Comp. Biochem. Physiol. Vol. 83B, No. 4, pp. 813-817, 1986 0305-0491/86 $3.00+0.00 Printed in Great Britain ((7 1986 Pergamon Press Ltd

PURIFICATION A N D C H A R A C T E R I Z A T I O N OF PURPLE ACID PHOSPHATASE FROM RAT BONE

TAKASHI KATO*, AKIRA HARA*, TOSHIHIRO NAKAYAMA*, HIDEO SAWADA*t, MICHIKO HAMATAKE~ and YASUHIRO MATSUMOTO +

*Department of Biochemistry, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502, Japan; and tDaiichi College of Pharmaceutical Sciences, Minami-ku, Fukuoka 815, Japan

(Received 18 June 1985)

Abstract--l. An acid phosphatase, which was immunochemically identical to splenic purple acid phosphatase, was purified to homogeneity from rat bone.

2. The enzyme was a two iron-containing monomeric glycoprotein with a mol. wt of 36,000. 3. The enzyme hydrolyzed aryl phosphates, nucleoside di- and triphosphates, thiamine pyrophosphate,

phosphoenolpyruvic acid and acidic phosphoproteins. 4. The enzyme was inhibited by ammonium molybdate, NaF and CuSO4 but not by tartrate and

SH-reagents.

INTRODUCTION

Rat bone exhibits significant tartrate-resistant acid phosphatase activity as compared with other organs (Vaes and Jacques, 1965; Wergedal, 1970). The en- zyme is abundant in the osteoclast (Burstone, 1959; Hammars t r6m et al. , 1971) and has been postulated to be involved in the osteolytic process (Vaes, 1965, 1968) and to serve as a biochemical marker for osteoclast function (Minkin, 1982). The tartrate- resistant enzyme has been partially purified from rat bone (Anderson and Toverud, 1979), and is also distinguished from another tartrate-sensitive enzyme in the tissue with respect to its low tool. wt of 34,000, broad substrate specificity over aryl phosphates, ATP, A D P and casein, and activation by ferrous ion and reducing agents (Anderson and Toverud, 1981, 1982). A recent histochemical study (Hammars t r6m et al. , 1983) which indicates that bone tartrate- resistant acid phosphatase is inactivated by dithionite and reactivated by the addition of ferrous ion has suggested that the enzyme contains iron.

We previously reported that the tartrate-resistant, Fe2+-activated acid phosphatase in rat bone showed electrophoretic mobility and affinity for Cibacron blue dye similar to those of spleen enzyme which was subsequently purified to homogeneity and character- ized as purple acid phosphatase (Hara et al. , 1983, 1984). A re-examination of the bone enzyme, partic- ularly in light of the studies of purple acid phos- phatases from other tissues (Schlosnagle et al. , 1974; Campbell e t al. , 1978; Davis e t al. , 1981; Hara et al. , 1984), suggested very strongly that this enzyme is also a purple acid phosphatase. In this paper, we report the purification and characterization of the enzyme from rat bone and demonstrate its identity with splenic purple acid phosphatase.

MATERIALS AND METHODS

Tibiae and femora were excised from 1-week-old Sprague-Dawley rats. Histone (Type II-S) was purchased

tTo whom all correspondence should be addressed.

from Sigma Chemical Co. Phosvitin and Blue-Sepharose were prepared as described by Mecham and Olcott (1949) and Heyns and De Moor (1974), respectively. Purple acid phosphatase was purified from adult rat spleen (Hara et al., 1984) and rabbit anti-IgG directed against it was prepared as described elsewhere (Hara et al., 1985). Other materials and reagents were obtained from sources listed in previous publications from our laboratory (Hara et al., 1983, 1984).

Enzyme purification

The procedure is adapted with certain modifications from that which we developed for isolation of splenic purple acid phosphatase (Hara et al., 1984). The modifications of a typical preparation starting with 40 g of tissue are briefly described. The following operations were performed at 0-4°C. The frozen bones were thawed, minced and homoge- nized with 60 ml of 50 mM Tris-HC1 buffer, pH 7.5, con- taining I M KCI and I mM phenylmethylsulfonyl fluoride in a Polytron homogenizer. The homogenate was stirred for 30 rain and centrifuged at 105,000g for 1 hr. The pellet was taken with 30 ml of the same buffer, and the stirring and centrifugation were repeated once. The supernatants were combined and the pH adjusted to 4.5 with 2 M acetic acid. The precipitated proteins were removed by centrifugation at 15,000g for 5 rain. The pH of the supernatant fraction (acid-treated KC1 extract) was adjusted to 7.5 with 1 M KOH and the supernatant diluted to 0.1 M KC1 with the same buffer without KC1. It was mixed for 4 hr with I00 ml of slurry containing Blue-Sepharose (packed volume 50 ml) in 50mM Tris-HCl, pH 7.5, supplemented with 1 mM phenylmethylsulfonyl fluoride and 0.1 M KC1, and the resin was then packed in a glass column. The column was washed with 11 of the same buffer, and activity then eluted with the buffer containing 2 M KCI. After concentration in an Amicon Diaflo concentrator with a YM-10 membrane, the enzyme was further purified by following the procedures for isolation of the spleen enzyme (Hara et al., 1984).

