Purification and Initial Characterization of Intrinsic Membrane-bound ...

5 downloads 0 Views 3MB Size Report
lipids, the extreme sensitivity to substrate inhibition, and the tetrameric ... inhibitors of this enzyme (15,. 16). We now report the isolation and initial characterization ... at this stage, with no apparent loss of activity. ..... 1 mM; D”c1,2 mM;. H,. 3 mM.
THEJOURNALOF

BIOLOGICAL CHEMISTRY

Vol. 256, No. 14, Issue of July 25, pp. 7262-7268, 1981 Printed in U.S.A.

Purification and Initial Characterizationof Intrinsic Membrane-bound Alkaline Phosphatasefrom Chicken Epiphyseal Cartilage* (Received for publication, November 5, 1980, and in revised form, March 2, 1981)

Grant W. Cyboron and Roy E. Wuthier From the Department of Chemistry, University of South CaroZina, Columbia, South CaroZina 29208

Alkaline phosphatasehas been purified from micro- calcification can be inhibited by compounds which inhibit somes of chicken epiphyseal cartilage by first selec- alkaline phosphatase (7, 10). tively extractingcertainadventitiousproteinswith Alkaline phosphatase in matrix vesicles appears to be very 0.25 M trichloroacetate. The membrane-bound enzyme tightly associated with the membrane as an intrinsic memwas thensolubilized by1% cholate in buffered 33% brane component. It has been isolated previously frommatrix saturated ammonium sulfate and purified by column vesicles and chondrocytes that were obtained by crude-collachromatography on Bio-Gel A-5m, extraction with 1genase digestion of epiphyseal cartilage (11).The extremely butanol, and ion exchange chromatographyon DEAE- low subunit molecular weight(M,= 18,000)and the instability Bio-Gel A. The purified alkaline phosphatase from the of the enzyme following treatment with metal ion chelators, cartilage membrane had a subunit molecular weight of however, suggests that the enzyme had been altered by pro53,000and aholoenzymeweight of 207,000-220,000, teases known to contaminate the crude collagenase (12, 13). indicating a tetramer. While enzyme activity is not destroyed by treatment of the The pHoptima forp-nitrophenylphosphate,ATP,and tissue with proteases, it is likely that this nevertheless dampyrophosphate hydrolysis were 10.3, 9.0, and 8.5, respectively.Values of Vmax(in micromoles/min/mg) ages the noncatalytic portion of the protein. This may affect were 220,3.1, and 0.8, respectively. Substrate inhibition characteristics critical to itsfunction in calcification Alkaline phosphatase has been isolated from rabbit fracwas pronounced at values of pH below 8.5. Inhibition ture-callus cartilage which had not been treated with proof p-nitrophenylphosphate hydrolysis at pH 10.3 teases (14). Two antigenically distinct forms were obtained. showed that phosphate and arsenate were competitive , and However, because of the method employed (ie.extraction of inhibitors (KI = 1.88 and0.15 m ~ respectively) levamisole was an uncompetitive inhibitor (KI = 0.32 whole tissue), it was impossible to ascertain the subcellular m),while L-phenylalanine and ZnClz were mixed in- source of the two forms. While one may have been derived hibitors (K, = 15.8 and 0.02 m ~ respectively). , Inhibition from chondrocytes and the other from matrix vesicles, it is bypreincubation in 1 m~ EDTA wasreversibleby equally possible that one formmay be a soluble and the other, readdition of 0.25 m~ MgClz and 20pM ZnClz. a membrane-associated form of the enzyme. This was not The data indicate that this membrane-bound alkaline explored. phosphatasefromchickenepiphysealcartilage is a Recently, we found that microsomes isolated from chicken Zn2+ andpossibly Mgz+-containing enzyme. Whilethe epiphyseal cartilage by non-protease-dependent methods not of the only were able to support rapid mineralization using physiosubunit molecular weight and kinetic properties enzyme are quite typical of vertebrate alkaline phos- logical synthetic cartilage lymph but also wereenriched &fold phatases, the tightness of binding to the membrane in alkaline phosphatase (15). Mineralization of this fraction lipids, the extreme sensitivity to substrate inhibition, was significantly retarded by inhibitors of this enzyme (15, of the holoenzyme are 16). We now report the isolation and initial characterization and the tetrameric conformation unusual. of this alkaline phosphatase, an integral protein of the microsomal membranes of chicken epiphyseal cartilage. This work represents the fwstsuccessful isolation of the membraneAlkaline phosphatase, at high levels of activity, has long bound enzyme from mineralizing cartilage, without resort to been associated with calcifying tissues; however, its role in the the use of proteases. The enzyme, unlike that isolated from calcification process remains unclear. Recently, it was discov- protease-treated tissue, has a signifcantly higher subunit moered that alkaline phosphatase is a major enzymatic activity lecular weight and different holoenzyme structure (17). The in extracellular matrix vesicles (1-4) which are known to be catalytic activity (pH optima, kinetic behavior, response to closely associated with the initial calcification of epiphyseal inhibitors, etc.) are, however, similar to that of other bone cartilage (5, 6 ) . Matrix vesicles isolated from crude collagen- alkaline phosphatases (14, 18, 19). The enzyme is unusual in ase-digested epiphyseal cartilage can accumulate 45Ca from that it is a tetramer, and displays physical characteristics metastable calcium/phosphate solutions (7-10). Such prepa- typical of an intrinsic membrane protein. rations, however, usually require either elevated pH (7), Ca MATERIALS AND METHODS X Pi ion products (7,8, IO), and/or the addition of high energy phosphate substrates (7-10) tosupport calcification. This Purification of Alkaline Phosphatase * This work was supported in part by Grant AM-I8983 from the United States Public Health Service, National Institute of Arthritis, Metabolism and Digestive Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

