THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255, No. 21, Issue of November 10, pp. 10338-10343, 1980 Printed in U S. A

Purification and Characterization of Acid Phosphatase-1 from Drosophila melanogaster*

(Received for publication, December 24,1979, and in revised form, May 29,1980)

Marc I. Feigen, Mitrick A. Johns$, John H. Postlethwait, and Ronald R. Sederom From the Department of Biology, University of Oregon, Eugene, Oregon 97403

Acid phosphatase-1 (orthophosphoric monoester phosphohydrolase, acid optimum, EC 3.1.3.2), the major phosphatase in adult Drosophila melanogaster, has been purified t o apparent homogeneity. The final prod- uct is a glycoprotein homodimer with a subunit molec- ular weight of about 50,000, as measured by its electro- phoretic mobility in denaturing conditions on poly- acrylamide gels containing sodium dodecyl sulfate. It has a turnover number of 1720 1-naphthyl phosphate molecules hydrolyzed/s by each acid phosphatase-1 molecule at 37”C, pH 5.0. An average fly contains about 5 ng of enzyme.

Pure acid phosphatase-1 displays heterogeneity in isoelectric focusing, with a major band at pH 5.3, The enzyme hydrolyzes a wide variety of phosphate mon- oesters, including AMP, glucose 6-phosphate, and 2- glyceryl phosphate, but not glucose 1-phosphate, ATP, choline phosphate, or phosphoproteins. The maximum reaction rates are different for all substrates, and some substrates appear to inhibit the reaction at high sub- strate concentrations. The Michaelis constants for 1- naphthyl phosphate and p-nitrophenyl phosphate are 79 p~ and 68 p ~ , respectively, at pH 5.0 and 37°C. The optimum pH level for 1-naphthyl phosphate is 4.5. Acid phosphatase-1 is inhibited by L(+)-tartrate (but not D(-)-tartrate), phosphate, and fluoride. The reaction rate increases 2.1-fold for every 10°C rise in tempera- ture. Above 48”C, the rate of thermal denaturation is greater than the rate of the enzyme reaction.

Acid phosphatases (orthophosphoric monoester phospho- hydrolases, acid optimum, EC 3.1.3.2) have been found in virtually every organism studied to date (1). In the fruitfly Drosophila melanogaster, the major acid phosphatase, called acid phosphatase-1, has mainly been studied from the genet- ical point of view. MacIntyre (2) identified and mapped the structural gene coding for acid phosphatase-1 using naturally occurring electrophoretic variants. Subsequently, Bell et al. (3) induced a series of “null activity” mutations at this struc- tural gene using the mutagen ethyl methanesulfonate. The acid phosphatase-1 in these strains is altered in enzymatic activity, electrophoretic mobility, and immunological proper- ties (4). However, the null mutant flies are perfectly viable and fertile under laboratory conditions (5).

* This work was supported by National Institutes of Health Grants GM 21548 (to J. H. P.) and GM 23334 (to R. R. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. + Supported by National Institutes of Health Predoctoral Training Grant GM 07413. To whom correspondence should be addressed.

5 Present address, Department of Genetics, North Carolina State University, Raleigh, NC 27607

Mammals have several acid phosphatases, a few of which have been purified. The two which seem most similar to Drosophila acid phosphatase-1 are the prostate acid phospha- tase, a dimer of total molecular weight 100,OOO inhibitable by L(+)-tartrate (6), and a liver acid phosphatase with similar but not identical properties (6). Acid phosphatases isolated from bovine liver (7) and bovine milk (8) have much lower molecular weights and are not inhibitable by tartrate. Other forms of acid phosphatase found in the liver as well as other organs may or may not be identical to these (9-11). In humans, a syndrome causing death in early infancy results from an inherited lack of lysosomal acid phosphatase in fibroblasts (and presumably other tissues as well) (12).

