THE J O U R N A L OF BIOLOGICAL CHEMISTRY Vol. 260, No. 8, Issue of April 25, pp. 4653-4660,1985 Printed in U.S.A.

Phosphotyrosyl-specific Protein Phosphatase Activity of a Bovine Skeletal Acid Phosphatase Isoenzyme COMPARISON WITH THE PHOSPHOTYROSYL PROTEIN PHOSPHATASE ACTIVITY OF SKELETAL ALKALINE PHOSPHATASE*

(Received for publication, October 15, 1984)

K.-H. William Lau, John R. Farley, and David J. Baylink From the Departments of Biochemistry and Medicine, Loma Linda University, and Mineral Metabolism Unit, Jerry L. Pettis Veterans Administration Hospital, Loma Linda, California 92357

A partially purified bovine cortical bone acid phos- phatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phos- photyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phos- phoseryl histones. The pH optimum was about 5.5 with p-nitrophenyl phosphate as substrate but was about 6.0 with phosphotyrosine and about 7.0 with phospho- tyrosyl histones. The apparent K,,, values for phospho- tyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, The p-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl pro- tein phosphatase activities appear to be a single protein since (a) these activities could not be separated by Sephacryl 5-200, CM-Sepharose, or cellulose phos- phate chromatographies, (b) the ratio of these activities remained relatively constant throughout the purifica- tion procedure, (c) each of these activities exhibited similar thermal stabilities and similar sensitivities to various effectors, and (d) phosphotyrosine andp-nitro- phenyl phosphate appeared to be alternative substrates for the acid phosphatase.

Skeletal alkaline phosphatase was also capable of dephosphorylating phosphotryrosyl histones at pH 7.0, but the activity of that enzyme was about 20 times greater at pH 9.0 than at pH 7.0. Furthermore, the affinity of skeletal alkaline phosphatase for phospho- tyrosyl proteins was low (estimated to be 0.2-0.4 mM), and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephospho- rylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphoty- rosyl protein phosphatase under physiologically rele- vant conditions.

Phosphorylation of protein tyrosyl residues has recently been suggested to be associated with cell proliferation and differentiation and with cell transformation (1-5). It has also been suggested that tyrosyl protein phosphorylation might be

* This work was supported by National Institutes of Health Grants AM31062 and AM32256 and a research grant from Loma Linda University, and received research support from the Veterans’ Admin- istration. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

an early event in the cellular response to growth factors and that the signal for cell proliferation might be initiated with the tyrosine phosphorylation and terminated with phospho- tyrosyl protein dephosphorylation (6). In order to regulate such a proliferative signal, it is necessary that a phosphoty- rosyl protein phosphatase can be activated, and the tyrosine kinases are inhibited when stimulated growth is no longer required. Since bone cells show a proliferative response to a variety of growth factors, it is reasonable to suspect that both tyrosine kinase and phosphotyrosyl protein phosphatase ac- tivities should be significant in these cells.

Skeletal acid phosphatase is one of the most abundant enzymes in bone. High levels of acid phosphatase activity can be found in both osteoclasts (7) and osteoblasts (8). Although this enzyme has been studied extensively since it was discov- ered many decades ago, its biochemical function is still un- defined. There are at least two forms of acid phosphatase in bone (9). Studies of Anderson and Toverud (10, 11) and of Wergedal (12, 13) have identified one isoenzyme, which is sensitive to tartrate inhibition, as the tartrate-sensitive skel- etal acid phosphatase. A second isoenzyme, which is sensitive to inhibitions by fluoride, molybdate, and diphosphonate, but not tartrate, is known as tartrate-resistant skeletal acid phos- phatase.

We have isolated a tartrate-resistant skeletal acid phospha- tase from bovine cortical bone and found that this partially purified acid phosphatase was strongly associated with a phosphotyrosyl protein phosphatase activity at neutral pHs. Because previous studies have suggested that alkaline phos- phatase also has phosphotyrosyl protein phosphatase activity, we have compared the activities of skeletal acid phosphatase and skeletal alkaline phosphatase toward phosphotyrosyl pro- teins under physiologically relevant conditions.

EXPERIMENTAL PROCEDURES

Materials Bovine bones were obtained from a local slaughterhouse. A-431

human epidermoid carcinoma cells were obtained from Dr. Stanley Cohen of Vanderbilt University and maintained in our laboratory. [3*P]Pi and [‘261]NaI were obtained from ICN Biochemicals, Inc. [y- 32P]ATP was synthesized from [32P]P, according to Walseth and Johnson (14). pNPP,’ DL-Tyr(P), L-Tyr(P), histones (Type IIA), ATP, bovine serum albumin, Folin-Ciocateu phenol reagent, CAMP- dependent protein kinase, DEAE-cellulose (DE52) CM-Sepharose CL-GB, and cellulose phosphate were obtained from Sigma. Sephacryl

The abbreviations used are: pNPP, p-nitrophenyl phosphate; Tyr(P), phosphotyrosine; Hepes, 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid; Tris, tris(hydroxymethy1)aminomethane; Mes, 2-(n-morpholino)ethanesulfonic acid; SDS, sodium dodecyl sulfate.

4653

4654 Skeletal Acid and Alkaline Phosphatases S-200 and phenylalanine-agarose were from Pharmacia. Bio-Gel A- 1.5, agarose-linked Cibacron blue, gel filtration molecular weight marker proteins, and materials for polyacrylamide gel electrophoresis were purchased from Bio-Rad. Dulbecco's modified Eagle's medium and fetal calf serum were obtained from Gibco Laboratory. Murine epidermal growth factor was purchased from Collaborative Research Inc. Dichloromethylene diphosphonate was a product of Procter and Gamble Company. Other reagents were of reagent grades and were obtained from Sigma.