All steps prior to the chromatography columns were performed a t 0-4 “C unless otherwise noted. The ammonium sulfate solution was

saturated a t room temperatureand all per cent saturations are relative to this. Chromatographic columns were operated at room temperature. Proteinconcentrations were estimated by the procedure of Lowry et al. (20).

7262

7263

Membrane-bound Epiphyseal Cartilage AlkalinePhosphatase Isolation of Microsomes-Slices of epiphyseal cartilage (100-120 g wet weight) from the proximal end of the metatarsus of 8- to 10week-old broiler-strain chickens were obtained by a published procedure (21). The slices were suspended in 175 ml of an isolation medium composed of10% (w/v) sucrose, 50 mM Tris-HC1 (pH 8.0) and 2.5 mM MgC12, and homogenized for 6 min with aTekmar Tissuemizer equipped with a type SDT-180EN probe. Differential centrifugation was employed as previously described (13). The pellet from the 85,000 X g centrifugation was resuspended in the isolation medium with the aid of a Potter-Elvehjemhomogenizer and adjusted to a total volume of 15 ml. Solubilization-To the microsomal pellet suspension was added, at room temperature and with vigorous stirring, 0.11 volume of 2.5 M sodium trichloroacetate (pH 8) to give a final concentration of 0.25 2. This was stirred for 30 min and then centrifuged a t 85,000 X g for 60 min. The supernatant was discarded and the pellet was resuspended to a total volume of 10 ml with isolation medium. The phosphatase was extracted by dropwise addition of 0.5 volume of saturated ammonium sulfate followed by0.08 volume of 20% (w/v) potassium cholate (pH 8). The mixture was stirred for 15 min and then centrifuged at 85,000 X g for 60 min. The pellets were discarded and the supernatants were saved. The enzyme can be frozen overnight at this stage, with no apparent loss of activity. Gel Filtration-The cholate-soluble material was precipitated by the addition of an equal volume of saturated ammonium sulfate. It was stirred in an ice bath for 15 min and then centrifuged a t 120,000 X g for 60 min. The subnatants were removed by puncturing the bottoms of the tubes. The coagulum was dissolvedin isolation medium (total volume, 5.5 ml), any insoluble material being removed by centrifugation at 120,000 X g for 30 min. The clear supernatant was applied to a Bio-Gel A-5m column (3.2 X 50 cm) which had been equilibrated with a solution composed of0.5% (w/v) cholic acid, 50 m~ Tris-HC1 (pH 8.0), and 2.5 mM MgCI,. The column was eluted with the same buffer and the peak activity fractions were pooled. Ion Exchange Chromatography-The pooled fractions from the Bio-Gel A-5m column were extracted at room temperature with an equal volume of I-butanol by vortexing seven times at 2-min intervals. The aqueous phase was collected and diluted with an equal volume of distilled water. The diluted enzyme was applied to a DEAE-BioGel A column (0.8 X 8 cm) which had been equilibrated with a 50 mM Tris-HC1 (pH 8.0), 2.5 m~ MgCL buffer. The column was washed with the equilibration buffer until the absorbance at 280 nm returned to the base-line, the phosphatase being eluted with a linear gradient composed of the above buffer and 1.0 M NaCl in the same buffer (50 ml each). The active fractions were pooled and concentrated using an Amicon YM-10 membrane.