We are studying the regulation of acid phosphatase-1 in Drosophila as a model system for understanding gene regu- lation in eukaryotes. Besides being tractable genetically, this system has the added advantage that acid phosphatase-1 gene expression is under the control of the steroid hormone ecdy- sone in the larval salivary gland (13) and under the control of juvenile hormone in the ovary (14). In this paper, the purifi- cation of acid phosphatase-1 to apparent homogeneity and the characterization of several of its structural and catalytic properties are reported. Acid phosphatase-1 is very similar to several well characterized acid phosphatases from mammals, and future work on the regulation of Drosophila acid phos- phatase-1 may shed light on the regulation of mammalian acid phosphatases and lysosomal enzymes in general.

EXPERIMENTAL PROCEDURES’

RESULTS

In polyacrylamide gel electrophoresis at pH 8.9, acid phos- phatase-1 migrates toward the anode with a mobility of 0.17 relative to the dye front (Fig. 2). Several other phosphatases with greater relative mobilities can be seen. These all appear to have higher pH optima than acid phosphatase-1. No phos- phatase migrating toward the cathode has been found. The alkaline phosphatases in adult Drosophila have been previ- ously described (15). We have shown that acid phosphatase-1 retains some activity up to pH 9.5 (Fig. 3), and the gel strips stained at high pH shown in Fig. 2b bear a resemblance to the Fig. Ik of Schneiderman et al. (15) even though their gels were run and stained under somewhat different conditions. The slowly migrating band that they call alkaline phospha- tase-1 and described as being weakly present in all develop-

’ Portions of this paper (including “Experimental Procedures,” some “Results,” Figs. 1, 3, 5, and 6, and Table 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockdle Pike, Be- thesda, Md. 20014. Request Document No. 79M2573, cite authorb), and include a check or money order for $2.25 per set of photocopies. Full sized photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

10338

Drosophila Acid Phosphatase- 1 10339

mental stages is probably acid phosphatase-1 judging by cor- responding mobilities and the fact that acid phosphatase-1 is also present throughout the life cycle (16).

The graph in Fig. 3 shows that acid phosphatase-1 is the predominant phosphatase in adult Drosophila. When phos- phatase activity in mutant null activity flies (which have mutations in the structural gene for acid phosphatase-1 that cause the fly to have no acid phosphatase-1 detectable by enzymatic or immunological techniques (4)) is compared to the phosphatase activity in wild type flies, the contribution of acid phosphatase-1 to the total phosphatase activity of the fly can be determined. This contribution can be confmed by measuring the phosphatase activity of pure acid phosphatase- 1 at various pH levels. I t can be seen that at pH 5.0, 90% of the phosphatase activity is due to acid phosphatase-1. Since both wild type and mutant null flies have more phosphatase activity in the range pH 7.5 to 9.0 than pure acid phosphatase- 1, other phosphatases must exist. The graph also shows that acid phosphatase-1 has a pH optimum of about pH 4.5 and that it retains some activity in the alkaline range.

Table I summarizes the results of the purification of acid phosphatase-1 from 287.1 g of flies. Material from the final step was judged to be pure because it showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 4). We estimate that an impurity of 0.5% of the mass of the acid phosphatase-1 would have been visible.

The final product of the purification had a specific activity of 1034 pmol of 1-naphthyl phosphate hydrolyzed/min/mg of protein. This corresponds to a turnover number of 1720 sub-

B A

v- ” A C P f i ,

FIG. 2. Phosphatases in adult Drosophila These electropho- resis gels were run as described under “Experimental Procedures.” The origin is at the bottom of the photograph. Acid phosphatase-1 (ACPH) is indicated. A, each strip represents bands staining at Yl pH unit intervals from pH 4 (on the left) to pH 10 (on the right). B, each strip represents bands staining a t ‘h pH unit intervals from pH 7 to pH 1 0 these strips were overstained to bring out faint bands with high pH optima.

strate molecules hydrolyzed/s by each acid phosphatase-1 molecule a t 37°C and pH 5.0, if one assumes a molecular weight of 100,000.