Methods

Enzyme Assays 1) Acid Phosphatase Assay-Skeletal acid phosphatase activity

was assayed in a reaction mixture (0.2 ml) containing 10 mM pNPP, 100 mM sodium acetate, pH 5.5, a t 37 "C. Assays were initiated with addition of enzyme and terminated by addition of 1 ml of 0.2 M NaOH. Appropriate controls in which NaOH was added prior to addition of enzyme were included with each experiment to correct the absorbance due to the color of the bone extracts. Nonenzymatic hydrolysis of pNPP was corrected by including control assays without added enzyme. The amount of product, p-nitrophenol, produced was calculated from the increase of absorbance at 410 nm using the molar extinction coefficient of 17,800 M" cm", which was determined with p-nitrophenol standards. One unit of enzyme is defined as the amount of enzyme that is required to hydrolyze 1 pmol of pNPP/min at 37 "C. Tartrate-resistant acid phosphatase was determined in the presence of 20 mM tartrate, adjusted to pH 5.5, and the amount of tartarte- sensitive activity was calculated from the difference between the total and the tartrate-resistant activities.

2) Phosphoamino Acid Phosphatase Assay-Tyr(P) phosphatase activity was assayed by the production of tyrosine from Tyr(P) in a reaction mixture containing 10 mM DL-Tyr(P) or L-Tyr(P) in 100 mM sodium acetate, pH 5.5. When the reaction pH was pH 7.0, 20 mM Hepes buffer was used instead of sodium acetate. Tyrosine concentration was determined according to the method of Lowry et al. (15). When other phosphoamino acids (i.e. phosphoserine or phosphothreonine) were used, the rate of dephosphorylation was determined by the rate of production of Pi. Pi concentration was measured with a colorimetric assay (16). One unit of enzyme activity defines as the amount of enzyme required to hydrolyze 1 pmol of phosphoamino acid/min at 37 "C.

3) Phosphotyrosyl Protein Phosphatase Assay-The phosphoty- rosy1 protein phosphatase activity was monitored by the release of [32P]P; from [3ZP]phosphotyrosyl histones or from [32P]phosphotyro- syl IgG. [32P]Phosphotyrosyl proteins were prepared according to the procedure described by Swarup et al. (17), in which the histones (Sigma Type IIA) or IgG were phosphorylated with an epidermal growth factor-stimulated protein kinase isolated from A-431 cells in the presence of [y-32P]ATP. The A-431 cells were cultured in Dul- becco's modified Eagle's medium in the presence of 10% fetal calf serum. The A-431 cell membrane fraction containing epidermal growth factor-stimulated tyrosine kinase activity was prepared as previously described (18). The phosphotyrosyl protein dephosphoryl- ation reaction was carried out under the following conditions. Reac- tion mixtures contained 5-10 p~ phosphoproteins (concentrations calculated from the amounts of [32P]Pi incorporation into proteins (about 20,000-30,000 cpm) based on the known specific activity of [y31P]ATP) in 20 mM Hepes, pH 7.0, 100 mM NaCl, and 1 mM dithlothreitol in a final volume of 0.2 ml. Reactions were initiated with the addition of the enzyme and terminated with addition of 0.1 ml of 80 mM phosphosilicotungstic acid to precipitate phosphotyrosyl proteins. After centrifugation (500 X g for 20 min) the supernatant was spotted and air dried on a Whatman ET-31 filter paper (2 X 2 cm), and the radioactivity was determined in a Beckman model liquid scintillation counter. Over 95% of the radioactivity released in the dephosphorylation reaction could be extracted with ammonium mo- lybdate-butyl acetate (19), confirming that the radioactivity released represented [3zP]Pi rather than 32P-containing phosphosilicotungstic acid-soluble peptides and eliminating the possibility that radioactivity was released by proteolysis. One unit of enzyme activity was defined as the amount of enzyme needed to release 1 nmol of ["'PlPi from the [3ZP]phosphotyrosyl protein/min at 37 'C. The same reaction condi- tions were used when phosphoseryl histones were evaluated as sub- strates. [32P]Phosphoseryl histones were prepared according to the procedure described by Swarup et al. (17), in which histones were phosphorylated with CAMP-dependent protein kinase.

4) Alkaline Phosphatase Assay-Alkaline phosphatase activity was measured according to Farley and Jorch (20). The enzyme activity was measured at room temperature in a reaction mixture consisting of 30 mM pNPP, 150 mM carbonate buffer, pH 10.3, and 1 mM MgC12. The reaction was initiated by addition of enzyme and monitored by the change in absorbance at 410 nm during a 15- to 60-min incubation. Controls, without skeletal alkaline phosphatase, were included with each assay to correct for the nonspecific hydrolysis of pNPP. The amount of product formed was calculated with an extinction coeffi- cient of 17,800 M" cm". One unit of skeletal alkaline phosphatase is defined as the amount of enzyme that is required to hydrolyze 1 pmol of pNPP/min at room temperature. Fifty mM Tris/carbonate and Hepes buffers were used to evaluate skeletal alkaline phosphatase activity at pH less than 10.3.

Purification of Skeletal Acid Phosphatase

The purification scheme for tartrate-resistant acid phosphatase from bovine cortical bones was adopted and modified from the pro- cedure described by Anderson and Toverud (10) for rat skeletal acid phosphatase isoenzymes. Briefly, the bovine long bones were sawed into small cubes, and the marrow was removed. After extensive washing with distilled water, the bone pieces were ground to powder in a Wiley mill and stored at -20 "C until use.