TABLEI Purification of membrane-bound aZkalinephosphatase from chicken epiphyseal cartilage microsomes Purification step

Protein

mg

Act+ Itya

Specific activity

~;1;;P!;. mg"

1.0 7.51 841 Microsomes 112 1.3 9.84 620 Trichloroacetate 63 insoluble 27.2 16 435 Cholate-soluble 9.2 2.7 69.4 187 Bio-Gel A-5m 0.56 222 124 DEAE-Bio-Gel A "p-Nitrophenylphosphate hydrolysis at pH 10.3 and Methods").

ad 96

~urification

-fold

100 74 52 22 15

3.6

29.6

(see "Materials

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis This was performed in slab gels (130 X 200 X 1.5 mm) essentially as described by OFarrell(24),using gelscontaining 12.5%acrylamide. Samples from each of the various purification steps were lyophilized and dissolved in 50 pl of sample buffer. The cholate-soluble and BioGel eluate fractionswere dialyzed overnight against 100 ml of sample buffer to remove excess salt. Electrophoresis was carried out at 35 mA until the tracking dye (bromphenol blue) was within 5 cm of the bottom. The gel was stained for protein by the method of Blakesley and Boezi (25). Nondissociative Polyacrylamide Gel Electrophoresis A discontinuous system, essentially as described by Davis (26)was used. Samples of purified alkaline phosphatase (10 pg of protein) were applied in the buffer with which they were eluted from the DEAEBio-Gel A column. Other proteins (hemoglobin, catalase, and aldolase) were dissolved in 50 mM Tris-HC1 (pH 8.0). The running buffer was0.192 M glycine, 25 mM Tris. Electrophoresis was carried out using the same type of slab gels, tracking dye (bromphenol blue), and protein staining as described above. Assay of Column Fractions Column fractions were assayed in 0.7 mMpNPP,' 0.125 M 2-amino2-methyl-I-propanol buffer (pH 10.3) a t 37 "C. Assays were started by addition of enzyme and absorbance at 410 nm was monitored continuously with a GCA/McPherson model EU-701 spectrophotometer.

Kinetic Determinations Hydrolysis ofp-Nitrophenylphosphate-Assays were performed at 37 "C in a GCA/McPherson model EU-701 spectrophotometer set at 410 nm. The instrument was operated in the single beam mode with the display updated every 5 s. Absorbance readings were entered into a Wang model 2200s microcomputer until 18 points (high activity) or 54 points (low activity) were collected and the slopes obtained by a linear least squares fit. Activities were converted from AA410/s to micromoles/min by the use of extinction coefficients determined by dilution of a stock solution of p-nitrophenol with the appropriate buffer. Hydrolysis of ATP-This was measured by determining P, released. Tubes containing 1.5 ml of buffer and about 0.5 pg of enzyme protein were preincubated at 37 "C for 10 min. A 5O-pl aliquot of an ATP solution was then added and the incubation continued for 30 rnin. The reaction was terminated by the addition of 50 p1 of 70% perchloric acid. Pi was separated and determined by the isobutanol/ benzene extraction method of Martin and Doty (22). Correction for nonenzymatic hydrolysis was made through the use of simultaneous incubations containing no enzyme. Hydrolysis of PPi-This was assayed by the method described for ATP substituting [32P]PPias substrate. The amount of 32Piproduced was determined by liquid scintillation spectrometry of an aliquot of the phosphomolybdic acid-containing organic phase. Plastictubes were used for all assays to minimize association of PPi and P,with the walls of the tubes. Estimation of Kinetic Constants-The data from the activity measurements were analyzed by a BASIC language translation of the HYPER fit program developed by Cleland (23). All fits were based on 5-8 substrate concentrations with velocities determined in triplicate at each concentration.

Sucrose Density Gradient Centrifugation Purified alkaline phosphatase was layered onto a continuous sucrose density gradient (5-20%, w/v, dissolved in 50 mM Tris-HC1 buffer, pH 8.0, containing 0.1 M NaCl and 2.5 mM MgC12). Centrifugation was carried out for 12 h a t 40,000 rpm using a SW 50.1 rotor. Standards (bovine serum albumin, catalase, and lactic acid dehydrogenase) were run on parallel gradients. Fractions of 0.1 ml were collected and assayed by standard procedures for catalase (27) or lactic acid dehydrogenase (28) activity. The bovine serum albumin peak was detected by protein concentration. Sedimentation data were analyzed by the method of Martin and Ames (29). Chemicals Levamisole, L-phenylalanine, p-nitrophenol (25 mM standard solution), cholic acid, acrylamide, N,N'-methylenebisacrylamide, N,N,N',N',-tetramethylethylenediamine, and 2-amino-2-methyl-lpropanol were obtained from Sigma;pNPP was from Technicon; BioGel A-5m and DEAE-Bio-Gel A were products of Bio-Rad; AcA34 was obtained from LKB Produkter; and [3zP]PP,was purchased from New England Nuclear. AU other chemicals used were of reagent grade and were supplied by Fisher. RESULTS

Purification of Membrane-bound Alkaline PhosphataseTable I summarizesa typical preparationof membrane-bound alkaline phosphatase from 97 g (wet weight) of chicken epiphyseal cartilage. An approximately 30-fold purificationfrom I

The abbreviation used is: pNPP, p-nitrophenylphosphate.

