Nucleoside monophosphoramidate hydrolase from rat liver: Purification and characterization

Inl. J. Biochem. Vol. 26, No. 2, pp. 235-245, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved

Pergamon

0020-71IX/94 $6.00+ 0.00

NUCLEOSIDE MONOPHOSPHORAMIDATE FROM RAT LIVER: PURIFICATION CHARACTERIZATION

HYDROLASE AND

MASAKO KUBA, TSUYOSHIOKIZAKI, H~TOSHIOHMOR~and AKIRA KUMON* Department of Biochemistry, Saga Medical School, Nabeshima 5-I-1, Saga-shi, Saga-ken 849, Japan (Received 6 July 1993)

Abstract-l. Adenosine S-phosphoramidate hydrolase of 29 kDa was isolated from rat liver cytosol. 2. It consisted of two subunits of 14 kDa. 3. It hydrolyzed nucleoside S-monophosphoramidates into nucleoside S-monophosphates and ammonia, while it did not hydrolyze adenylyl phosphoramidate, adenylyl imidodiphosphate and N-phosphorylated compounds like phosphocreatine, N”-phosphoarginine, 6-phospholysine and 3-phosphohistidine. 4. Divalent cations and cyclic AMP had no effect on the hydrolytic activity.

INTRODUCTION

Compounds having a nitrogen-phosphorus bond are divided into two classes, I and II (Rossomando and Hadjimichael, 1986). Compounds of class I are characterized by the presence of terminal phosphate, and phosphocreatine and NW-phosphoarginine belong to this group. On the other hand, N-adenylyl protein, a compound having terminal AMP is a member of class II compounds. The phosphorus of N-adenylyl protein is linked to protein moiety and adenosine moiety via nitrogen and oxygen, respectively. N-Adenylyl protein occurs in membrane fraction of Dictyostelium discoideum (Rossomando et al., 198 1; Hadjimichael and Rossomando, 1991), in RNA ligase of wheat germ (Pick et al., 1986), and in DNA ligase of Escherichia coli (Gumport and Lehman, 1971). The simplest form of class II compound is adenosine 5’-phosphoramidate (AMPN). It is synthesized in vitro from adenosine 5’-phospho*To whom correspondence should be addressed. Abbreviations: PNP, imidodiphosphate; AMPPNP, adenylyl imidodiphosphate; GMPPNP, guanylyl imidodiphosphate; AMPPN, adenylyl phosphoramidate; AMPN, adenosine 5’-phosphoramidate; GMPN, guanosine Sphosphoramidate; IMPN, inosine 5’-phosphoramidate; TMPN, thymidine 5’-phosphoramidate; CMPN, cytosine 5’-phosphoramidate; UMPN, uridine S-phosphoramidate; AMPHMD, adenylyl hexamethylenediamine; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

sulfate and ammonia by an adenylyl transferase that is distributed in a wide variety of organisms including bacteria, algae, fungi and higher plants (Frankhauser et al., 1981). Concerning enzymatic hydrolysis of class II compounds, one phosphoamidase has been purified from Dictyostelium discoideum with AMP-linked affinity column chromatography (Rossomando and Hadjimichael, 1986). It requires a magnesium ion for the full activity and 0.1 mM cyclic AMP inhibits the activity. However, there is no report of phosphoamidase specific for class II compounds from mammalian tissues. In this manuscript, authors isolated and characterized AMPN hydrolase from rat liver cytosol. MATERIALS AND METHODS

Materials NW-Phosphoarginine, phosphocreatine, methylene-diphosphonic acid, imidodiphosphate (PNP), adenylyl imidodiphosphate (AMPPNP), guanylyl imidodiphosphate (GMPPNP), adenylyl phosphoramidate (AMPPN), and AMPN were purchased from Sigma. Various nucleoside 5’-phosphoramidates, that is, guanosine S-phosphoramidate (GMPN), inosine 5’-phosphoramidate (IMPN), thymidine 5’-phosphoramidate cytosine 5’-phosphoramidate (TMPN), (CMPN) and uridine 5’-phosphoramidate (UMPN), were prepared according to the method of Chambers and Moffatt (1958). 235

236

MASAKO KUBA et al.