About 70 g of the bovine bone powders were homogenized in 5 volumes of 0.1% Triton X-100,0.3 M KC1 with the Polytron homog- enizer. The resulting extract was frozen and thawed, and precipitates were removed by centrifugation (15,000 X g for 20 min). Protamine sulfate was then added to a final concentration of 0.05% (v/v). Precipitates were again removed by centrifugation (15,000 X g for 20 min), and the supernatant was dialyzed overnight at 4 "C against 5 mM sodium acetate, pH 7.0. After this dialysis, the bone extract was adjusted to pH 4.8 with glacial acetic acid. Precipitates were removed, and the supernatant was loaded on a CM-Sepharose column (20 X 2 cm, inner diameter), which has been pre-equilibrated with 10 mM sodium acetate, pH 4.8. The column was washed with 100 ml of the same buffer, and the tartrate-sensitive acid phosphatase was eluted with a 300-ml gradient from 10 mM sodium acetate, pH 4.8, to 200 mM sodium acetate, pH 6.5. Only small amounts of tartrate-sensitive skeletal acid phosphatase were obtained by this procedure (i.e. less than 10% of total acid phosphatase applied). Tartrate-resistant skel- etal acid phosphatase activity was eluted with a second 200-ml gradient from 200 mM to 1 M sodium acetate, pH 6.5. The fractions corresponding to the tartrate-resistant acid phosphatase were pooled and concentrated in an Amicon ultrafiltration concentrating system with a YM-10 membrane (M, cutoff of approximately 10,000). En- zyme activity from this stage of purification was used for most of the experiments described in this study. Tartrate-resistant skeletal acid phosphatase was further purified on a cellulose phosphate column (20 X 1 cm, inner diameter), which was pre-equilibrated with 100 mM sodium acetate, pH 6.5. The enzyme activity was eluted with a 200- ml gradient from 0 to 2 M NaCl in the same equilibrating buffer. Fractions corresponding to tartrate-resistant acid phosphatase were pooled and concentrated with the Amicon as above. The concentrated tartrate-resistant skeletal acid phosphatase was then further purified on a Sephacryl S-200 gel filtration column (100 X 2.5 cm, inner diameter) with an eluting buffer of 100 mM sodium acetate, pH 5.5, 0.1% Triton X-100, and 0.2 M KCl. The gel filtration column was calibrated with thyroglobulin ( Vo), immunoglobulin G, ovalbumin, cytochrome c and vitamin B12 (V,). The size of bovine tartrate- resistant skeletal acid phosphatase estimated by this method was approximately M, 40,000. Native polyacrylamide gel electrophoresis revealed that the preparation of bovine tartrate-resistant acid phos- phatase showed one band of enzyme activity which could be classified as a band 5 acid phosphatase isoenzyme based on the nomenclature suggested by Lam et al. (21). Fractions from Sephacryl S-200 column containing tartrate-resistant acid phosphatase activity were pooled and concentrated to a final volume of about 3 ml. The purified enzyme was stored at 4 "C and was stable for at least 4 months.

Partial Purification of Skeletal Alkaline Phosphatase from Human Bone

Human skeletal alkaline phosphatase was partially purified ac- cording to the procedure previously described by Farley and Jorch (20). Briefly, human femoral heads were obtained at hip replacement surgery, frozen, crushed, rinsed (to remove contaminating marrow and serum), and extracted with 20% butanol (v/v) in 25 mM carbonate

Skeletal Acid and Alkaline Phosphatases (pH 8.3) for 24 h at 4 "C. Skeletal alkaline phosphatase activity was obtained in the aqueous phase, and after dialysis against the carbon- ate buffer, the activity was purified by ammonium sulfate precipita- tion, DEAE-cellulose column chromatography, and filtration through a column of agarose-linked Cibacron blue. This material was further purified by hydrophobic chromatography on a column of phenylala- nine-agarose and by preparative polyacrylamide gel electrophoresis on a 4-20% gradient gel. The resulting preparation of human skeletal alkaline phosphatase migrated as a single band on polyacrylamide gel electrophoresis after labeling with [1251]NaI.

Partial Purification of Skeletal Alkaline Phosphatase from Chick Bone

Chick skeletal alkaline phosphatase was partially purified from embryonic chick calvaria as previously described (20). Briefly, cal- varia were rinsed and extracted with 20% butanol (v/v) in 50 mM Tris/carbonate buffer (pH 6.5) for 24 h at 4 "C. The extract was dialyzed against the same buffer, and the insoluble material was removed by centrifugation (15,000 X g for 20 min). Chick skeletal alkaline phosphatase activity was then partially purified by DEAE- cellulose column chromatography and molecular sieve chromatogra- phy on a column of Bio-Gel A-1.5.

Other Analytic Methods Protein concentration was determined according to Lowry et al.

(15) using bovine serum albumin to construct the standard curves. Native polyacrylamide gel electrophoresis for skeletal acid phospha- tase was performed as described by Lam e t al. (21). SDS-polyacryl- amide gel electrophoresis was performed according to Laemmli (22). The kinetic constants (i.e. apparent K,, apparent K;) were determined by the Lineweaver-Burk double reciprocal plots and replots.