7264

Cartilage Alkaline Phosphatase

Membranebound Epiphyseal

the microsomal pellet was achieved. Recovery of activity from the microsomal pellet ranged from15-25%. The fact that only a 30-fold purification was required to achieve homogeneity suggests that a substantial proportion of the protein in the microsomal pellet was alkaline phosphatase. Since the microsomal pellet itself provided a 5-fold enrichment of alkaline phosphatase activity relative to thesupernatant from the 600 X g centrifugation ( E ) , the total purification from the tissue was greater than 150-fold. A typical elution profile from Bio-GelA-5m is shown in Fig. 1. The hydrolytic activity did not correspond to any discrete peak with absorbance at 280 nm. A large degree of purifkation was obtained at this stage, however. Extraction with 1-butanol could not precede this step as the enzyme was then inactivated. In addition, it was critical that cholate be included in the eluting buffer to prevent irreversible aggregation of the enzyme and concomitant loss of activity. Ion exchange chromatography on DEAE-Bio-Gel A (Fig. 2) resulted in the removal of the final contaminating proteins. Dilution of the 1-butanol-extracted aqueous phase was necessary in order for the enzyme activity to bind to thecolumn. The inability of the enzyme to bind appeared to be caused by 0.15 r

1

II

the presence of the saturating levels of butanol in the aqueous phase. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of aliquots from each of the purification steps are shown in Fig. 3. There was no obvious reduction in the number of protein bands following extraction of microsomes with 0.25 M trichloroacetate; however, incorporation of this step wasfound to becrucial. Its omission resulted in the presence of several contaminating proteins after the DEAEBio-Gel A step and these could not be removed by varying the conditions for elution from the columns or by inclusion of a concanavalin A-Sepharose chromatography step. Physical Characterization-Calibration of the sodium dodecyl sulfate-polyacrylamidegel electrophoresis yielded a molecular weight (M,) for the subunits of this alkaline phosphatase of53,000, a value near the range reported for other vertebrate alkaline phosphatases (Mr = 55,000-70,000) (17). The molecular weightof the holoenzyme wasestimated to be 207,000 by sucrose-density gradient ultracentrifugation, and 220,000 by gel filtration on AcA34 (data not shown). These findings suggest that the enzyme exists as a tetramer in its native configuration. Electrophoresis of the enzyme under nondissociative conditions was also performed usinga graded series of acrylamide concentrations. When these results were analyzedthrough the use of Ferguson plots (30), an apparent molecular weight of approximately 80,000 was obtained (Fig.4). The low molecular weight observed by this method may be caused by a high charge density for the enzyme and/or the loss of metal ions 1

2 ””

3

4

5

6

7

.

Y

Y

m

2

0.05

m

0.00

Iln

FRACTION N u a m

FIG. 1. Elution of alkaline phosphatase from Bio-Gel A-5m column (3.2 X 50 cm). Absorbance a t 280 run was monitored continuously with an ISCO Model UA-5 monitor with a type6 optical unit. Sample was applied and eluted, and fractions were assayed for alkaline phosphatase activity as described under “Materials and Methods.”

0.075

t FRACTIONWvlara

FIG. 2. Elution of alkaline phosphatase from DEAE-Bio-Gel A column (0.75X 8 cm). Absorbance at 280 nm was monitored and fractions assayed for alkaline phosphatase as described in the legend to Fig. 1. Sample application and elution were as described under “Materials and Methods.” The arrows indicate the fractions where the wash and gradient, respectively, were started.

FIG. 3. Sodium dodecyl sulfate-polyacrylamide electrophoresis of samples from various stages of alkaline phosphatase purification.Samples were prepared and handled as described under “Materials and Methods.” Samples and amounts loaded were: Lane 1, standards (phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme, 2.5 pg each); Lane 2, microsomal fraction, 100 pg of protein; Lane 3, trichloroacetate-insoluble fraction, 100 pg of protein; Lane 4, cholatesoluble fraction, 100 pg of protein; Lane 5, Bio-Gel A-5m eluate, 50 pg of protein; Lane 6, DEAE-Bio-Gel A eluate, 40 pg of protein; Lane 7, standards. The arrow designates the tracking dye front. The principle band in Lane 6 (M,= 53,000) is the membrane-bound alkaline phosphatase. Also detectable in Lane 6 are several minor bands which sometimes appear in final preparations.

