

- P-Arg

- Arg

+

AMP.

-Pi

- P-His -P-Lys

ADP ATP 0-

-0

1

2

3

4

5

6

Fig. 2. Product analysis after the incubation of N-phosphorylated amino acids with PAPase. Reaction mixture (100 pl) containing 5 mM Tris/ HCI, pH 8.0, 2 mM N-phosphorylated amino acid and 0.01% polidocanol was incubated for 40 min at 30°C in the absence (lanes 1 , 3 and 5) and presence (lanes 2 , 4 and 6) of 5 pg PAPase. Substrates: lanes 1 and 2, 6-phospholysine; lanes 3 and 4, 3-phosphohistidine; lanes 5 and 6, N"-phosphoarginine. The reaction was terminated by the addition of 1 p1 10 mM sodium vanadate. An aliquot (5 pl) of various reaction mixtures was applied to a filter paper (Toyo filter paper, No. 51C, 20 cm x 20 cm) and high-voltage paper electrophoresis was carried out at 15°C for 30 min, according to the method of Hultquvist et al. [8]. (A) Ninhydrin staining; (B) Rosenberg's staining. 0, P-Lys, P-His, P-Arg, Arg and Pi denote the positions of origin, 6-phospholysine, 3-phosphohistidine, N"-phosphoarginine, arginine and inorganic phosphate, respectively.

pounds containing a N-P bond, an 0 - P bond or a C-P bond. The present enzyme released inorganic phosphate from NOphosphoarginine and the apparent K, for N"-phosphoarginine was 100 pM. Another hydrolysis product from NOphosphoarginine was arginine as shown in lanes 5 and 6 of Fig. 2. Other N-phosphorylated amino acids, 6-phospholysine and 3-phosphohistidine, were not cleaved by the same enzyme (Fig. 2). N-phosphorylated compounds like phosphocreatine, phosphoramidate, imidodiphosphate and adenylyl imidodiphosphate were not hydrolyzed, either. Moreover, O-phosphorylated compounds (ATP, ADP, AMP, IDP, phosphoserine, phosphothreonine, phosphotyrosine, 4-nitrophenyl phosphate, glucose 6-phosphate and inorganic pyrophosphate), a C-phosphorylated compound (methylenediphosphonic acid) and phosphoproteins (phosvitin and casein) were not substrates for the PAPase enzyme. One possibility of phosphotransfer from N"-phosphoarginine to ADP was examined in Fig. 3. Whereas the enzyme hydrolyzed Nu-phosphoarginine to arginine and inorganic phosphate (Fig. 3), PAPase of the same activity in the mixture containing 5 mM ADP and N"-phosphoarginine was not able to synthesize ATP (Fig. 3A). Similarily, PAPase did not produce N"-phosphoarginine from 5 mM ATP and 5 mM arginine (Fig. 3B). These results indicate that rat liver PAPase was different from arginine kinase [2] or phosphoramidate/ adenosine diphosphate phosphotransferase [14].

-0

- Pi

1

2

3

4

5

6

7

Fig. 3. A possibility of phosphotransfer from N"-phosphoarginine to ADP, or from ATP to arginine. 5 mM N"-phosphoarginine (P-Arg) or arginine was incubated for 60min in the absence or presence of 5 mM ADP or ATP in 100 p1 50 mM Tris/HCl, pH 7.0, 0.01% polidocanol, 43 pg PAPase, 10 mM MgClz and 10 mM 2-mercaptoethanol. The reaction was terminated by 1 pl 10 mM sodium vanadate, and an aliquot (5 pl) of the reaction mixture was subjected to thin-layer chromatography or electrophoresis. (A) After the sample was applied to a sheet of PEI-cellulose F (Eastman-Kodak 5725), the plate was chromatographed with 1 M LiCI. The migration position of nucleotides was detected with an ultraviolet lamp. (0)The position where each sample was spotted. Lane 1, standards (AMP, ADP, ATP); lane 2, P-Arg/ADP; lane 3, P-Arg/ADP/enzyme; lane 4, Arg/ ATP; lane 5 , Arg/ATP/enzyme. (B), (C) A cellulose sheet (Kodak 13265) spotted with 5 p1 each of various reaction mixtures was electrophoresed for 20 min at 50 V/cm in 0.86 M pyridine/acetate, pH 7.5. After drying it, the sheet was sprayed with ninhydrin reagent (B) or Rosenberg's reagents (C). (0)Position where each sample was applied. Lane 1, P-Arg; lane 2, P-Arg/enzyme; lane 3, P-Arg/ADP; lane 4, P-Arg/ADP/enzyme; lane 5, Arg/ATP; lane 6, Arg/ATP/enzyme; lane 7, standards (Arg, P-Arg and Pi).