RESULTS

Tyr(P) and Phosphotyrosyl Protein Phosphatase.Actiuities in Tartrate-resistant Skeletal Acid Phosphatase ' Prepara- tions-We have partially purified a tartrate-resistant acid phosphatase from bovine cortical bone using pNPP as sub- strate according to a procedure modified from one described for the purification of rat skeletal acid phosphatase (10). Our acid phosphatase preparation migrated as a single band of activity on acidic polyacrylamide gel electrophoresis (Fig. 1) and can be classified as a band 5 acid phosphatase isoenzyme according to the nomenclature suggested by Lam et al. (21). The bovine tartrate-resistant skeletal acid phosphatase showed activity toward nucleotide triphosphates but not to- ward nucleotide monophosphates or @-glycerol phosphate (Table I). We have also found that our preparations of par- tially purified tartrate-resistant skeletal acid phosphatase ex- hibited phosphatase acitivities toward Tyr(P) and phospho- tyrosyl proteins (Table I and Fig. 2). Both of these phospha- tase activities were time dependent (Fig. 2, A and C) and concentration dependent (Fig. 2, B and D).

The phosphoamino acid phosphatase activity in the tar- trate-resistant skeletal acid phosphatase preparations ap- peared to be relatively specific for Tyr(P). The enzyme was 7-10-fold more active toward Tyr(P) than either phospho- serine or phosphothreonine under our assay conditions (Table I). The protein phosphatase activity in our acid phosphatase preparation also appeared to be relatively specific for phos- photyrosyl phosphoproteins. Under our assay conditions, [32P]phosphotyrosyl histones were dephosphorylated, but no significant [32P]phosphoseryl histones dephosphorylation was detected (Table I). The tartrate-resistant acid phosphatase enzyme preparation also dephosphorylated [32P]phosphoty- rosy1 IgG.

Fukami and Lipmann (23) have recently reported that a Tyr(P) phosphatase activity purified from larvae had very strict substrate stereospecificity for L-Tyr(P) and did not show activity on D-Tyr(P). We have examined the substrate stereospecificity of the Tyr(P) phosphatase activity in our

sACP-

Acidic PAGE A B

5'-

I j

!

!i

4655

SDS PAGE

320k*F - C D

158k+ Q

46 k+(3

1 7 k - a

FIG. 1. Acidic and SDS-polyacrylamide gel electrophoreses of the partially purified bovine tartrate-resistant skeletal acid phosphatase. Bovine tartrate-resistant skeletal acid phosphatase was purified as described under "Methods." The specific activity of the enzyme was about 500 milliunits/mg. Columns A and B are the acidic native gel electrophoresis of this enzyme. Thirty milliunits (or 60 pg of protein) was applied to each tube gel. Column A is stained for enzyme activity according to Lam et al. (21), and column B is stained for protein with Coomassie Brilliant Blue. Columns C and D are 10% SDS gels stained with silver stain technique. Column C is molecular weight standards, and column D is the SDS gel for 20 pg of partially purified tartrate-resistant skeletal acid phosphatase (SAW). PAGE, polyacrylamide gel electrophoresis.

TABLE I Relative activities of the partially purified tartrate-resistant skeletal

acid phosphatase on phosphoamino acids, phosphohistones, and nucleotide phosphates

The enzyme used in these experiments was purified up to the CM- Sepharose chromatography.

Substrate Enzyme Relative activity activity

Reaction pH 5.5 10 mM pNPP 10 mM phosphotyrosine 10 mM phosphoserine 10 mM phosphothreonine

10 mM GTP 25 mM ATP

25 mM ADP 25 mM AMP 10 mM GMP 5 mM cGMP 25 mM @-glycerol phosphate

Reaction pH 7.0 10 mM pNPP 5 PM phosphotyrosyl histones

rniUiunits/ml

57.6 100.0 20.0 34.7 3.7 6.4 3.6 5.6

24.9 43.2 23.6 41.0 17.3 30.0 1.0 1.7 4.3 7.5

<0.1 <0.2 3.1 5.4

<0.1" <0.2 4.2* 100

5 ;M phosphoserylhistones <0.7" <17 a The activitv on DNPP and DhosDhoservl histones at DH 7.0 was

" . . below the detection limits of our assays.

reaction mixture for 30 min at 37 "C. This activity represented about 3000 cpm released from a 0.2-ml

bovine tartrate-resistant skeletal acid phosphatase prepara- tions. As indicated in Fig. 3A, this activity dephosphorylated both DL-Tyr(P) and L-Tyr(P) equally well, with apparent K , values of approximately 0.6 mM for each at pH 5.5. Assuming that DL-Tyr(P) was not predominantly L-Tyr(P), this obser- vation implies that our Tyr(P) phosphatase activity was not strictly stereospecific. We have also determined the affinity of the phosphotyrosyl protein phosphatase activity for phos-

4656 Skeletal Acid and Alkaline Phosphatases

Time (mid

0 10 20 30 40

PNPP Phosphatase (mU/ml)

FIG. 2. Time and concentration dependence of the Tyr(P) phosphatase and phosphotyrosyl protein phosphatase activi- ties in the tartrate-resistant skeletal acid phosphatase prep- aration. The tartrate-resistant skeletal acid phosphatase used in this experiment was purified up to the CM-Sepharose chromatography step with a specific activity of 113 milliunits/mg protein (assayed with pNPP). Panels A and C are the time courses of the Tyr(P) phosphatase (A) and phosphotyrosyl IgG phosphatase (C) activities. The concentration of the enzyme used in these studies was 28 milli- units/ml and 23.3 milliunits/ml for the time study of Tyr(P) phos- phatase and phosphotyrosyl IgG phosphatase, respectively. Panels B and D show the concentration dependence of the Tyr(P) phosphatase ( B ) and the phosphotyrosyl IgG phosphatase (D) activities. Concen- tration of [32P]phosphotyrosyl IgG in the phosphotyrosyl protein phosphatase assay was 300 nM.

photyrosyl histones. The apparent K, value for the phospho- tyrosyl protein phosphatase activity was approximately 300 nM protein phosphate groups at pH 7.0 (Fig. 3B).