Membrane-bound Epiphyseal Cartilage Alkaline Phosphatase 0

7265

100

-0.5 80

-4

-1.0 Y

f

w 0 _1

60

Y

0

-1.5

M 40 5

-2.0

"

r Q

20 % ACRYLAIIIDE

PH

FIG. 5. Effect of pH on V,, of chicken epiphyseal cartilage alkaline phosphatase. Assay conditions were as described under

0

0.1

0.2

-

0.3

KR

.,

FIG. 4. Ferguson plot for determination of molecular weight by polyacrylamide gel electrophoresis. Electrophoresis was performed as described under "Materials and Methods." A, mobilities were determined relative to the migration of bromphenol blue as thyroglobulin (Mr = 660,000); A, catalase (M, = tracking dye. 244,000); 0, aldolase ( M , = 158,000);0, hemoglobin (M, = 64,000); and 0,alkaline phosphatase. B , plots of the slopes of lines in A versus

"Materialsand Methods" for the three substrates shown. Assays were buffered with 0.25 M AMP (2-amino-2-methyl-I-propanol)for pH9.011.0 and with Tris for pH 7.0-9.0. There was no significant difference between the two buffers at pH 9.0.Points represent V,, at indicated pH, expressed as a percentage of that observed at the pH of highest pH 8.5, 0.8 pmol of PPi activity. Maximal values were: PP, (M), pH 9.5, 3.1 pmol of Pi rehydrolyzed.min".mg"; ATP (A-A), pH 10.3, 220 pmol hydrolyzed. 1eased.min-l.mg"; pNPP (-), min" mg of protein".

.

I

I

I

I

I

. 3 ' 2 "

normally part of the enzyme structure (17), allowing the subunits to dissociate. Kinetics-The pH profiles for ATP, PPi and pNPP hydrolyses are shown in Fig. 5. As expected, the pH optimum was dependent on the substrateused. With pyrophosphate the pH optimum was 8.5. A t this pH the Vmaxwas 0.8 pmol of PPi hydrolyzed/min/mg of protein, the K, was 85 p ~ The . most alkaline pH optimum (10.3) was obtained with pNPP. For this substrate the Vmaxwas 220 pmol/min/mg of protein, with a K,,, of 0.7 mM. The optimum for ATP was intermediate at pH 9.0, its values for V,,, (3.1 pmol/min/mg of protein) and K,,, (0.23 mM) also being intermediate between those for PP, and pNPP. Substrateinhibition with PPi was quite marked substrate concentrations had to be maintained below 100 p~ to obtain linear reciprocal plots. With ATP, concentrations up to 0.5 mM could be used. When pNPP hydrolysis was measured, substrate inhibition was not observed with 1 mM substrate, except when the pH was below 8.5. At these lower pH values the highest concentration used was 50 p ~ . The apparent optimum pH was affected by the concentration of the substrate, as is shown in Fig. 6 for hydrolysis of pNPP. Thiswas due to several effects, primarily the increase in substrate inhibition and the decrease in K,,, at decreasing pH. In Fig. 7 , A and C, the log ( Vmax/Kn)and (-)log K,, respectively, are plotted as functions of pH. The K , forpNPP hydrolysis decreased by almost 4 orders of magnitude between pH 10.5 and 7.5. In the same range Vmaxchanged byonly slightly more than 2 orders of magnitude (Fig. 7 B ) . These effects combine to shift the apparent pH optimum toward neutrality at low substrate concentrations. Plots of log ( VmaX/ K,) uessus pH are generally considered to reflect two features

I

I

9.0

9.5

,

1

10.0 PH

10.5

I

11.0

FIG. 6. Effect of substrate concentration on apparent pH optima ofpNPP hydrolysis by cartilage alkaline phosphatase. Hydrolysis was measured in 0.25 M AMP buffer as described under 5.0 "Materials and Methods." Concentrations ofpNPP were: o"-o, 1.0 a; H, 0.6 mM; A-A, 0.4 mM; O " - O , 2.5 mM; C l " - U , mM; and A-A, 0.1 mM.

of the interaction between the substrate and the enzyme: 1) the inflection points occur at the apparent pK, of the functional groups involved, and 2) the magnitude of the slope reflects the number of groups involved in substrate binding. The observed plots (Fig. 7A) are consistent with the involvement of two ionizable groups. One is evident in the plots for all three substrates with an apparent pK, between 8 and 8.5. A second group with a pK, between 9.5 and 10.0 can be seen in the plot for pNPP. Since these two apparent pK, values are near each other, the values should be considered to be upper and lower limits for the lower and upper pK, values, respectively. The effects of various inhibitors of pNPP hydrolysis are summarized in Table 11. Sodium phosphate was a competitive inhibitor (KI = 1.88 mM, Fig. 8 ) as was sodium arsenate, a