Adenylyl hexamethylenediamine (AMPHMD) was prepared according to the method of Rossomando et al. (1987), and a compound that had absorbance at 260 nm and that was stained with ninhydrin reagent was purified with Dowex 1 x 8 (bicarbonate form). Phosphoramidate, 6-phospholysine and 3-phosphohistidine were synthesized according to methods of Sheridan et al. (1971) Zetterqvist and Engstriim (1967) and Hultquist et al. (1966), respectively. Every synthetic compound was free of inorganic phosphate on high voltage paper electrophoresis (Hultquist et al., 1966). Hydrolase assays

A 100 pl-reaction mixture for AMPN hydrolase contained 50 mM Tris-HCl (pH 7.0), 10 mM 2-mercaptoethanol, 2mM AMPN and the enzyme preparation. After the reaction was terminated by boiling the mixture for 15 min, the amount of ammonia released from AMPN was measured by the method of Tabor (1970). When AMPHMD was employed as a substrate, the amount of AMP released was measured by using mini-columns of Dowex 1 x 2 as follows: the reaction was terminated by an addition of 10~1 of 10% SDS, and 90 ~1 of the mixture were applied to 0.3 ml of Dowex 1 x 2 (Clform) equilibrated with 50 mM Tris-HCl (pH 8.5). After washing the resin with 3 ml of 50 mM Tris-HCl (pH 8.5), the column was eluted with 1.2 ml of 50 mM HCl. The absorbance of AMP in the eluate was measured at 260 nm. When class I compounds were used as substrates for AMPN hydrolase, malachite green assay was employed to measure the amount of inorganic phosphate released (Kuba et al., 1992). Ultracentrifugation in a linear gradient of sucrose (5-20%)

According to the conventional method (Martin and Ames, 1961), 400~1 of the enzyme preparation at step 8 were overlaid with or without internal standard proteins including ovalbumin (3.7 s), carbonic anhydrase (3.1 s), myoglobin (1.9 s) and cytochrome c (1.8 s) on a 11 ml-linear gradient of sucrose (5-20%) containing 50 mM Tris-HCl (pH 7.0) and 10 mM 2-mercaptoethanol. The tube was centrifuged for 72 hr at 120,000 g in a SW40 rotor in a Beckman ultracentrifuge. After centrifugation, fractions of 15 drops each were collected from the bottom. 15 ~1 of fractions were subjected to SDS-PAGE (Laemmli, 1970). Protein

concentration was determined by the method of Bradford (1976). High voltage paper electrophoresis and thin layer chromatography

After incubation of various nucleotide analogs or AMPHMD with pure hydrolase (step 9) products were analyzed by high voltage paper electrophoresis (Hultquist et al., 1966) and thin layer chromatography of PEIIcellulose 5725) (Randerath and (Eastman-Kodak, Randerath, 1967). RESULTS

Purtfication of AMPN hydrolase

All operations were carried out at 4°C. Step 1. Extraction. Livers (1 kg) of Wistar rats were homogenized with a Waring blender in 3 1 of buffer A (50mM Tris-HCl, pH 7.5 and 10 mM 2-mercaptoethanol) containing 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 1 mM benzamidine hydrochloride. The homogenate was centrifuged for 40 min at 15,000 g. Step 2. Treatment with DEAE-cellulose. The supernatant was mixed with 500ml of DEAEcellulose equilibrated with buffer A and stirred gently for 30 min. After filtration of DEAEcellulose suspension in a funnel, the resin remaining on a sheet of filter paper was washed with another 1.5 1 of buffer A. All filtrates were combined together. Step 3. Fractionation with ammonium sulfate.