An average adult fly contains 5 nmol/min of acid phospha- tase-1 activity; this corresponds to 5.0 ng of enzyme/fly. Well fed females can contain up to three times this amount. We calculate that about 0.02% of the total protein soluble in 0.05 M sodium acetate at pH 5.0 is acid phosphatase-I.

The fact that acid phosphatase-1 bound to concanavalin A in the final step of the purification and was eluted with 3.0 M D(+)-InannOSe suggests that it is a glycoprotein.

On sodium dodecyl sulfate-polyacrylamide electrophoresis gels, acid phosphatase-1 runs as a band of molecular weight 50,000, which is consistent with the data of MacIntyre and Dean (17), showing that acid phosphatase-1 is a homodimer, and MacIntyre’s (18) estimation (using velocity centrifugation in a sucrose gradient) of 100,000 for the molecular weight of the native enzyme. Also, Feigen (19), using a calibrated Seph- adex G-200 Superfine column, showed that native acid phos-

A B C D E FIG. 4. Sodium dodecyl sulfate-polyacrylamide gel electro-

phoresis conducted on aliquots of the purification steps. The gels were run as described under “Experimental Procedures” and stained for protein with Coomassie Brilliant Blue R. The origin is at the top of the photograph. A, phosphocellulose peak; B, hydroxyapa- tite peak; C, DEAE-Sephadex peak; D, concanavalin A-agarose peak. The single band is acid phosphatase-1; E , ovary extract. The 3 prominent bands are the yolk proteins with molecular weights of 45,000,46,000, and 47,000.

TABLE I Summary ofpurification steps for acid phosphatase-I

Each purification step was assayed for acid phosphatase-1 and protein as described under “Experimental Procedures” after dialysis overnight against 5 mM sodium acetate, pH 5.0.

Step Total acid phosphatase-1’’ protein activity” purification purification Lase-1 yield

Specific Within-step Cumulative Acid phospha-

mg .fold .fold 04 Homogenate 1500 5900 0.25 1 .o 100 Ammonium sulfate 970 1500 0.65 2.6 2.6 65

730 Phosphocellulose 290 2.5 3.9 9.9 49 Hydroxyapatite 410 7.5 55. 21.8 216 27 DEAE-Sephadex 156 0.76 207 3.8 820 10.4 Concanavalin A-agarose 99 0.096 1030 5.0 4100 6.6

“ Expressed as micromoles of 1-naphthol released/min. ’ Expressed as micromoles of 1-naphthol released/min/mg of protein.

10340 Drosophila Acid Phosphatase-1

phatase ran as a peak of molecular weight 90,OOO while acid phosphatase-1 subunits dissociated by raising the pH level to 10.8 ran at a molecular weight of 45,000.

The Michaelis constants for two important artificial sub- strates were determined by the method of Lineweaver and Burk (20). The K,,, value for 1-naphthyl phosphate was 79 f 18 p~ (average of five determinations) and the K,,, value for p- nitrophenyl phosphate was 68 k 6 p~ (average of five deter- minations). Several specific acid phosphatase inhibitors have been tested for their effects on acid phosphatase-1 in 0.05 M sodium acetate, pH 5.0, at 37°C. Inhibition constants were calculated for competitive inhibitors using the equation given in Dixon (21). I,(+)-Tartrate was a competitive inhibitor of acid phosphatase-1 with a KI value of 64 p ~ ; D(-)-tartrate at 5 mM had no effect. Phosphate ion inhibited acid phosphatase- 1 in a competitive fashion with a KI value of 520 p ~ . Sodium fluoride also inhibited acid phosphatase-1 with a KI value of