Membrane-bound Epiphyseal Cartilage Alkaline Phosphatase

7266

FIG. 8. Inhibition of p-nitrophenylphosphate hydrolysis by inorganic phosphate. Hydrolysis was determined and kinetic parameters were estimated as described under “Materials and Methods.” A, Lineweaver-Burk plots of hydrolysis data. Phosphate concentrations used were: -, 0 mM; -, 1 mM; D ” c 1 , 2 mM; H, 3 mM. B, slopes of Lineweaver-Burk plots uersus phosphate concentration. The line is drawn according to a linear least squares fit. 7.0

9.0 PH

8.0

10.0

11.0

FIG. 7. Effect of pH on kinetic parameters of cartilage alkaline phosphatase. Kinetic parameters were estimated by a nonlinear least squares fitting procedure as described under “Materials and Methods.” A, log (V,,/K,,,) as a function of pH. V, values are in micromoles. min” .mg”; IC, values are in millimolar concentrations except where marked. -, p-nitrophenylphosphate hydrolysis; M, pyrophosphate hydrolysis (K, in micromolar concentrations); A-A, ATP hydrolysis. All values for ATP have been increased by 1 to permit separation from pyrophosphate values. B, Log V,,,, as a function of pH. Values of Vmaxare in micromoles. min” . mg”. To avoid overlap, as above, values of log VmaX+ 1 and log V, + 2, respectively, were plotted for pyrophosphate and ATP. C, effect of pH on -log IC,,,.All K , values are in molar units. For clarity, to prevent overlap with data for other substrates, -log K , + 2 was plotted for pyrophosphate.

TABLE I1 Reversible inhibitors of membrane-bound alkaline phosphatase from chicken epiphyseal cartilage Inhibitor ~~~~~~~

~~

G ”

Type of inhibition

-5x

mM

1.88 0.15 0.32 15.8 0.019

50

Competitive Competitive Uncompetitive Mixedb Mixedb

Sodium phosphate Sodium arsenate L-Tetramisole L-Phenylalanine Zinc chloride a Inhibition of p-nitrophenylphosphate hydrolysis at pH 10.3 (see “Materials and Methods”). Mixed uncompetitive and noncompetitive inhibition.

structural analog of Pi. It was a considerably more potent inhibitorthan Pi ( K I = 0.15 mM; Fig. 9). Levamisole (Ltetramisole) was an uncompetitive inhibitor (Fig. 10). Plots of 1/Vmaxuersus [levamisole] were linear and yielded KI a of 0.32 KIM. Inhibition by L-phenylalanine was of mixed non- and uncompetitive type. A plot of l/Vmaxuersus [phenylalanine] was also linear and extrapolatedto a K I of 15.8 mM. The most

x

40

30

-

x

>

3

20-

/*’ */*

10 I

-0.25

0

1

I

I

I

0.25

0.50

0.75

1.0

[ARSENATE]

(M!)

FIG. 9. Inhibition of pnitrophenylphosphate hydrolysis by arsenate. For details of hydrolysis conditions and methods for estimating kinetic parameters, see “Materials and Methods.” A, Lineweaver-Burk plots of p-nitrophenylphosphate hydrolysis. Arsenate concentrations were: w, 0 mM; 17”--0,0.25 mM; M, 0.50 mM; t”., 0.75 mM. B. slopes of Lmeweaver-Burk plots uersus arsenate concentration. The line is drawn using linear least squares tit.