Effects of various metals and compounds on PAPase Cation chlorides

Concentrations of NaC1, KCl and LiCl for 50% inhibition were 0.87, 1.00 and 0.70 M, respectively. 1 mM divalent cations, like Mg2+,Ca2+,Mn2+ and Zn2+ had no effect on the activity, while some multivalent cations, e.g. 0.5 mM vanadium and 0.5 mM molybdenum, repressed 40% and 35% of the control activity, respectively. Phosphatase inhibitors

5 mM EDTA, tetramisole, sodium tartrate, sodium sulfate and sodium fluoride, and 1 mM okadaic acid had no inhibi-

750 None Polylysine Cytochrome c Protamine Histone 111 Histone II Ca-Calmodulin 5-100 BSA

0

0

10

20 30 Concentration, pM

40

50

Fig.4. Dose effects of sodium molybdate and sodium vanadate on PAPase. Various concentrations of sodium molybdate and sodium vanadate were added to the assay mixture containing 2.8 pg PAPase. Incubation was carried out for 45 min. Sodium molybdate (0); sodium vanadate (13).

2

4 6 8 10 Pi Released, nmoV30 min

12

Fig. 6. Effects of proteins on PAPase activity in the presence and absence of polidocanol. PAPase activities were assayed in reaction mixtures containing 3.5 pg PAPase and 0.1 mg/ml proteins in the presence (closed) and absence (open) of 0.01% polidocanol. When calcium was used, the final concentration was 1 mM.

polidocanol, while these stimulatory effects were abolished by addition of polidocanol, suggesting that these stimulatory proteins and polidocanol interacted with the same site on the enzyme molecule. Concentrations of albumin, histone IIS and S-100 protein for half-maximal activation in the absence of polidocanl were 10 - 20 pg/ml.

DISCUSSION

0

10

20 30 40 [Phospholipid], pg/ml

50

Fig. 5. Dose effects of some phospholipids on PAPase. PAPase activities were assayed in reaction mixtures containing 1.4 pg PAPase, 0.01 % polidocanol and various concentrations of lipids, that is, lysophosphatidic acid (0),phosphatidic acid ( O ) , lysophosphatidylinositol ( A ) and phosphatidylinositol (A).

tory effect. However, 1 mM sodium vanadate and 1 mM sodium molybdate inhibited PAPase completely, with concentrations for 50% inhibition of 12 pM and 8 pM for vanadate and molybdate, respectively (Fig. 4). Phospholipids

While 50 pg/ml phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine and lysophosphatidylethanolamine had no effect on PAPase activity, some acidic phospholipids inhibited PAPase activity. Concentrations of phosphatidic acid, lysophosphatidic acid, and lysophosphatidyl-inositol for 50% inhibition were 12, 25 and 30 pg/ml, respectively. However, 50 pg/ml phosphatidylinositol had no effect on PAPase activity (Fig. 5). Proteins

Fig. 6 shows effects of various proteins on PAPase activity. Several proteins stimulated PAPase activity in the absence of