The above flow-through fraction was saturated with solid ammonium sulfate to 40%, and was centrifuged for 30 min at 15,OOOg. The supernatant was saturated with further addition of ammonium sulfate to 60% and proteins were precipitated by centrifugation. Precipitate was suspended in 150 ml of buffer A and dialyzed overnight against buffer A containing 0.15 M NaCl. Step 4. 1st Sephadex G - 75 column chromatog raphy. After insoluble materials were removed

by centrifugation, dialyzate was applied to a Sephadex G-75 column (5 x 95 cm) equilibrated with buffer A containing 0.15 M NaCl, and the gel was eluted with the same buffer. Fractions of 17 ml each were collected. Fractions from 48 to 80 had the hydrolytic activity for AMPN. After proteins of these fractions were collected by precipitation with 80% ammonium sulfate, the precipitate was dissolved in 150 ml of buffer A. Dialysis was carried out against buffer A containing 0.15 M NaCl.

Nucleoside phosphoramidate

hydrolase

231

Fraction Number, 10 ml/tube 0.8

20

0.6

15

0.4

10

0.2

5

0.0 20

30 40 50 60 Fraction Number, 5 ml/tube

70’

0.0 0

20 40 60 Fraction Number, 6 ml/tube

Fig. 1. Elution profiles of AMPN hydrolase on various column chromatographies during purification. (Panel a) DEAE-cellulose column chromatography (step 6). Dialyzed fraction of step 5 was applied to a DEAEcellulose column (2.7 x 95 cm) equilibrated with buffer A. The column was eluted with only buffer A and fractions of 10 ml each were collected. AMPN hydrolase assay was carried out for 30 min at 30°C by employing 2 ~1 of the eluate. Fractions of the latter peak were combined and precipitated with 80% ammonium sulfate. The precipitate was collected by centrifugation and dialyzed against buffer A containing 0.15 M NaCl. (Panel b) The 3rd Sephadex G-75 column chromatography (step 7). Dialyzed fraction of step 6 was applied to a Sephadex G-75 column (2.7 x 95 cm) equilibrated with buffer A. Fractions of 5 ml each were collected. Hydrolytic activity for AMPN was assayed by a 10 min-incubation of 2 ~1 of the fraction. Carbonic anhydrase (29 kDa) was eluted at fraction 54. (Panel c) Hydroxylapatite column chromatography (step 8). Fractions from 50 to 57 of the 3rd Sephadex G-75 column chromatography was applied to a hydroxylapatite column (1.6 x 5 cm) equilibrated with 50 mM Tris-HCl (pH 7.0) and 10 mM 2-mercaptoethanol. After washing the column, elution was performed with a 450 ml-linear gradient of potassium phosphate buffer (PH 7.0) from 0 to 60 mM. Fractions of 6 ml each were collected. AMPN hydrolytic activity was assayed by 2 ~1 of each fraction. Open circles and closed circles in each panel indicate the absorbance at 280 nm and the activity for hydrolysis of AMPN, respectively.

Step 5. 2nd Sephadex G- 75 column chromatography. The dialyzate was applied again to a

Sephadex G-75 column (5 x 90 cm) equilibrated with buffer A containing 0.15 M NaCl. Fractions of 17 ml each were collected. Fractions from 45

to 60

had the hydrolytic activity for AMPN and were combined together. Proteins of combined fractions were precipitated with 80% ammonium sulfate, were dissolved in 20 ml of buffer A and were dialyzed against buffer A.

MASAKO KUBA et al.

238

kDa

2

1

3

4

5

6

7

8

-

68

-

58

-

20

-

12

9

Step Fig. 2. SDS-PAGE of AMPN hydrolase preparation at each purification step. 1Opg of enzyme preparation at each purification step were subjected to SDSlS% PAGE. After electrophoresis, the gel was stained with Commassie brilliant blue. Standard proteins employed were phosphorylase b (100 kDa), bovine serum albumin (BSA) (68 kDa), catalase (58 kDa), ovalbumin (43 kDa), glyceraldehyde 3-phosphate dehydrogenase (36 kDa), soybean trypsin inhibitor (20 kDa) and cytochrome c (12 kDa).

Table 1. Summary

of purification

Total protein Steps 1. 2. 3. 4. 5. 6. 7. 8. 9.