Membrane- bound Epiphyseal Cartilage Alkaline Phosphatase

7267

alkaline phosphatase from pig kidney with subunits of M, = 39,000 has been described by Wachsmuth and Hiwada (33). However, Ramaswamy and Butterworth later reported this enzyme to be a dimer with a total molecular weight of about 185,000 (34). While it is possible that aggregation of our cartilage enzyme may have occurred, as is known to happen with the placental enzyme (35), this appears unlikely considering the low protein concentrations employed (less than 50 pg/ml). Further, 0.1 M NaCl was included in the buffers during determinations of molecular weights by the hydrodynamic method to minimize the possibility of aggregation. In an experiment where the salt was omitted, alkaline phosphatase activity eluted from a Bio-Gel A-15m column in several distinct peaks corresponding approximately to multiples of the tetrameric enzyme, the largest appearing to be of 16 subunits. In the presence of 0.1 M NaCl the enzyme activity eluted as a single symmetrical peak corresponding to thetetrameric configuration. As noted in the introduction, we feel that several characteristics of matrix vesicles isolated from crude collagenase-digested epiphyseal cartilage indicate that they have been damaged by this protease treatment. These include the necessity of high Ca X Pi ion products ( 7 ) and/or high pH (7, 8, 10) and/or the presence of alkaline phosphatase substrates (7-10) to initiatesignificant 45Cauptake by the isolated vesicles. The alkaline phosphatase isolated from such vesicles also revealed FIG. 10. Inhibition ofpnitrophenylphosphate hydrolysis by evidence of proteolytic damage (11). For example, the active Levamisole. For details of hydrolysis conditions and methods for estimating kinetic parameters, see “Materialsand Methods.” A, Line- subunits had a very low molecularweight (Mr = 18,000), weaver-Burk plots of the hydrolysis data. Levamisole concentrations although the total weight of the active and inactive subunits were: H, 0 mM; o ” - o , 0.25 mM; C+-U, 0.50 mM; M,(63,000, assuming a 1:1 relationship) would be well within the 1.0 mM. B, plot of the reciprocal of the apparent VmaX as a function of range of values for mammalian alkaline phosphatases. FurLevamisole concentration.The line is drawn using linear least squares ther, the instability reported for the enzyme when Zn2+ was fit. removed by EDTA treatment (31-33) probably also stems from proteolytic scission of the enzyme. Removal of the metal potent inhibitor tested, however, based on plots of l/Vmax ion apparently allows the enzyme fragments to irreversibly versus inhibitor concentration, was ZnCl2 with a Kr of 0.02 denature, much as occurs with insulin whenits disulfide bonds mM. are cleaved. It should be recalled that the membrane-bound Neither MgC12 nor CaClz wasfound to have any significant enzyme we report in this paper couldbe renaturedafter effect on the activity of the purified epiphyseal alkaline phos- EDTA treatment by the readdition of Zn2+and M$’. Our phatase at concentrations up to 1 mM (data not shown). findings thus suggest that themolecular weightof the epiphyPreincubation of the enzyme (approximately 25 pg protein/ seal cartilage alkaline phosphatase is not greatly different ml) with 1 mM EDTA at pH 8.0 for 15 min at room tempera- from that of enzymes isolated from other vertebrate sources, ture resulted in the loss of greater than 95% of the activity. in agreement with findings of Arsenis et al. (14). However, approximately 70% of the activity could berestored In several respects the phosphatase I from rabbit cartilage by the addition of 20 PM ZnClp (an amount equimolar with reported by Arsenis et al. (14) is similar to the membranethe EDTA transferred from the preincubation) and 0.25 mM bound enzyme whichwe have isolated from chickencartilage. MgC12to theassay mixture. MgC12alone was not sufficient to Assuming a dimeric structure for their enzyme (no subunit restore activity. This observation is in marked contrast with determination was reported), aM , of 63,000would beobtained the studies of Fortuna et al. (31,32)in which their chondrocyte for the subunits of their enzyme. This would be inreasonable and matrix vesicle alkaline phosphatases were irreversibly agreement with the value reported here, the small difference inactivated by EDTA. perhaps being a species variation. Substrate specificities of both enzymes are similar in that both are active toward PP,, DISCUSSION ATP, and pNPP with similar pH optima. Nevertheless, a This paper describes the isolation and characterization of a significant difference is apparent in that their enzyme required membrane-bound alkaline phosphatase from microsomes pre- the addition of M$+ to the assay medium, while the alkaline pared from mineralizing epiphyseal cartilage. The enzyme is phosphatase we isolated from chicken epiphyseal cartilage tightly bound and can only be solubilizedby treatment of the was fully active in the absence of exogenous Mg2’ ions. membranes with detergents. Since neither treatment with This membrane-bound enzyme showed kinetics quite typihigh ionic strength salt solutions nor the chaotrope, trichlo- cal of other alkaline phosphatases. For example, the pH roacetate, solubilizes it, thisenzyme is properly classed as an optimum varied with the type of substrate, ranging from pH intrinsic membrane protein. 10.3 for pNPP to about 8.5 for PPi. Inactivation by preincuThe epiphyseal cartilage alkaline phosphatase which we bation with EDTA and reconstitution by the addition of ZnCl2 describe here is a tetramer composed of apparently identical suggest that the enzyme is a Zn2+-containingprotein. As is subunits with a M , of 53,000. While this subunit molecular generally true for alkaline phosphatases, substrate inhibition weight is similar to thatreported for other vertebratealkaline was noted with this enzyme. It appears, however, that this phosphatases (17), the oligomeric structure is significantly alkaline phosphatase is more sensitive to inhibition by PPi different in that thetypical structure is a dimer. A tetrameric than are the other reported cartilage alkaline phosphatases.