The optimum pH for each dephosphorylation reaction was also determined (Fig. 4). The pH optimum for the pNPP phosphatase activity was pH 5.5, but the Tyr(P) phosphatase activity exhibited optimum activity at pH 6, and the phos- photyrosyl histone phosphatase activity was maximal at pH 7.0.

Co-purification of Tyr(P) Phosphatase and Phosphotyrosyl Protein Phosphatase Activities wi thpNPP Phosphatase Actiu- ity-To further examine the identity of these phosphatase activities in our partially purified tartrate-resistant skeletal acid phosphatase preparation, we attempted to separate the activities by (i) gel filtration (Sephacryl S-200) chromatogra- phy (Fig. 5A) , (ii) ion exchange (CM-Sepharose) chromatog- raphy (Fig. 5B) , and (iii) affinity (cellulose phosphate) chro- matography (Fig. 5C). As indicated in Fig. 5, the Tyr(P) and phosphotyrosyl protein phosphatase activities co-migrated with the pNPP phosphatase activity during the gel filtration, ion exchange, and affinity chromatographies, suggesting that

' r 8' 1'

0.5 1.0 1.5 2.0 1 /(P-Tyrosine), mM - '

l/(P-Tyrosyl Histones), 1.1h4-l FIG. 3. Lineweaver-Burk plots for partially purified bovine

tartrate-resistant skeletal acid phosphatase using phospho- tyrosine (panel A ) and phosphotyrosyl histones (panel B) as substrates. InpanelA, the following symbols areused A, DL-Tyr(P); @, L-Tyr(P).

3 4 5 6 7 8 9

Reoction p H

FIG. 4. Relative pH optima for the pNPP, Tyr(P), and phos- photyrosyl histones dephosphorylation reactions. pNPP (10 mM), DL-Tyr(P) (10 mM), and [32P]phosphotyrosyl histones (5 PM) were used to assay for pNPP phosphatase (e), Tyr(P) phosphatase (U), and phosphotyrosyl protein phosphatase (A) activities, respec- tively. Sodium acetate (100 mM) was used for reactions with pH 3 to 6, Mes buffer (100 mM) for reactions from pH 5.5 to 7, and Hepes buffer (100 mM) for reactions with pH 6 and above. There were no aparent effects of buffers on the enzyme activities.

these activities are closely associated and might be mediated by the same enzyme.

To further investigate the possibility that these three activ- ities were mediated by the same protein, we compared the relative recovery of these activities during the preparation of the bovine tartrate-resistant skeletal acid phosphatase. If

Skeletal Acid and Alkaline Phosphatases 4657

oval cytc 7 (ratio B/A) and that of the phosphotyrosyl protein phospha- 1 1 tase activity to the acid pNPP phosphatase activity (ratio C/ '? A) remained relatively constant throughout the preparation, P? but the ratio of skeletal alkaline phosphatase to skeletal acid a

phosphatase (pNPP phosphatase) activities (ratio D/A) de- creased with purification (Table 11). This suggests that (a) the contaminating skeletal alkaline phosphatase in the prep- aration did not contribute significant Tyr(P) phosphatase or phosphotyrosyl protein phosphatase activity in our assays

SaLh- and that (b) the relative recovery of the pNPP phosphatase, Tyr(P) phosphatase, and phosphotyrosyl protein phosphatase - activities was approximately equal at each stage of our puri-

E fication, further suggesting that these three activities were closely associated and might be medicated by the same pro- 8 tein. Relative Thermal Stability of Tartrate-resistant Skeletal

8 Acid Phosphatase and Sensitivity to Various Effectors-We 0.6 a next examined the thermal stabilities of the pNPP phospha-

0.4 tase, Tyr(P), and phosphotyrosyl protein phosphatase activi- ties in our enzyme preparations. The three phosphatase activ- ities had very similar stabilities to thermal denaturation (Fig. 6). Preincubation of the enzyme preparation at 60 "C for 30 min only resulted in loss of about 20% of each of the original activities. Many enzymes would have been denatured under these conditions. Similar results were obtained when the enzyme fraction was preincubated at 70 " C , suggesting that

-2.0 n these activities were very heat stable. This is in contrast to .""- skeletal alkaline phosphatase which is rapidly inactivated at

- temperatures greater than 50 "C (data not shown). The sensitivities of the three phosphatase activities toward

- 0.4 9 various agents known to affect tartrate-resistant skeletal acid

- 0-4 phosphatase activity have also been examined (Table 111). Since the optimum pH of the three activities was quite differ- ent and since differences in pH might account for different effects with respect to both the substrates and the enzyme, we compared the actions of the effectors at two different pHs. We compared the pNPP phosphatase and Tyr(P) phospha- tase activities at DH 5.5. and we comuared the Tvr(P) Dhos-

40 20

20 40 60 80 100 W

1.0

1.6

10 20 30 40 50 Fraction Number

FIG. 5. Elution profiles for pNPP phosphatase (O), Tyr(P) phosphatase (a), and phosphotyrosyl protein phosphatase (A) activities on Sephacryl S-200 gel filtration column (panel A), on CM-Sepharose column (panel B) , and on cellulose phos- phate column (panel C). The procedures for these column chro- matographies are as described under "Methods," except that only the second sodium acetate gradient was used for the CM-Sepharose chromatography shown here. The gradients of sodium acetate and sodium chloride in the CM-Sepharose and cellulose phosphate chro- matographies, respectively, were determined by measuring the con- ductivities. Vo, thyroglobulin; Vi, vitamin BIZ; Cyt c, cytochrome c; Oval, ovalbumin.

these activities were attributed to the same protein, the rela- tive ratio of these activities should remain relatively constant during the preparation, providing that no other contaminating phosphatase activities were present. Table I1 summarizes the results of a typical preparation of bovine tartrate-resistant skeletal acid phosphatase.