Table I1 lists several phosphate monoesters which are sub- strates for acid phosphatase-1 and their relative reaction rates at two substrate concentrations. The Michaelis constants for all substrates are well below 1 mM, because a 10-fold increase in substrate concentration caused at most a %fold rise in reaction velocity. However, the changes in reaction velocity between the two substrate concentrations allows a rough ordering of the K,,, values: glucose 6-phosphate and 2-glyceryl phosphate probably have the highest K,,, values, followed by p-nitrophenyl phosphate and AMP. UMP probably has the lowest K,,, value because the reaction velocity was virtually unchanged between 1 mM and 10 mM. For pyridoxal phos- phate and 1-naphthyl phosphate, evidence exists for inhibition by high substrate concentrations: the reaction velocity for these substrates was less at 10 mM than at 1 mM. At 1 mM, 1- naphthyl phosphate was a slightly better substrate than p- nitrophenyl phosphate. The evidence strongly suggests that the maximum reaction velocities for different substrates are quite different.

In isoelectric focusing gels, pure acid phosphatase-1 displays heterogeneity (Fig. 5). The major band has an isoelectric point at pH 5.3. A secondary band has a more basic isoelectric point, and two more acidic bands can be seen here; on the other gels, up to seven bands which were more acidic than the main band have been observed, but only one band more basic than the main band is ever seen.

10.9 pM.

DISCUSSION

We have found that acid phosphatase-1 is a tartrate-inhibit- able homodimeric enzyme of molecular weight -100,000 with a pH optimum of about pH 4.5. Very similar enzymes have been described in mammals, and it is interesting to see how certain properties are apparently conserved in the long period of time since the ancestors of the arthropods and the verte- brates diverged in evolution. Acid phosphatases, which can accept many different substrates, commonly have several distinct alleles present in natural populations, and for this reason they are considered to be less subject to natural selec- tion than those enzymes which have only a single specific substrate (22). Apparently, the low degree of selection for properties like electrophoretic mobility is not reflected in the functional properties of the enzyme, which appear to be highly conserved in evolution.

At least two different acid phosphatases with properties similar to Drosophila acid phosphatase-1 have been isolated from mammalian tissues: from the prostate (6) and from the liver (6). Both enzymes are tartrate-inhibitable homodimers of molecular weight -100,OOO and low pH optima. In contrast to these enzymes, other acid phosphatases purified from mam-

mals, such as one from bovine liver (7) and another from bovine milk (a), have lower molecular weights, are not in- hibited by tartrate, and have pH optima close to neutrality. The residual phosphatase activity at pH 5.0 in Drosophila strains lacking detectable acid phosphatase-1 is resistant to tartrate, which makes it likely that similar enzymes exist in Drosophila.

The turnover number for acid phosphatase-1 is fairly high compared to several mammalian acid phosphatases (6,23-24). However, differences in enzyme and protein assay conditions can seriously affect the calculation of turnover number: for pure human prostate acid phosphatase, Choe et al. (24) re- ported a value of 2230 pmol of p-nitrophenyl phosphate hy- drolyzed/min/mg of protein, while Van Etten and Saini (6) reported a value of 267 pmol of p-nitrophenyl phosphate hydrolyzed/min/mg of protein.

Drosophila acid phosphatase-1 is very similar to one of the mammalian enzymes in its inhibition constant for L(+)-tar- trate: 64 p~ for Drosophila uersus 140 p~ for human prostate and 0.43 p~ for human liver. In this case, the Drosophila enzyme is more similar to the prostate acid phosphatase than to the liver enzyme. All three enzymes are inhibited about equally by inorganic phosphate: 520 p~ for Drosophila, 860 p~ for human liver (25), and 960 p~ for human prostate (6).

Acid phosphatase-1 hydrolyzes a variety of phospho- monoesters, and the data indicate that the maximum reaction velocities are different for different substrates. This is unlike the case of human prostate acid phosphatase, where the maximum velocities are said to be the same for all substrates (26). In this respect, acid phosphatase-1 is more like the human liver enzyme (23), and it is unlikely that the rate- limiting step for acid phosphatase-l is the release of phosphate ion from the enzyme.