Extraction 1st DEAEcellulose Ammonium sulfate 1st Sephadex G-75 2nd Sephadex G-75 2nd DEAEcellulose 3rd Sephadex G-75 Hydroxylapatite Phenyl Sepharose

(mg) 168,300 86,020 32,460 6525 2442 80 29 12 9

of AMPN

Total activity (pmol/min) 715 753 617 604 503 180 84 44 33

hydrolase

from rat liver

Specific activity (nmol/min/mg) 4 9 19 93 210 2250 2900 3670 3670

(%)

Purity (fold)

100 105 86 84 70 25 12 6 5

1 2 5 33 49 536 690 874 874

Yield

Nucleoside phosphoramidate

hydrolase

239

Fraction number, 15 drops/tube

kDa

100

68 58

20

12 1

3

5

7

9

10

11

12

13

14

15

16

17

18

19

20

Fraction number Fig. 3. Ultracentrifugation of rat liver AMPN hydrolase in a linear gradient of sucrose. 400 _ulof AMPN hydrolase preparation at step 8 was overlaid with and without internal standard proteins including ovalbumin (3.7 s), carbonic anhydrase (3.1 s), myoglobin (1.9s) and cytochrome c (1.8 s) on the 11 ml-sucrose gradient (5-20%) and centrifuged for 72 hr at 120,OOOgat 4°C with a rotor of SW40 Ti in a Beckman ultracentrifuge. After centrifugation, fractions of 15 drops each were collected from the bottom. Fractions of 10 pl and 1.5~1 were served for AMPN hydrolase assay (Panel a) and SDS-IS% PAGE (Panel b), respectively. Open circles and closed circles in Panet a indicate the protein con~ntration and the activity for AMPN hydrolase, respectively. The gel on SDS-PAGE was stained with Commassie brilliant blue (Panel b). The elution positions of internal standard proteins were fractions 12, 14.5, 17 and 17.5 for ovalbumin, carbonic anhydrase, myoglobin and cytochrome c, respectively (data not shown).

MASAKO KUA

240

PH Fig. 4. The effect of pH on AMPN hydrolase. Reaction mixtures containing 2 mM AMPN, 10 mM 2-mercaptoethanol, 0.1 mg/ml BSA and 0.3 pg of the pure enzyme (step 9) in 50 mM acetate buffer (pH 4.0-5.0) 50 mM T&buffer (PH 5.5-9.0) or 50mM glycine buffer (pH 9.5-11.0) were incubated for 30 min.

Step 6. DEAE-cellulose column chromatography. The dialyzate was applied to a

DEAE-

cellulose column (2.7 x 90 cm) equili-

Table 2. Substrate specificity of nucleoside 5’-phosphoramidate hydrolase from rat liver Phosphorylated compounds AMPN GMPN IMPN TMPN CMPN UMPN AMPHMD

V mm ( pmol/min/mg)

0.70 0.76 0.68 0.90 0.28 0.50 0.24

15 12 17 1.7 5.3 5.9 5.9

A 100 pl-reaction mixture consisted of 50 mM Tris-HCl (pH 7.0). 10mM 2-mercaptoethanol, 0.1 mg/ml BSA, 1 pg of purified enzyme (step 9), and 2 mM phosphorylated compound. Compounds assayed for the release of inorganic phosphate were ATP, ADP, AMP, cyclic AMP, glucose 6-phosphate, phosphothreonine, phosphoserine, phosphotyrosine, p-nitrophenyl phosphate, inorganic pyrophosphate, methylenediphosphonic acid, phosphocreatine, N”-phosphoarginine, 6-phospholysine, 3-phosphohistidine, AMPPNP, GMPPNP, phosphoramidate and PNP. Compounds assayed for the release of ammonia were AMPN, GMPN, IMPN, TMPN, CMPN, UMPN and AMPPN. A compound assayed for release of AMP was AMPHMD. The analysis of reaction products from AMPN, AMPPN, AMPPNP, GMPPNP and AMPHMD was also carried out by high voltage paper electrophomsis and thin layer chromatography as shown in Figs 5 and 6. These analyses all revealed that only the compounds listed above were hydrolyzed. V,, and K,,,of these compounds were determined by measuring reaction velocity under various concentrations of substrate and by the analysis of double reciprocal plot.