Since it has been previously reported that both bacterial and mammalian alkaline phosphatases could also dephos- phorylate phosphotyrosyl proteins at neutral pH (17), we were concerned that contaminating skeletal alkaline phosphatase activity in the preparations might interfere with the deter- mination of the activities of Tyr(P) and phosphotyrosyl pro- tein phosphatases associated with the tartrate-resistant skel- etal acid phosphatase. Thus, we measured the total activity and specific activity of skeletal alkaline phosphatase at each of the purification steps. We found that the ratio of Tyr(P) phosphatase activity to the acid pNPP phosphatase activity

phatase with the phosphotyrosyl protein phosphatase activi- ties at pH 7.0, since tartrate-resistant skeletal acid phospha- tase did not show good activity with pNPP at the latter pH. As summarized in Table IV, both pNPP phosphatase and Tyr(P) activities showed similar responses to effectors of tartrate-resistant skeletal acid phosphatase at pH 5.5, and similarly, both the Tyr(P) phosphatase and phosphotyrosyl protein phosphatase activities exhibited similar sensitivities to the same effectors at pH 7.0.

Tyr(P) Is an Alternative Substrate for Tartrate-resistant Skeletal Acid Phosphatase-If the Tyr(P) phosphatase and pNPP phosphatase activities are mediated by the same en- zyme, Tyr(P) should be an alternative substrate for pNPP phosphatase activity and, therefore, should behave as a com- petitive inhibitor with respect to pNPP. Furthermore, the apparent value of K, for Tyr(P) as an inhibitor of pNPP phosphatase activity should be equal to the apparent value of K, for the Tyr(P) dephosphorylation reaction. Indeed, as shown in Fig. 7, we found that Tyr(P) was a competitive inhibitor for the pNPP phosphatase activity with an apparent Ki of about 0.6 mM at pH 5.5, a value nearly equal to the apparent K, (0.6 mM) for Tyr(P) in the Tyr(P) phosphatase reaction at the same pH (Fig. 3A).

Skeietal Alkaline Phosphatase as a Phosphotyrosyl Protein Phosphatase-Both highly purified skeletal alkaline phospha- tase from human bone and partially purified skeletal alkaline phosphatase from chick bones were capable of dephosphoryl- ating both the phosphoseryl and phosphotyrosyl histones in

~~ -_ I \ 1

4658 Skeletal Acid and Alkaliw Phosphatases TABLE I1

A typical purification table for bovine tartrate-resistant skeletal acid phosphatase

Step Tota l sACP protein $ALPb P-tyrosine

( B )

P-tyrosyl Ratio

protein ( A ) phosphatase phosphatase ( D ) ( C ) BfA' CIA' DfA'

mg rnvd mU/mg m u mU/rng mU rnUfng rnU mufmg Crude extract 394 2632 6.68 1265 3.21 78.2 0.20 4311 10.9 0.48 0.030 1.63 Protamine sulfate-treated fraction 493' 2456 4.99 1546 3.13 85.0 0.17 3726 7.6 0.63 0.034 1.52 Dialyzed and pH-adjusted fraction 477 1972 4.14 914 1.91 33.5 0.17 680 1.4 0.46 0.041 0.34 CM-Sepharose pool 12 524 43.3 151 12.5 18.6 1.54 55 4.6 0.29 0.035 0.11 Cellulose phosphate pool 0.87 194 218 64 72.0 6.4 7.40 0 0 0.33 0.034 0 Sephacrrl S-200 pool 0.35 116 330 40 113.7 4.0 11.55 0 0 0.34 0.035 0

"Skeletal acid phosphatase (sACP) activity represented the pNPP phosphatase activity assayed at pH 5.5 determined as described under "Methods."

sALP, skeletal alkaline phosphatase. B / A = Tyr(P) phosphatase activity/pNPP phosphatase activity; CIA = phosphotyrosyl protein phosphatase

activity/pNPP phosphatase activity; and D / A = skeletal alkaline phosphatase activity/skeletal acid phosphatase (pNPP phosphatase) activity.

mu, milliunits. e A significant amount of protamine sulfate was left in the extract; therefore, the total amount of protein was

increased and the specific activities were decreased.

a

5 I O 15 20 25 Incubation Time IMinI

- 8

I

30

FIG. 6. Comparison of the thermal stability of the pNPP phosphatase (O), Tyr(P) phosphatase (0), and phosphotyrosyl protein phosphatase (A) activities. Partially purified bovine tar- trate-resistant skeletal acid phosphatase (50 milliunits/ml), purified up to CM-Sepharose chromatography, was incubated at 60 "C. At the indicated time intervals, aliquots of enzyme were withdrawn to assay for the three phosphatase activities.