Acid phosphatase-1 does not seem to hydrolyze phosphate groups linked to macromolecules or phosphodiester bonds. In addition, the enzyme does not hydrolyze several phospho- monoesters, including choline phosphate. The ability to hy- drolyze this substrate is perhaps the characteristic which most clearly distinguishes prostate acid phosphatase from liver acid phosphatase in mammals (23). In this respect, Drosophila acid phosphatase-1 is like the liver enzyme.

Acid phosphatase-1 appears to be glycosylated, a property it shares with acid phosphatases from widely different species, such as Neurospora (27) and human prostate (24). Glycosyl- ation may be a general property of lysosomal enzymes (28). Differential glycosylation might provide an explanation for the several acid phosphatase-1 bands seen after isolectric focusing of pure acid phosphatase-1; a similar phenomenon was shown to have such a cause in human prostate acid phosphatase (24).

In conclusion, we have shown that the major phosphatase in adult Drosophila, acid phosphatase-1, is similar to several mammalian acid phosphatases in molecular weight, subunit composition, turnover number, inhibition by tartrate and phosphate, and wide substrate specificity. Kilsheimer and Axelrod (29) have reported that acid phosphatases inhibitable by L(+)-tartrate are widespread through the animal kingdom, although “unequivocal plants” seem to lack such enzymes. This report demonstrates that acid phosphatases in such widely separated groups as insects and mammals are very similar in several properties. This suggests an evolutionary relationship between the enzymes, but further work will be necessary to prove this.

Acknowledgments-We would like to thank Douglas Sears for doing the sodium dodecyl sulfate-gel electrophoresis, Robert Kas- chnitz for suggesting the concanavalin A purification step, Diana B.

Drosophila Acid Phosphatase-1 10341

Smith for access to unpublished data on isoelectric focusing of acid phosphatase-I, Loran Waldron for growing the flies, and Leah Cawvey for doing the typing.

REFERENCES 1. Hollander, V. P. (1971) in The Enzymes (Boyer, P. D., ed) Vol. 4,

2. MacIntyre, R. J. (1966) Genetics 53,461-474 3. Bell, J. B., MacIntyre, R. J., and Olivieri, A. P. (1972) Biochem.

4. Bell, J., and MacIntyre, R. (1973) Biochem. Genet. 10,39-55 5. Ogah, F., and MacIntyre, R. J. (1974) Drosophila Inf. Seru. 49,

6. Van Etten, R. L., and Saini, M. S. (1978) Clin. Chem. 24, 1525-

7. Heinrikson, R. L. (1969) J. Biol. Chem. 244,299-307 8. Andrews, A. T. (1976) Biochim. Biophys. Acta 434,345-353 9. Rehkop, D. M., and Van Etten, R. L. (1975) Hoppe-Seyler’s 2.

10. DiPietro, D. L., and Zengerle, F. S. (1967) J. Biol. Chem. 242,

11. Lundin, L.-G., and Allison, A. C. (1966) Acta Chem. Scand. 20,

12. Nadler, H. L., and Egan, T. J. (1970) N. Engl. J. Med. 282,302-

13. Aizenzon, M. G., Korochkin, L. I., and Zhimulev, I. F. (1975) Dokl.

14. Postlethwait, J. H., and Gray, P. (1976) Deu. Biol. 47, 1216-205 15. Schneiderman, H., Young, W. J., and Childs, B. (1966) Science

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16. Yasbin, R., Sawicki, J., and MacIntyre, R. J. (1978) Dew Biol. 63,

17. MacIntyre, R. J., and Dean, M. R. (1967) Nature 214, 274-275 18. MacIntyre, R. J. (1971) Biochem. Genet. 5,45-56 19. Feigen, M. I. (1978) Master’s thesis, University of Oregon 20. Lineweaver, H., and Burk, D. (1934) J. Am. Chem. SOC. 56,658-

21. Dixon, M. (1953) Biochem. J. 55, 170-171 22. Johnson, G. B. (1974) Science 184, 28-37 23. Saini, M. S., and Van Etten, R. L. (1978) Arch. Biochem. Biophys.