et al.

brated with buffer A. The column was eluted with buffer A and fractions of 10ml each collected. Hydrolytic activity for were AMPN appeared in two peaks, fractions 29-35 and 82-90 (Fig. la). The activity of the latter peak was 6-fold larger than the activity of the former peak, and only the latter enzyme was purified further in this manuscript. Step 7.3rd Sephadex G- 75column chromatography. Proteins in the latter peak of Fig. la were precipitated with 80% ammonium sulfate and dissolved in a minimum volume of buffer A. The dissolved sample was applied to the 3rd Sephadex G-75 column (2.5 x 95 cm) equilibrated with buffer A containing 0.15 M NaCl. Fractions of 5 ml each were collected. The peak of AMPN hydrolase was eluted in the fraction 54, where one standard protein, carbonic anhydrase (29 kDa) also had the elution peak (Fig. lb). Step 8. Hydroxylapatite column chromatography. Fractions containing hydrolytic activity for AMPN on step 7 were applied to a hydroxylapatite column (1.6 x 5 cm) equilibrated with buffer A. After washing the column with 30 ml of buffer A, the column chromatography was performed with a 450ml-linear gradient of potassium phosphate buffer (pH 7.0) from 0 to 60mM. Fractions of 6 ml each were collected. Hydrolytic activity for AMPN was eluted in fractions 10 to 13 (Fig. lc) and these fractions were combined. Step 9. Phenyl-Sepharose column chroma tography. Combined fractions were applied to a phenyl-Sepharose column (1.6 x 30 cm) equilibrated with buffer A and the column was eluted with a 600 ml-gradient of ethyleneglycol from 0 to 60% in 50mM Tris-HCl (pH 7.0) and 10 mM 2-mercaptoethanol. Fractions of AMPN hydrolase eluted at 45% ethyleneglycol contained a single peptide of 14 kDa on SDS-PAGE (step 9 in Fig. 2). The eluate was dialyzed against 50% glycerol containing 50 mM Tris-HCl, pH 7.0 and 10 mM 2-mercaptoethanol, and was stored at -20°C. Above results were summarized in Table 1. Finally, the enzyme was purified 870-fold and the yield was 5%. 1 mg of the final step preparation could hydrolyze 3.7 pmol of AMPN/min at 30°C. The SDS-15% PAGE of each step revealed that 14 kDa protein band increased, according to the progression of purification (Fig. 2).

Nucleoside

phosphoramidate

hydrolase

241

AMPPN AMPN

AMP

ADP

AMPPNP GMPPNP

ATP

Origin

1

2

3

4

5

6

I

8

9

Fig. 5. Analysis of reaction mixtures containing AMPN, AMPPN, AMPPNP and GMPPNP by thin layer chromatography. A 25 +reaction mixture containing 5 mM nucleotide analog was incubated for 30 min in the presence or absence of 2 pg of the pure enzyme (step 9). An aliquot (5 ~1) of the reaction mixture was applied to a thin layer sheet of PEI-cellulose F (Eastman-Kodak 5725). The plate was chromatographed with 1 M LiCl. The migration positions of nucleotide analogs were detected at 254 nm. Lane 1, standard nucleotides (ATP, ADP and AMP); lane 2, AMPN/enzyme (-); lane 3, AMPN/enzyme (+); lane 4, AMPPN/enzyme (-); lane 5, AMPPN/enzyme (+); lane 6, AMPPNP/enzyme (-); lane 7, AMPPNP/enzyme (+); lane 8, GMPPNP/enzyme (-); lane 9, GMPPNP/enzyme (+).