a time- and concentration-dependent manner (data not shown). However, unlike the phosphotyrosyl protein phos- phatase activity in the tartrate-resistant skeletal acid phos- phatase preparation, the phosphotyrosyl protein phosphatase activity of the skeletal alkaline phosphatase was more active at alkaline than at neutral pH (Fig. 8). The relative activity of this highly purified human skeletal alkaline phosphatase was about 22-fold higher at pH 9 than at pH 7, with 20 nM phosphotyrosyl histones as the substrate. Similarly, the rela- tive velocity of the same enzyme at pH 9, with either pNPP or DL-Tyr(P) as substrate, was about 23-fold higher than it was at pH 7 (data not shown). The apparent K, values for pNPP, Tyr(P), and phosphotyrosyl histones were also deter- mined at three different pHs, using this highly purified human skeletal alkaline phosphatase. As summarized in Table IV, we found that the three phosphatase activities of the skeletal

I / o

/// I I I I

1 2 3 4

1 / P N P P (mM")

FIG. 7. Inhibition of the pNPP dephosphorylation reaction by DL-Tyr(P). Partially purified bovine tartrate-resistant skeletal acid phosphatase, after the CM-Sepharose chromatography, was used in this experiment. The inset is the replot of the Lineweaver-Burk plots.

alkaline phosphatase exhibited similarly increased affinity at lower pH ( i e . 7) than at higher pH (i.e. pH 9), suggesting that although these three activities might be attributed to the same protein, they were distinct from the activities we had mea- sured at neutral pH and attributed to our tartrate-resistant skeletal acid phosphatase.

DISCUSSION

In the present study, we have partially purified a skeletal acid phosphatase from bovine cortical bone, using pNPP as substrate. This skeletal acid phosphatase shared many similar properties with a rat bone acid phosphatase isoenzyme that has been classified as tartrate-resistant skeletal acid phospha- tase (10-13). The activity we have characterized is sensitive to inhibitions by fluoride, molybdate, and diphosphonate, but not tartrate. Our acid phosphatase will hydrolyze nucleotide triphosphates but has no activity on &glycerol phosphate or nucleotide monophosphates.

Our experimental evidence supports the proposition that this bovine tartrate-resistant skeletal acid phosphatase is a

Skeletal Acid and Alkaline Phosphatases 4659

TABLE 111 Relative sensitivity of pNPP phosphatase, Tyr(P) phosphatase, and phosphotyrosyl protein phosphatase activities to tartrate-resistant

acid phosphatase effectors Relative enzyme activities

At pH 5.5 At pH 7.0

Effector pNPP Tyr(P) Tyr(P) P-tyrosy'

phospha- phospha- phospha- phospha- protein

tase' tase" taseb taseb

No addition 5 mM Pi 5 mM PPi 5 mM ATP 5 mM C12MDPd 1 mM fluoride 2 mM vanadate 20 mM molybdate 20 mM tartrate

100 100 100 56.2 71.5 57.7 50.3 49.9 45.8 33.5 36.5 63.4 56.0 56.7 68.3 20.4 18.6 19.6 38.9 31.6 29.6 10.1 0 2.8 95.4 95.3 122.5

100 47.3 44.9 71.2 76.6 24.2 25.4 10.1

104.6

"pNPP phosphatase activity was assayed with 10 mM pNPP in 100 mM sodium acetate buffer, pH 5.5.

* Tyr(P) phosphatase activity was assayed with 10 mM DL-Tyr(P) in 100 mM sodium acetate, pH 5.5, for the pH 5.5 reactions but with 50 mM Hepes, pH 7.0, for the pH 7.0 reactions.

Phosphotyrosyl protein phosphatase activity was assayed with 20 nM [32P]phosphotyrosyl IgG in a buffer containing 50 mM Hepes, pH 7.0, and 100 mM NaCl.

CI,MDP, dichloromethylene diphosphonate.

TABLE IV Apparent K,,, values for a highly purified human skeletal alknline phosphatase toward pNPP, Tyr(P), and phsphotyrosyl histones

Substrate Apparent K," at pH

7 8 9

P M

pNPP 14 64 440 DL-Tyr(P) 12 83 360 Phosuhotvrosvl histones 200 450 1370

Apparent K,,, for pNPP and Tyr(P) determined from kinetic plots. Apparent K,,, for phosphotyrosyl histones calculated by assuming that Vmax of phosphotyrosyl histone phosphatase activity was approxi- mately equal to that of pNPP phosphatase activity, since Vmax for Tyr(P) phosphatase activity was equal to V,. for pNPP phosphatase activity.

phosphotyrosyl-specific protein phosphatase. We have dem- onstrated that preparations of partially purified bovine tar- trate-resistant skeletal acid phosphatase were capable of de- phosphorylating both Tyr(P) and phosphotyrosyl proteins. The same enzyme preparations, however, did not have appre- ciable activities toward other phosphoamino acids or toward phosphotyrosyl proteins, indicating that the activity is rela- tively specific for the Tyr(P) moiety. The relatively high affinity (low K,) for phosphotyrosyl proteins is also consis- tant with this specificity.