Analyt ical methods

Acid phosphatase activity was assayed with p-nitrophenyl phosphate as a substrate and 0.1 mM FeSO 4 as the activator as described by Hara et al. (1984). A unit of activity corresponds to the amount required to release 1.0 #tool of p-nitrophenol per min at 30°C. The enzymatic hydrolysis of other phosphoesters and phosphoproteins was determined by measuring the release of orthophosphate into the reac- tion mixture by the method of Heinonen and Lahti (1981).

813 C.B,P. 83/41~-H

814 TAKASHI KATO et al.

Table 1. Purification of purple acid phopshatase from rat bone

Step

Total Specific

Protein activity activity Yield Purifi-

(mg) (units) (units/mg) (%) cation

Acid-treated 891 974 1.09 lO0 l.O KCI extract

Blue-Sepharose 282 737 2.60 76 2.4

Concanavalin A- 7.40 802 I08 82 99 Sepharose

Sephadex G-IO0 1.14 551 483 57 440

CM-Sephadex 0.27 388 1440 40 1300

Protein analyses were performed as described by Lowry et al. (1951) using bovine serum albumin as the standard.

Electrophoresis of the native enzyme at pH 4.0 and activity staining of the gels were performed as described by Axline (1968), and sugar was stained by the method of Zacharius et al. (1969). The mol. wt of the native enzyme was estimated by a high performance liquid chromatograph (Waters Ltd., Model 204A) with a TSK gel-G3000 SW (0.75 × 60cm) in 50mM sodium acetate buffer, pH 5.5, containing 0.5 M KCI. The column was calibrated with bovine serum albumin, ovalbumin, chymotrypsinogen and myoglobin at a flow rate of 0.5 ml/min. The mol. wt of the denatured enzyme with 1% sodium dodecyl sulfate (SDS) and 2% 2-mercaptoethanol was determined by SDS- electrophoresis (Weber and Osborn, 1969) with the same standards except that trypsin was employed instead of chymotrypsinogen. Isoelectric focusing on polyacrylamide gel was performed as described by Hara et al. (1982, 1985). Iron assay was performed as described by Campbell and Zerner (1973). Phosphorus concentration was determined as described by Bitte and Kabat (1974). Ultraviolet absorption was recorded using a Hitachi 100-50 spectrophotometer.

lmmunochemical analysis

Double diffusion test was performed on 1.2% agarose containing 0.14M NaCI and 20 mM Tris-HCl buffer, pH 7.5. The enzyme activity of the precipitin line was stained in 50 mM sodium acetate buffer, pH 5.5, containing 1 mg/ml of 1-naphthyl phosphate and Fast Garnet GBC salt after the agarose was washed with 20 rnM Tris-HC1 buffer, pH 7.5, containing 0.5M KC1. Immunoprecipitation procedures with the enzymes and IgG were as follows. Constant amounts of the enzyme were incubated at 4°C overnight with an increasing range of amounts of IgG in 10mM Tris-HCl buffer, pH 7.5, containing 0.5% bovine serum albumin and 0.5 M KCI, in a final vol of 0.2 ml. The mixture was centrifuged at 6000g for 10min at 4°C. The residual activities of the enzyme in the supernatant and the sus- pension of the precipitate in the buffer were determined by assaying 50/~1 samples. The IgG from a non-immunized rabbit was used as a control.

patible with the values of splenic purple acid phos- phatases (Davis et al., 1981; Hara et al., 1984).

Physicochemical properties

The enzyme was stained red with perchloric acid-Schiff 's reagent (Fig. 1). The mol. wt of the denatured enzyme calculated from SDS- electrophoresis was 36,000 and the value of the native enzyme estimated by high pressure exclusion chro- matography was 37,000. The pI value of the enzyme was 7.6 when analyzed on gel isoelectric focusing. These results indicate that the enzyme is a monomeric and basic glycoprotein.