Ultracentrifugation of AMPN linear sucrose gradient (5-20%)

hydrolase in a

Upon ultracentrifugation, the peak of AMPN hydrolase migrated together with carbonic anhydrase (29 kDa, 3.1 s) to fractions 14 and 15 (Fig. 3a) and the result of SDS-PAGE showed that these fractions contained a 14 kDa-protein (Fig. 3b). These results indicated that the enzyme of 29 kDa consisted of two identical subunits of 14 kDa. Optimal pH

Hydrolytic activity for AMPN was assayed in the pH range from 4 to 11. Optimal activity was observed at pH 7.0 (Fig. 4).

Substrate spec$city

Various compounds containing an oxygenphosphorus bond were not hydrolyzed (Table 2). AMPPN (Fig. 5) and N-phosphorylated compounds of class I like 6-phospholysine, N”phosphoarginine, PNP and AMPPNP (Table 2 and Fig. 5) were not hydrolyzed either, while class II compounds like nucleoside 5’-monophosphoramidates (Table 2 and Fig. 5) and AMPHMD (Table 2 and Fig. 6) were hydrolzyed. Purine nucleoside 5’-phosphoramidates like IMPN, AMPN and GMPN seems to be better substrates than pyrimidine nucleotide derivatives like UMPN, CMPN and TMPN. On the other hand, the extent of AMPHMD

MASAKO KUBA et al.

242

Front

AMPHMD HMD

AMP

AMPHMD 0

-0

Fig. 6. Analysis of reaction mixtures containing AMPHMD by high voltage paper electrophoresis (A) and thin layer chromatography (B). A 25 PI-reaction mixture containing 5 mM AMPHMD was incubated for 30 min in the absence (lanes 1 in A and B) or presence (lanes 2 in A and B) of 2 pg of the pure enzyme at step 9. 5~1 of the reaction mixture were subjected to high voltage paper electrophoresis (A) and to PEIWhin layer chromatography (B). The paper electrophoreiogram was stained with ninhydrin reagent (A) and the nucleotide spot of the thin layer sheet was visualized at 254nm (B). Lanes I in A and B, AMPHMD/enzyme (-); lanes 2 in A and B, AMPHMD/enzyme (+).

hydrolysis was 40% of that of AMPN (Table 2).

hydrolysis

Eflects of various chemicals on AMPN

hydrolase

The hydrolytic activity for AMPN was stimulated by thiol compounds (Fig. 7). Dithiothreitol was the most effective among four thiol compounds employed and the extent of the activation was the range from 180 to 250%. SDS at 0.1% inhibited 85% of the activity, while other detergents including

Triton x 100, Lubrol, Tween 20, Nonidet P 40, digitonin and sodium cholate had no effect on the activity. Among various nucleotides, GTP had the inhibitory effect on the enzyme and its concentration necessary for 50% inhibition was lO,uM, while 2 mM cyclic AMP had no inhibitory effect on the activity (data not shown). MgCl,, MnCl,, CoCl,, CuCl,, ZnC1, and EDTA at 2 mM concentration had no effect on the activity. Various phosphatase inhibitors,

Nucleoside phosphoramidate

01 . 0

1

8

2

8

I

2

4

6

6

10

hydrolase

243

0

50

0

1

30

40

100 D3SAl, vg

150

200

I ml

[SH Compound], mM

Fig. 7. Effects of thiol compounds on AMPN hydrolase. Reaction mixture containing 50mM Tri-HCI (pH 7.0) 10 mM 2-mercaptoethanol, 0.1 mg/ml BSA, 2 mM AMPN, purified enzyme (0.3 pg) and various concentrations of 2-mercaptoethanol (O), glutathione (a), cysteine (A), or dithiothreitol (A) was incubated for 30min.

4

5

&lPNl, 3mM

2 mM sodium vanadate, 2 mM sodium molybdate, 20 ~1M okadaic acid, 5 mM sodium fluoride and 5 mM sodium tartrate, had no effect on the activity (data not shown). Stimulation

by various proteins

Hydrolytic activity for AMPN of the pure enzyme was 4-fold higher in the presence of BSA than in the absence of BSA (Fig. 8a). Concentration of BSA for maximal stimulation was 0.1 mg/ml. This stimulation was caused by V,,, increase as shown in Fig. 8b. Similar stimulation was observed by other proteins like S-100 protein, calmodulin, myelin basic protein, histones, and cytochrome c. When the enzyme was incubated for 5 min at various temperatures in the absence of BSA, one-half activity was lost at 48°C. However, loss of one-half activity occurred at 70°C in the presence of BSA (Fig. 8~).

