addition of enzyme and terminated by addition of 1 ml of 0.2 M. NaOH. Appropriate .... gradient from 200 mM to 1 M sodium acetate, pH 6.5. ..... 0.20 4311 10.9.
Vol. 260,No. 8,Issue of April 25, pp. 4653-4660,1985 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Phosphotyrosyl-specific Protein PhosphataseActivity of a Bovine Skeletal Acid Phosphatase Isoenzyme COMPARISON WITH THE PHOSPHOTYROSYLPROTEINPHOSPHATASE ALKALINE PHOSPHATASE*
ACTIVITY OF SKELETAL
(Received for publication, October 15, 1984)
K.-H. William Lau, John R. Farley, andDavid 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 phosphatase, which shared similar characteristics with a class of acid phosphatase known as tartrate-resistant acid phosphatase, was found to dephosphorylate phosphotyrosine and phosphotyrosyl proteins, with little activity toward other phosphoamino acids or phosphoseryl 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 phosphotyrosyl histones. The apparentK,,, values for phosphotyrosyl histones (at pH 7.0) and phosphotyrosine (at pH 5.5) were about 300 nM phosphate group and 0.6 mM, respectively, Thep-nitrophenyl phosphatase, phosphotyrosine phosphatase, and phosphotyrosyl protein phosphatase activities appear to be a single protein separated by since (a)these activities couldnotbe Sephacryl 5-200, CM-Sepharose, or cellulose phosphate chromatographies,(b)the ratioof these activities remained relatively constant throughout the purification procedure, (c) each of these activities exhibited similar thermal stabilities and similar sensitivities to (d)phosphotyrosine andp-nitrovarious effectors, and phenyl phosphate appeared tobe alternative substrates for theacid 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 phospho0.2-0.4 mM), tyrosyl proteins was low (estimated to be and its protein phosphatase activity was not specific for phosphotyrosyl proteins, since it also dephosphorylated phosphoseryl histones. In summary, these data suggested that skeletal acid phosphatase, rather than skeletal alkaline phosphatase, may act as phosphotyrosyl protein phosphatase under physiologically relevant conditions.
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 phosphotyrosyl protein dephosphorylation (6). In order to regulate such a proliferative signal, it is necessary that a phosphotyrosyl 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 activities 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 discovered many decades ago, its biochemical function is still undefined. 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 skeletal acid phosphatase. A second isoenzyme, whichis sensitive to inhibitions by fluoride, molybdate, and diphosphonate, but not tartrate, is known as tartrate-resistantskeletal acid phosphatase. We have isolated a tartrate-resistantskeletal acid phosphatase 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 phosphatase also has phosphotyrosyl protein phosphatase activity, we have compared the activities of skeletal acid phosphatase and skeletal alkaline phosphatase toward phosphotyrosyl proteins under physiologically relevant conditions. EXPERIMENTALPROCEDURES
Materials
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’ Administration. The costs of publication of this article were defrayed in part by the payment of page charges. This article musttherefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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]Piand [‘261]NaI wereobtained from ICN Biochemicals, Inc. [y32P]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, CAMPdependent 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-piperazineethanesulfonic acid; Tris, tris(hydroxymethy1)aminomethane; Mes, 2-(n-morpholino)ethanesulfonicacid; SDS, sodium dodecyl sulfate.