7268

Membrane-bound Epiphyseal Cartilage AlkalinePhosphatase

We observed obvious inhibition at pH 8.5 when PPi concentrations exceeded 100 PM. By contrast, Fortuna et al. (31, 32) saw inhibition only at concentrations above 10 mM, and Arsenis et al. (14) routinely assayed pyrophosphatase activity using 2 mM PPi at pH 8.5. Under these conditions, our enzyme showed barely detectable activity. This difference may be of physiological significance since PPi is a known inhibitor of hydroxyapatite crystal growth (36). In conclusion, because of the apparent close association between the membrane-bound cartilage alkaline phosphatase and the mineralization induced by matrix vesicles (15), any studies of the physical properties of this enzyme must be done on an enzyme that is fully preserved during the isolation procedure. Unfortunately, in the previously reported matrix vesicle alkaline phosphatases (11, 14, 18), the proteases present in the crude collagenases required for release of the matrix vesicles from which the enzyme was isolated make it almost certain that these had undergone some degree of proteolysis. That this is indeed true is suggested by differences we now report in the molecular weight,stability, and kinetic behavior of this membrane-bound cartilage enzyme. 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. Kessler, E., and Yaron, A. (1973) Biochem. Biophys. Res. Commun. 50,405-412 14. Arsenis,C., Rudolph, J., andHackett, H. M. (1975) Biochim. Biophys. Acta391,301-315 15. Wuthier, R. E . , Linder, R. E., Warner, G. P., Gore, S. T., and Borg, T. K. (1978) Metabolic Bone Disease and Related Research 1. 125-136 16. Hubbard, H. L., Warner, G. P., and Wuthier, R.E. (1980) 2nd

Annual Meetingof the American Society for Bone and Mineral Research, Abstr. 87, p. 21A, Washington, D. C. 17. McComb, R. B., Bowers, G. N., Jr., and Posen, S. (1979)Alkaline Phosphatase, Plenum Press, New York 18. Kahn, S. E., Jafri, A.M.,Lewis,N. J., and Arsenis,C. (1978) Calcif. Tissue Res.25, 85-92 19. Farley, J. R., Ivey, J. L., and Baylink, D. J. (1980) J. Biol. Chem.

255,4680-4686 20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275 21. Wuthier, R. E. (1979) in Skeletal Research (Simmons, D. J., and Kunin, A. S., eds) pp. 121-138, Academic Press, New York 22. Martin, J. B., and Doty, D. M. (1949) Anal. Chem. 21,965-967 23. Cleland, W.W. (1979) Methods Enzymol. 63, 103-138 24. O'Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021 25. Blakesley, R. W., and Boezi, J. A. (1977) Anal. Biochem. 82,580582 REFERENCES 26. Davis, B. J. (1964) Ann. N . Y. Acad. Sci. 121, 404-427 Matsuzawa, T., and Anderson, H. C. (1971) J. Histochem. Cyto- 27. Beers, R. F., Jr., and Sizer, I. W. (1952) J. Biol. Chem. 195, 133140 chem. 19,801-808 Ali, S. Y., Sajdera, S. W., and Anderson, H. C. (1970) Proc. Natl. 28. Schwert, G. W., and Winer, A. D. (1963) in The Enzymes (Boyer, P. D., Lardy, H., and Myrback, K., eds) 2nd Ed, Vol. 7, pp. 127Acad. Sci. U.S. A. 67,1513-1520 148, Academic Press, New York Maieska. R. J.. and Wuthier. R. E. (1975) Biochim. Bioohrs. Acta 29. Martin, R. G., and Ames, B. N. (1961) J. Biol. Chem. 236, 1372391.5i-60 1379 Felix. R.. and Fleisch. H. (1976) Calcif. Tissue Res. 22. 1-7 30. Ferguson, K. A. (1964) Metab. Clin. Exp. 13,985-1002 Anderson, H.C. (1969) J. 'Cell Biol. 41,59-72 31. Fortuna, R., Anderson, H. C., Carty, R. P., and Sajdera, S. W. Bonnucci, E. (1970) Z. Zellforsch. 103, 192-217 (1979) Biochim. Biophys. Acta 570,291-302 Ali, S. Y., and Evans, L. (1973) Biochem. J. 134,647-650 Hsu, H. H. T., and Anderson, H. C. (1977) Biochim. Biophys. 32. Fortuna, R., Anderson, H. C., Carty, R. P., and Sajdera, S. W. (1978) Calcif. Tissue Znt. 30, 217-225 Acta 500, 162-172 Felix, R., Hemnann, W., and Fleisch, H. (1978) Biochem. J. 170, 33. Wachsmuth, E. D., and Hiwada, K. (1974) Biochem. J. 141,273282 681-691 34. Ramasamy, I., and Butterworth, P. J. (1974) Biochim. Biophys. Vaananen, H. K. (1980) Calcif. Tissue Znt. 30,227-232 Acta 370,477-486 Fortuna, R., Anderson, H.C., Carty, R. P., and Sajdera, S . W. (1978) Metabolic Bone Disease and Related Research 1, 161- 35. Gottlieb, A. J., and Sussman, H. H. (1968) Biochim.Biophys. Acta 160, 167-171 168 Peterkofsky, B., and Diegelmann, R. (1971) Biochemistry 10, 36. Fleisch, H., Russell, R. G . G., and Straumann, F. (1966) Nature (Lond.)212,901-903 988-994 "