The concentrated enzyme exhibited a purple color with absorption maxima at 280nm and 540nm (Fig. 2). This spectrum is similar to those of iron- containing acid phosphatases from other tissues (Davis et al., 1981; Hara et al., 1984). The iron content of the bone enzyme was 2.1 atoms per 36,000 enzyme molecule.

Catalytic properties

Table 2 summarizes the substrate specificity. The enzyme hydrolyzed aryl phosphates, nucleoside tri- and diphosphates, thiamine pyrophosphate and phosphoenolpyruvic acid at an optimal pH of around 6.0. The enzyme also dephosphorylated acidic phos- phoproteins such as phosvitin and casein at relatively low rates, but was inactive towards histone. The bone enzyme was activated by Fe 2+, ascorbic acid, cysteine, dithiothreitol and 2-mercaptoethanol as described previously (Anderson and Toverud, 1982). The highest activation rate, 5-times the activity with- out an activator, was observed by the addition of Fe z+ at concentrations of 0 .05-0 .5mM, whereas higher concentrations of more than 1 mM and about 10 min preincubation were required for activation of

1 I,

B

A

C

O

O

!

RES L S D | Purification

About 70% of Fe2+-activated acid phosphatase activity of the bone homogenate was recovered in the acid-treated KC1 extract. The enzyme was purified by the same procedures as described for isolation of purple acid phosphatase of rat spleen (Hara et aL, 1984). The purification resulted in 40% recovery of enzyme purified to homogeneity with respect to poly- acrylamide gel electrophoresis (Table 1 and Fig. 1). The specific activity of the purified enzyme is c o r n -

O

Fig. I. Polyacrylamide gel electrophoresis of the purified acid phosphatase from rat bone. About 1, 10 and 50/~g of the enzyme were run at pH 4.0 without SDS and stained for (a) activity, (b) protein and (c) sugar, respectively. (d) Protein staining on SDS-gel electrophoresis. Protein was stained with Coomassie brilliant blue. The origin of the

separating gel is indicated by the arrow.

0.2

Bone purple acid phosphatase

i | i

250 300 350 400 SO0

Wavelength (nm)

0,[ $,. o

I !

600 700

Fig. 2. Absorption spectrum of rat bone acid phosphatase.

O.OS

0.04

0.03

0.02

0.01

8 g e g

815

the enzyme in the case of the reducing reagents, of which ascorbic acid at 10-20 mM gave the highest activation rate, similar to that with Fe 2÷. The effects of inhibitors, which have been described with crude enzyme extract (Vaes and Jacques, 1965; Wergedal, 1970) and with the partially purified preparation (Anderson and Toverud, 1979) from bone, on the present enzyme were examined. Ammonium molyb- date and NaF were competitive and noncompetitive inhibitors of the bone enzyme with K~ values of 0.9/~M and 0.7 mM, respectively, and CuSO4 showed a mixed type of inhibition with a Ki value of 6/t M. L-Tartrate (10mM), p-chloromercuriphenylsulfonic acid (10mM), N-ethylmaleimide ( lmM) , HgCI~ (1 mM) and p-chloromercuribenzoic acid (1 mM) were without effect. Almost the same results were obtained with purple acid phosphatase from rat spleen. It should be noted that these SH-reagents did not inhibit the enzymes from bone and spleen of rat when the enzymes were preincubated with them for 10 min at pH 5.5 at 30°C before assay.

Irnmunochemical properties

The immunochemical relationship between the en- zymes from rat bone and spleen was first examined on immunodiffusion test with the anti-splenic purple acid phosphatase IgG (Fig. 3). The IgG formed a single precipitin line not only against the enzyme antigen but also against the bone enzyme, and the lines completely fused with each other. The precipitin line exhibited acid phosphatase activity when the agarose plate was stained for activity. Second, the immunoreactivity of the antibody to the two enzymes was compared by immunoprecipitation experiment. When the immune complex was removed by centrif- ugation, the residual phosphatase activity of the enzymes in the supernatant similarly decreased with increasing amounts of the antibody and the loss of activity from the supernatant was partly recovered in the pellets (Fig. 4).