24. Choe, B. K., Pontes, E. J., McDonald, I., and Rose, N. R. (1978)

25. Van Etten, R. L., Waymack, P. P., and Rehkop, D. M. (1974) J.

26. Van Etten, R. L., and McTigue, J. J . (1977) Biochim. Biophys.

27 Jacobs, M. M., Nyc, J. F., and Brown, D. M. (1971) J. Biol. Chem.

28. Barrett, A. J. (1972) Lysosomes, pp. 40-109, North-Holland, Am-

29. Kilsheimer, G. S., and Axelrod, B. (1958) Nature 182, 1733-1734 30. Chandrahjan, J., and Klein, L. (1976) Anal. Biochem. 72, 407-

31. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 32. O’Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021 33. Leaback, D. H., and Wrigley, C. W. (1976) in Chromatographic

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10342 Drosophila Acid Phosphatase1

Step 5 . DEAE-Sephadex chromatoeraphv. A 2.5 x 100 M column was f l l l e d 2/3 f u l l wlth DEAE-Sephadex IPhamacia A-501 whrch had been swelled over- n i g h t ~n 0 . 0 1 M Trl4-HC1 pH 8 . 0 and washed wl th severa l colvmn volumes o f

e l u t e d w l t h a llnedr g r a d l e n t o f 0 - 0 . 5 M NaCl I n 0 .01 I Tr15 H C I pH 8 . 0 this b u f f e r . The d i a l y s a t e f r o m S t e p 4 was l oaded onto t h e colywi and

(125 m 1 o f each stare~nq s o l v t i o n l . fracflons of 8 m l were c o l l e c r e d . Acid o h o s o h a t a s e - 1 e l u t e d at abou t 0 . 4 0 M N d C l lF19. 1 c l . The Peak o f

: I

Drosophila Acid Phosphatase-1 10343

crlbed 1" "Experimental Procedures' and the gel was scanned after s t a m m q F l q . 5 . Isoelectrlc focusing. PUTe aCld phosphatase-1 was run a6 des-

Ylth a llnear transport and a chart recorder at 5 4 0 m. The pH of 1 an for ackd phosphatase-1 In a B e c h ~ model DUR spectrophotometer equipped

slices of a parallel gel soaked ~n water for 60 m n 2s shown.

Temperature Effects. The response of acxd phosphatase-1 actxvzty to temperature I" 0.05 U sodlm acetate pH 5 . 0 is shown I" F l q . 6 . In the lover temloerature ranae acld ohosohatase-1 a c t l v l t v increases 2.1-fold for

C .- I I l I I I 1

\ V

E a8 10-

-

- -

10.2 1 I I I I I I a 10 20 30 40 50 60 70 2 Temperoture, O C

phosphatase-1 ~n 0 . 0 5 H sodlvm acetate pH 5.0 plus 1 mq/ml bovine serm F l q . 6 . Effect of temperature On acld phosphatase-1 activlty. Pure acld

albmln was assayed usmq the method of ~aclntyre 1181 Wlth 1-naphthyl phos- phate as substrate. The data 1s plotted sem~-loqarlthmlcally.

ethyl phosphate. phenolphthalein dlphasphate and IMP are also subs tra tes for acld phosphatase-1 and unpublished 1esu1cs of M O I T I S O ~ and lacintyre reported I" Yasbzn et al. 116) say that adenosme 2'-mnophosphate, adeno- 51ne 3'-mo"~ph~~ph.~,~yt..~~~ 3'-monophosphare, uridine 3'-monaphosphare and tyrOSlne 0-phosphate are BYbStrateS. However we have fovnd that ADP, ATP, sodlum pyrophosphate, chollne phosphate, qlucose 1-phosphate, fructose 1-phosphate, threonlne phosphate, phoIphoCelluloSe, phosvlrln and casem

enzyme IS lmlted to s o r e ohosohate monoesters and does not seem caoable are not substrates for acld phosphatase-1 (data not shovni. Thus, the

Other SUbStTateS. We have found that naphthol AS-BI phosphate, cyano-