We have also shown that (a) the Tyr(P) phosphatase and phosphotyrosyl protein phosphatase activities could not be separated from the acid pNPP phosphatase activity by a variety of chromatographic procedures, (b) the relative ratios of these three activities remained constant throughout the purification of the bovine tartrate-resistant skeletal acid phos- phatase, ( c ) the three phosphatase activities exhibited similar sensitivities toward effectors of tartrate-resistant skeletal acid phosphatase and similar thermal stabilities, and (d) Tyr(P) was an alternative substrate for tartrate-resistant skeletal acid phosphatase. Together, these findings suggest a tight association between the pNPP phosphatase, Tyr(P) phospha- tase, and phosphotyrosyl protein phosphatase activities in our bovine tartrate-resistant skeletal acid phosphatase prepara-

8o t

c " ' e o 0 * O k - 7 8 9

Reaction pH FIG. 8. Effect of pH on the phosphotyrosyl protein phospha-

tase activity of human skeletal alkaline phosphatase. A highly purified human skeletal alkaline phosphatase (1.6 milliunits/ml, as- sessed with pNPP at pH 9) was assayed for phosphotyrosyl histones phosphatase activity at the indicated pH values in a titrated mixture of 25 mM carbonate, 25 mM Tris. The concentration of [32P]phospho- tyrosyl histones was 20 nM, and the reaction was allowed to proceed at 37 "C for 3 h.

tion and are consistent with the possibility that all three activities are mediated by the same enzyme.

Brautigan et al. (24, 25) have identified a membrane-asso- ciated phosphotyrosyl protein phosphatase in membrane ves- icles derived from A-431 cells and Rous sarcoma virus-trans- formed rat cells which was inhibited by micromolar concen- trations of Zn2+, but not by fluoride. Foulkes et al. (26) also described a fluoride-insensitive phosphatase activity in ex- tracts of both rat skeletal muscle and rat liver which dephos- phorylates [32P]phosphotyrosyl IgG. Their work clearly in- diated that the phosphotyrosyl protein phosphatase present in these tissue extracts was distinct from the mammalian phosphoseryl/phosphothreonyl protein phosphatases. Based on these findings, it appears that a different class of protein phosphatase, which is specific for the phosphotyrosyl pro- teins, is responsible for the removal of the Pi group from the tyrosine residues.

The possible existence of acid phosphatase-like phospho- tyrosyl protein phosphatase activity has been suggested by several recent studies. Li et al. (27) reported that a phospho- tyrosyl protein phosphatase activity is strongly associated with the human prostatic acid phosphatase, which, however, is sensitive to tartrate inhibition. Leis and Kaplan (28) have also previously reported that the phosphotyrosyl protein phos- phatase activity in the plasma membrane of human astrocy- toma was an acid phosphatase-like activity. This phospha- tase-like membrane-associated phosphotyrosyl protein phos- phatase also has many properties similar to those of our bovine tartrate-resistant skeletal acid phosphatase. Both ac- tivities are insensitive to tartrate but sensitive to fluoride and vanadate, and both show relatively high specificity for phos- photyrosyl proteins and Tyr(P) and similar pH-dependent activity profiles. Fukami and Lipmann (23) have also recently reported the purification of a specific Tyr(P) phosphatase from the larvae of Drosophila. This Tyr(P) phosphatase also appeared to be an acid phosphatase-like enzyme, but unlike the Tyr(P) phosphatase activity in our preparations of bovine tartrate-resistant skeletal acid phosphatase, their activity had a strict substrate stereospecificity for L-Tyr(P). Together, these findings strongly suggest that a class of acid phospha-

4660 Skeletal Acid and Alkaline Phosphatases

tase-like Tyr(P)/phosphotyrosyl protein phosphatase exists in eucaryotic cells.

Although we found that purified skeletal alkaline phospha- tase could also dephosphorylate phosphotyrosyl proteins at neutral pH, the activity was about 20 times higher at pH 9 than that at pH 7. Furthermore, the apparent K , of skeletal alkaline phosphatase for phosphotyrosyl proteins was ex- tremely high compared to skeletal acid phosphatase. Two additional lines of evidence also argue against the possibility that alkaline phosphatase accounts for significant phospho- tyrosyl protein phosphatase activity in our preparations. First, unlike skeletal acid phosphatase, skeletal alkaline phospha- tase demonstrated significant activity toward [32P]phospho- seryl histones; the protein phosphatase activity of skeletal alkaline phosphatase was not specific for phosphotyrosyl pro- teins. Second, the ratios of phosphotyrosyl protein phospha- tase activity to tartrate-resistant skeletal acid phosphatase activities were constant throughout our purification proce- dure, despite the fact that significant amounts of skeletal alkaline phosphatase activity were removed at each stage of the purification. Together, these observations suggest that skeletal alkaline phosphatase might not function as a phos- photyrosyl protein phosphatase under physiologically rele- vant conditions.

Although we have not demonstrated a physiological role for tartrate-resistant skeletal acid phosphatase as a phosphoty- rosy1 protein phosphatase in bone, our data are consistent with that possibility. Recent studies have suggested that many tissues may contain multiple forms of phosphotyrosyl protein phosphatases (29-31), and it is possible that skeletal tissues also contain multiple forms of phosphotyrosyl protein phos- phatase activity. Our preliminary data indicate that tartrate- sensitive skeletal acid phosphatase might also associate with a phosphotyrosyl protein phosphatase activity. Furthermore, other proteins, e.g. calmodulin-dependent calcineurin (32), have also been reported to have phosphotyrosyl protein phos- phatase activity. In the case of phosphoseryl protein phos- phatases, a similar multiplicity of activities has been linked with in uiuo substrate specificity (33, 34). Thus, it is possible that the physiological relevance of the phosphotyrosyl protein phosphatase activity we have identified in bone will not be resolved until its physiological substrate(s) and its in vivo role has been identified.

Acknowledgments-We wish to thank Tim Freeman and Julie Vickers for their technical assistance and Penny Nasabal and the Medical Media staff of the Jerry L. Pettis Veterans’ Administration Hospital for their assistance in the preparation of this manuscript.

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