DISCUSSION Two acid phosphatases, which differ in sensitivity

to tartrate and substrate specificity, have been par- tially purified from rat bone by Anderson and Toverud (1979). In the present study, by procedures almost identical to those developed for isolation of splenic purple acid phosphatase (Hara et al., 1984), we have purified a purple acid phosphatase which is

Table 2. Substratespecificity of rat bone purple acid phos- phatase

Relative Km Substrate

activlty(%) (~)

p-Nitropheny] phosphate 100 1.5

GTP 98 0.91

4-Methylumbelli feryl phosphate 89 2.2

ATP 84 0.38

CTP 73 0.90

CDP 65 - -

Thiamine Pyrophosphate 64 2.5

Phenyl phosphate 62 ].2

Phenolphthalein diphosphate 54 1,3

1-Naphthyl phosphate 46 3.4

ADP 46 - -

Phenolphthalein n~nophosphate 45 2.5

Phosphoenolpyruvic acid 33 0.9]

a-Casein lO 2.0

Phosvitin 7 0.50

The activity for various substrates is relative to that with p-nitrophenyl phosphate as a substrate. The concentrations of the substrate were 5 mM except that phosphoproteins were 1.0mM which is expressed as a concentration of phosphorus in the proteins. Histone, glucose-l-phosphate, fl-gly- cerophosphate and IMP were hydrolyzed at rates less than 1% of the p-nitrophenyl phosphatase activity.

816 TAKASHI KATO et al.

Ca)

(b)

B

s

O

O Fig. 3. Immunodiffusion test of purple acid phosphatases from bone and spleen of rat. Center wells contained anti- IgG against spleen purple acid phosphatase and peripheral wells contained approx 0.1 unit of (B) the bone enzyme and (S) the spleen enzyme• (a) Precipitin line; (b) activity

staining•

electrophoretically homogeneous from rat bone. Ex- amination of molecular weight, substrate specificity and sensitivity to tartrate of the enzyme suggests that the purple acid phosphatase of the present study is the tartrate-resistant enzyme by Anderson and To- verud (1979). The enzyme was here further character- ized as a monomeric and two iron-containing gly- coprotein with a tool. wt of 36,000 and pI value of 7.6. Anderson and Toverud (1979) have suggested the probable presence of sulflaydryl groups in the enzyme because of stimulation by dithiothreitol and inhibition by p-chloromercuribenzoate with their partially purified enzyme preparation. However, dithiothreitol is a much less efficacious activator for the enzyme than Fe z+ and ascorbic acid, as described subsequently by Anderson and Toverud (1982). The inhibition of the present enzyme by

p-chloromercuribenzoate was negligible. The insen- sitivity to the SH-reagent is in agreement with the earlier observations with crude tartrate-resistant acid phosphatase (Wergedal, 1970). Moreover, other SH- reagents such as p-chloromercuriphenylsulfonic acid and N-ethylmaleimide did not influence the enzyme activity of purple acid phosphatases from bone and spleen of rat. Therefore, we have concluded that purple acid phosphatases from the two rat tissues do not have free sulfhydryl groups, at least around their catalytic site.

In addition to the physicochemical properties of the enzyme, its substrate and activator specificity and inhibition by NaF and ammonium molybdate are almost identical to those reported with splenic purple acid phosphatase (Hara et al., 1984). The immu- nochemical identities of the two enzymes also indi- cate that they resemble one another closely.

Acid phosphatases with properties similar to the bone purple acid phosphatase have been purified from tooth (Anderson et al. , 1984), spleen (Hara et al., 1984) and epidermis (Hara et al. , 1985) of rat, bovine spleen (Davis et al. , 1981; Campbell et al., 1978) and pig allantoie fluid (Schlosnagle et al., 1974). However, the physiological roles of purple acid phosphatases in these tissues are unknown except that the enzyme in pig uterus has been proposed to be implicated in iron transfer (Buhi et al., 1982). Histochemical studies have thought that acid phos- phatase in bone tissues is related to bone resorption (Vaes, 1965, 1968; Minkin, 1982). Some acids, such as citric acid and lactic acid, are produced in bone resorption and accumulated within osteoclasts, which must provide the acid environment in which the enzyme works optimally (Marks and Walker, 1976). From the substrate specificity of the enzyme, it may be involved in removal of various organic phos- phoesters in bone resorption. Alternatively, since the enzyme dephosphorylated acidic phosphoproteins but not basic phosphoprotein, and since bone and tooth contain some acidic phosphoproteins which are believed to be important for mineralization (Veis, 1977; Glimcher, 1981; Lee and Glimcher, 1981), the enzyme may play a role in bone resorption by dephosphorylating the endogenous phosphoproteins.

oll i (a)

°°st

/', i 0.06 0.12

IgG concentration (mg)

0,,t~ i (b)

o.os &

k J,

0.06 O.1Z

IgG concentration (mg) Fig. 4. Immunoprecipitation of purple acid phosphatases from spleen and bone with anti-spleen purple acid phosphatase IgG. (a) Spleen enzyme; (b) bone enzyme. • • , activity in the supernatant;

0 O, that in the precipitate.

Bone purple acid phosphatase 817

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