DISCUSSION

Nucleoside 5’-phosphoramidate hydrolase was purified from rat liver. It did not hydrolyze class I compounds just like phosphoamidase from Dictyostelium discoideum (Rossomando and Hadjimichael, 1986). However, the present enzyme was not stimulated by 2 mM MgClr and was active even in the presence of 2 mM EDTA, while the phosphoamidase specific for class II

50

60

70

00

Temperature on preincubation, “C

Fig. 8. Effects of BSA on AMPN hydrolase. (Panel a) Various concentrations of BSA from 0 to 0.2 mg/ml were added to the reaction mixture containing 2 mM AMPN and purified enzyme (0.3 yg), and the incubation was performed for 30 min. (Panel b) Reaction mixtures containing 0.3 pegof the pure enzyme and AMPN of various concentrations from 0.1 to 5 mM were incubated for 30 min in the presence (0) and absence (0) of 0.1 mg/ml BSA. (Panel c) Pure enzyme (0.3pg) in 50mM Tris-HC1 (pH 7.0), and IOmM 2mercaptoethanol was preincubated for 5min at indicated temperatures in the presence (a) and absence (0) of 0.1 mg/rnl BSA. The hydrolytic activity of these preincubated enzyme was determined by an addition of 2 mM AMPN.

compounds from slime mold needs Mg*+ for full activity. Moreover, the present enzyme was not inhibited by 2 mM cyclic AMP, contrary to the inhibition of slime mold phosphoamidase by 0.1 mM cyclic AMP (Rossomando and Hadjimichael, 1986). Class I compounds are known to be hydrolyzed by phosphoamidase (EC 3.9.1.1) that also cleaves oxygen-phosphorus bonds of phosphoserine (Singer and Fruton, 1957), inorganic

MASAKOKUBA er al.

244

pyrophosphate (Sundarajan and Sarma, 1959) and glucose 6-phosphate (Parvin and Smith, 1969). Recently, two hydrolases specific for N”-phosphoarginine and 6-phospholysine have been detected from rat liver (Kuba et al., 1992; Yokoyama et al., 1993) and rat brain (Ohmori et al., 1993), respectively. When the present enzyme is compared with these phosphoamidases for class I compounds, the former was insensitive to various phosphatase inhibitors. On the other hand, N”-phosphoarginine hydrolase and 6-phospholysine hydrolase are sensitive to sodium vanadate. However, there is no report about the occurrence of AMPN or N6(adenylyl) lysine residues of proteins in mammalian tissues. Consequently, the physiological significance of the present enzyme remains to be resolved.

Chambers R. W. and Moffatt H. G. (1958) The synthesis of adenosine-5’ and uridine-5’ phosphoramidates. J. Am. Chem. Sot. 80, 3752-3756.

Frankhauser H., Berkowitz G. A. and Schiff J. A. (1981) A nucleotide with the properties of adenosine 5’-phosphoarmidate from Chlorella cells. Biochem. biophys. Res. commun. 101, 524-532.

Gumport R. I. and Lehman I. R. (1971) Structure of the DNA ligase-adenylate intermediates: lysine (s-amino)linked adenosine-monophosphoramidate. Proc. natn Acad. Sci. U.S.A. 68, 2559-2563.

Hadjimichael J. and Rossomando E. F. (1991) Isolation and characterization of the protein phosphoamidates formed by a membrane bound adenylyl transferase reaction in Dictyostelium discoideum. Int. J. Biochem. 23, 535-539.