4653
AcidSkeletal
4654
and Alkaline Phosphatases
S-200 and phenylalanine-agarose were from Pharmacia. Bio-Gel A1.5, 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 of0.2 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 a t 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 theamount of enzyme that is required to hydrolyze 1pmol of pNPP/min at37 "C. Tartrate-resistant acid phosphatase was determined in the presence of 20 mM tartrate, adjusted to pH 5.5, and the amount of tartartesensitive activity was calculated from the difference between the total and the tartrate-resistantactivities. 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). Whenother 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 phosphotyrosy1 protein phosphatase activity was monitored by the release of [32P]P;from [3ZP]phosphotyrosylhistones or from [32P]phosphotyrosyl IgG. [32P]Phosphotyrosylproteins 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 Dulbecco's modified Eagle's medium in the presence of 10% fetal calf serum. The A-431cell membrane fraction containing epidermal growth factor-stimulated tyrosine kinase activity was prepared as previously described (18).The phosphotyrosyl protein dephosphorylation reaction was carried out under the following conditions. Reaction mixtures contained 5-10 p~ phosphoproteins (concentrations calculated from the amounts of [32P]Piincorporation 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 of0.2 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 theradioactivity was determined in aBeckman model liquid scintillation counter. Over 95% of the radioactivity released in the dephosphorylation reaction could be extracted with ammonium molybdate-butyl acetate (19), confirming that the radioactivity released represented [3zP]Pirather than 32P-containingphosphosilicotungstic acid-soluble peptides and eliminating the possibility that radioactivity was released by proteolysis. One unit of enzyme activity was defined as theamount of enzyme needed to release 1nmol of ["'PlPi from the [3ZP]phosphotyrosylprotein/min a t 37 'C. The same reaction conditions were used when phosphoseryl histones were evaluated as substrates. [32P]Phosphoserylhistones 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 1mM 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 coefficient of 17,800 M" cm". One unit of skeletal alkaline phosphatase is defined as theamount of enzyme that is required to hydrolyze 1pmol of pNPP/min at room temperature. Fifty mM Tris/carbonate and Hepes buffers were used to evaluate skeletal alkaline phosphatase activity at pHless 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 procedure described by Anderson and Toverud (10) for rat skeletal acid phosphatase isoenzymes. Briefly, the bovine long bones were sawed into small cubes, andthe marrow wasremoved. 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 homogenizer. 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 pH4.8 with glacial acetic acid. Precipitates were removed, and the supernatantwas 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, pH6.5. 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 skeletal 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-resistantacid phosphatase were pooled and concentrated in an Amicon ultrafiltration concentrating system with a YM-10 membrane (M, cutoff of approximately 10,000). Enzyme 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 1cm, inner diameter), which waspre-equilibrated with 100 mM sodium acetate, pH 6.5. The enzyme activity was eluted with a 200ml 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 tartrateresistant skeletal acid phosphatase estimated by this method was approximately M,40,000. Native polyacrylamide gel electrophoresis revealed that thepreparation of bovine tartrate-resistant acid phosphatase showed one band of enzyme activity which could beclassified 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 according 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 carbonate buffer, the activity was purified by ammonium sulfate precipitation, 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 phenylalanine-agarose and by preparative polyacrylamide gel electrophoresis on a4-20% gradient gel. The resulting preparation of human skeletal alkaline phosphatase migrated as asingle band on polyacrylamide gel electrophoresis after labeling with [1251]NaI.
Partial Purificationof Skeletal Alkaline Phosphatase from Chick Bone Chick skeletal alkaline phosphatase was partially purified from embryonic chick calvaria as previously described (20). Briefly, calvaria were rinsed and extracted with 20% butanol (v/v) in 50 mM Tris/carbonate buffer (pH 6.5) for 24 h a t 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 DEAEcellulose column chromatography and molecular sieve chromatography 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 phosphatase was performed as described by Lam et al. (21). SDS-polyacrylamide 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
4655
Acidic PAGE A B
SDS PAGE C D
320k*F 158k+ Q
5'-
46 k+(3
sACP-
I
j
17k-a
!
!i 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.
Tyr(P) and Phosphotyrosyl Protein Phosphatase.Actiuities PreparaTABLE I tions-We have partially purified a tartrate-resistant acid Relative activities of the partially purified tartrate-resistant skeletal phosphatase from bovine cortical bone using pNPP as subacid phosphatase on phosphoaminoacids, phosphohistones, and strate according to a procedure modified from one described nucleotide phosphates for the purification of rat skeletal acid phosphatase (10). Our The enzyme usedin theseexperiments was purified up to theCMacid phosphatase preparation migrated as a single band of Sepharose chromatography. activity on acidic polyacrylamide gel electrophoresis (Fig. 1) Relative Enzyme Substrate and can be classified as a band 5 acid phosphatase isoenzyme activity activity according to the nomenclature suggested by Lam et al. (21). rniUiunits/ml The bovine tartrate-resistant skeletal acid phosphatase Reaction pH 5.5 showed activity toward nucleotide triphosphates but not to10 mM pNPP 57.6 100.0 10 mM phosphotyrosine 34.7 20.0 ward nucleotide monophosphates or @-glycerolphosphate 10 mM phosphoserine 6.4 3.7 (Table I). We have also found that our preparations of par10 mM phosphothreonine 5.6 3.6 tially purified tartrate-resistant skeletal acid phosphatase ex43.2 24.9 25 mM ATP hibited phosphatase acitivities toward Tyr(P) and phospho41.0 23.6 10 mM GTP tyrosyl proteins (Table I and Fig. 2). Both of these phospha30.0 17.3 25 mM ADP 1.7 1.0 25 mM AMP tase activities were time dependent (Fig. 2, A and C) and 7.5 4.3 10 mM GMP concentration dependent (Fig. 2, B and D).