PAPase, presented in this study, released inorganic phosphate from one N-phosphorylated compound and is categorized as a hydrolase acting on phosphorus-nitrogen bonds. Phosphoamidase is the only hydrolase belonging to this class, and only a few studies have been reported using purified enzyme preparations [3 - 51. Phosphomonoesterase, which releases inorganic phosphate from 0-phosphorylated compounds, has been extensively studied and characterized with specific stimulators and activators. Accordingly, it seemed interesting to compare the properties of the present PAPase with those of phosphatases for 0-phosphorylated compounds. PAPase has a narrow substrate spectrum, hydrolyzing only N"-phosphoarginine among 20 phosphorylated compounds tested. Other N-phosphorylated compounds including phosphocreatine, 6-phospholysine, 3-phosphohistidine, imidodiphosphate, adenylyl imidodiphosphate and phosphoramidate were not used as substrates. Among these N phosphorylated compounds, phosphocreatine has a structure very similar to NO-phosphoarginine. That is, their phosphorylation sites are a guanidino moiety that is methylated in phosphocreatine, and phosphocreatine was not hydrolyzed by PAPase. Therefore, PAPase seemed to require an N-phosphorylated guanidino structure without methyl residues. Its strict specifity for N-phosphorylated substrate was a distinctive property, as phosphoamidases [3 - 51 and conventional phosphomonoesterases show relatively broad substrate specificities for phosphorylated compounds. A second feature of PAPase was its stimulation by detergents. As the stimulatory effect of some proteins disappeared in the presence of polidocanol (Fig. 6), these proteins and polidocanol seemed to have a common site of interaction

751 in the PAPase molecule. There are reports that non-ionic detergents also stimulate phosphoprotein phosphatase as follows: protein-tyrosine-phosphatase of the particulate fraction from rat spleen is stimulated by 4-%fold by Triton X-100, Nonidet P-40, Lubrol and Tween 80, but the enzyme present in the soluble fraction is not stimulated by Triton X-100 [15]. These findings indicate that the detergent solubilizes proteintyrosine-phosphatase from the membrane structure of the particle enhancing the ease of interaction with the substrate. The action of the detergent on protein-tyrosine-phosphatase is an indirect effect on the enzyme molecule, contrary to the direct effect of detergents on PAPase from the cytosolic fraction. The third feature of PAPase was that its activity was not affected by addition of EDTA and was relatively stable to many metal ions. Alkaline phosphatase is known to be a metalloprotein requiring Zn2+ or Mg2+ for its full activity [16, 171. Moreover, two protein-serine/threonine-phosphatases PP2B and PP2C, have absolute requirements for C a 2 + / calmodulin and Mg2+,respectively [18]. Recently, a proteintyrosine-phosphatase dependent on Mg2 and inactivated by micromolar concentrations of C a 2 + , has been partially purified from bovine brain cytosol [19]. Furthermore, it has been reported that 100 1 M Znz+ inhibits the activity of many protein-tyrosine-phosphatases [15, 19, 201. The fourth feature of PAPase was that it was inhibited in the presence of micromolar concentrations of sodium vanadate and sodium molybdate (Fig. 4). These salts are also known to have a potent inhibitory effect on acid phosphatase of 18 kDa [21] and protein-tyrosine-phosphatases [15, 20231. While sodium fluoride and okadaic acid are known to inhibit some protein-serine/threonine-phosphatases [18, 241 and sodium tartrate is an inhibitor of lysosomal acid phosphatase [25], all these salts had no effect on PAPase. Finally, contrary to the natural occurrence of phosphocreatine, there is no free N"-phosphoarginine in mammalian tissues and the physiological significance of PAPase remains to be elucidated. However, N"-phosphoarginine is known to be present in the form of NO-phosphoarginine residues of some proteins, such as myelin basic protein [26] and a capsidic protein of granulosis virus [27, 281. Moreover, two protein kinases specific for phosphorylation of arginine residue have been reported in rat liver nuclei [29,30]. Therefore, one possibility is that the present PAPase functions as a protein-arginine-phosphatase. This idea might be supported by the following evidence; a phosphomonoesterase, alkaline phosphatase has been known to react with phosphoserine and phosphotyrosine residues of phosphoproteins [31, 321. Furthermore, another phosphomonoesterase, acid phosphatase from various tissues exhibits the activity of protein-tyrosine-phosphatase [33 - 351. If the same situation is applicable to PAPase, the present enzyme might have protein-arginine-phosphatase activity as well as phosphoarginine-hydrolase activity. This hypothesis has been the motivation for us to study PAPase in rat liver. +

This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education. Science and Culture of Japan. We want to express our gratitude to Miss Tomoko Inoue for her excellent secretarial assistance.

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