Hultquist D. E., Moyer R. W. and Boyer P. D. (1966) The preparation and characterization of I-phosphohistidine and 3-phosphohistidine. Biochemistry 5, 322-33 I. Kuba M., Ohmori H. and Kumon A. (1992) Characterization of NW-phosphoarginine hydrolase from rat liver. Eur. J. Biochem. 208, 747-752.

Laemmli U. K. (1970) Cleavage of structural proteins during the assay of the head of bacteriophage T4. Nature 227, 680-685.

SUMMARY

Adenosine S-phosphoramidate hydrolase was purified from rat liver cytosol to apparent homogeneity on SDS-PAGE, employing column chromatographies of Sephadex G-75, hydroxylapatite and phenyl Sepharose. The holoenzyme of 29 kDa was composed of two subunits of 14 kDa. It hydrolyzed nucleoside S-monophosphoramidates into nucleoside 5’-phosphates and ammonia, or adenylyl hexamethylenediamine into AMP and hexamethylenediamine, while it did not hydrolyze adenylyl phosphoramidate, adenylyl imidodiphosphate and N-phosphorylated compounds like phosphocreatine, N”-phosphoarginine, 6-phospholysine and 3-phosphohistidine. It was distinguished from slime mold phosphoamidase, based on different effects of cyclic AMP and MgCl, on both enzymes.

Acknowledgements-This

work was supported in part by Grants-in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. We are indebted to Miss Tomoko Inoue for her secretarial assistance.

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Martin G. R. and Ames B. N. (1961) A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J. bioi. Chem. 236, 137221379. Ohmori H., Kuba M. and Kumon A. (1993) Two phosphatases for 6-phospholysine and 3-phosphohistidine from rat brain. J. biol. Chem. 268, 762557627. Parvin R. and Smith R. A. (1969) Phosphoamidates V. Probable identity of rat microsomal glucose 6-phosphatase, phosphoamidase, and phosphoamidate-hexose phosphotransferase. Biochemistry 8, 1748-l 755. Pick L., Furneaux H. and Hurwitz J. (1986) Purification of wheat germ ligase. II. Mechanism of action of wheat germ RNA ligase. J. biol. Chem. 261, 6694-6704. Randerath K. and Randerath E. (1967) Thin-layer separation methods for nucleic acid derivatives. Meth. Enzym. ltA, 323-347.

Rossomando E. F., Crean E. V. and Kestler D. P. (1981) Isolation and characterization of an adenylyl-protein complex formed during the incubation of membranes from Dictyostelium discoideum with ATP. Biochim. biophys. Acla 678, 386-39 1. Rossomando E. F. and Hadjimichael J. (1986) Characterization and CAMP inhibition of a lysyl- (N-s-5’-phospho) adenosyl phosphoamidase in Dictyostelium discoideum. Int. J. Biochem. 18, 481-484.

Rossomando E. F., Hadjimichael J., Varnum-Finney B. and Sol1 D. R. (1987) HLAMP- a conjugate of hippuryllysine and AMP which contains a phosphoamide bondstimulates chemotaxis in Dictyosfelium discoideum. Differentiation 35, 88-93.

Sheridan R. C., McCullough J. F. and Wakefield Z. T. (1971) Phosphoamidic acid and its salt. Inorg. Syntheses 13, 23-26. Singer M. F. and Fruton J. S. (1957) Some properties of beef spleen phosphoamidase. J. biol. Chem. 229, Ill119. Sundarajan T. A. and Sarma P. S. (1959) Substrate specificity of phosphoprotein phosphatase from spleen. Biochem. J. 71, 537-544.

Tabor C. W. (1970) The determination of NH, with the use of glutamic dehydrogenase. Meth. Enzym. 17A, ^__ 955.

Nucleoside phosphoramidate Yokoyama K., Ohmori H. and Kumon A. (1993) Isolation of N”-phosphoarginine hydrolase from rat liver and its physical properties. J. Biochem. 113, 236-240.

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Zetterqvist 0 and Engstriim L. (1967) Isolation of N-E-[~*P] phosphoryllysine from rat liver cell sap after incubation with [‘*PI adenosine triphosphate. Biochim. biophys. Acta 141, 523-532.