The structural basis of the anomalous kinetics of rabbit liver aryl sulfatase A

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 203, No. 1, August, pp. 11-24, 1980

The Structural Basis of the Anomalous Kinetics of Rabbit Liver Aryl Sulfatase A1

ABDUL WAHEED AND ROBERT L. VAN ETTEN2

Chemistry Department, Purdue University, West Lafayette, Indiana 47.907

Received September 26, 1979

Rabbit liver aryl sulfatase A (aryl sulfate sulfohydrolase, EC 3.1.6.1) is inactivated during the hydrolysis of nitrocatechol sulfate and the rate of formation of turnover-modified aryl sulfatase A depends on the initial velocity of the enzymatic reaction. Organic solvents such as ethanol and dioxane favor the anomalous kinetic behavior. The turnover-modified enzyme can apparently be reactivated by arsenate, phosphate, pyrophosphate, and sulfate in the presence of nitrocatechol sulfate. The apparent dissociation constants of these ions in the reactivation of the enzyme are similar to their K, values. Sulfite, which is a competitive inhibitor, does not reactivate the turnover-modified enzyme. Thus, all known activators are competitive inhibitors but not all competitive inhibitors are effective as activators. Inactivation of aryl sulfatase A during hydrolysis of 35S-labeled substrate at pH values near the pH optimum (pH 5-6) is accompanied by the incorporation of radioactivity into the protein molecule and the turnover-modified enzyme is thereby covalently labeled. The stoichiometry of the incorporation of radioactivity corresponds to 2 g atom of sulfur per mole of enzyme monomer, or 1 g atom of sulfur per equivalent peptide chain. It is also shown that isolated turnover-modified rabbit liver aryl sulfatase A has lost approximately 76% of its secondary structure as compared to the native enzyme. The specific activity of the inactive enzyme is also decreased by 82%. Turnover-modified rabbit liver aryl sulfatase A is partially reactivated by sulfate ions in the presence of nitrocatechol sulfate. However, circular dichroism measurements and fluorescence spectra of the isolated “reactivated” turnover-modified enzyme indicate only a further loss of secondary structure. The specific activity of this “reactivated” enzyme is in fact decreased. The loss in secondary structure and the enzyme activity of the “reactivated” aryl sulfatase A is prevented in the presence of sulfate ions. Turnover-modified rabbit liver aryl sulfatase A behaves as a very fragile molecule.

Mammalian sulfatases (aryl sulfate sulfo- delayed at lower temperatures. Natural hydrolase EC 3.1.6.1) are slowly inactivated substrates such as cerebroside sulfate or during the catalytic reaction and the inac- several other substrates of aryl sulfatase tive enzyme can apparently be partially such as L-ascorbic acid. sulfate and methyl- reactivated by either of the reaction products umbelliferone sulfate having turnover rates (nitrocatechol or sulfate ion) in the pres- lower than nitrocatechol sulfate, apparently ence of substrates (l-9). The anomalous do not give rise to the anomalous kinetic kinetic behavior of aryl sulfatase A is pre- behavior (6, 13-15). vented in the presence of sulfate, phosphate, Based on kinetic data, Baum and Dodgson and pyrophosphate ions (2, 4, S-10) which (2), Nicholls and Roy (9), and Stinshoff are competitive inhibitors of the enzyme (3) proposed a mechanistic scheme in an molecule (5, 11). Baum and Dodgson (2) and attempt to rationalize the anomalous kinetics Roy (12) observed that the substrate- induced inactivation of aryl sulfatase is

of the enzyme. According to their hypothesis, a new site of the enzyme molecule is exposed during hydrolysis of substrate and

1 Supported by Research Grant GM 22933 from this site can bind substrate, reaction prod- the USPHS National Institute of General Medical ucts, or certain other ions. The enzyme Sciences. molecule becomes inactive if substrate

* To whom correspondence should be addressed. binds to the new site. Nicholls and Roy,

11 0003-9861/80/090011-14$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

12 WAHEED AND VAN ETTEN

using spectrophotometric methods, could not detect the presence of substrate or phenolic product on the modified enzyme (9). Roy (12) has recently advanced a new hypothesis based on kinetic data. He suggested that the inactivation of the enzyme proceeds through an enzyme-sulfate ion complex during the catalytic reaction. How- ever, a chemical basis for such an enzyme- sulfate ion reaction was not proposed.

Comparatively little is known about the structure of the turnover-modified aryl sulfatase A and the “reactivated” enzyme. Lee and Van Etten (5) concluded from their results of reactivation of turnover-modified enzyme at different temperatures that only a small net structural change took place during the reactivation of the modified enzyme. Roy (12) observed that ox liver aryl sulfatase A bound to Sepharose is inactivated more slowly than the soluble enzyme. This suggested that structural changes occurred during the inactivation of the enzyme and these changes are minimized in Sepharose- bound aryl sulfatase A. More recently Rybarska-Stylinska and Van Etten (16) reported the loss of antigenic determinants accompanying formation of the turnover- modified aryl sulfatase A and it was concluded that either conformational changes in the protein molecule or else covalent modifications of residues associated with the antigenic determinant sites were occurring. The present work describes in detail experiments which establish that an incorporation of 35S from labeled substrate takes place during the formation of turnover-modified aryl sulfatase A and that there is an accompanying loss of secondary structure from the enzyme molecule. A communication describing related experiments has been published (17).

EXPERIMENTAL PROCEDURES

Materials. Rabbit liver aryl sulfatase A was isolated by the general procedure of Lee and Van Etten (5) with some modifications (18). Sephadex G-25 was obtained from Pharmacia Fine Chemical Company. Aquacide was obtained from Calbiochem. Other reagents were of analytical grade. Distilled deionized water was used throughout.

The dipotassium salt of nitrocatechol was purchased from Sigma Chemical Company. The potassium salt

of nitrocatechol [35S]sulfate was synthesized by the procedure of Fendler and Fendler (19) with some modification. Chlorosulfonic acid (3.8 mmol) containing 0.38 mmol of [Yl]chlorosulfonic acid (3.8 mCi) was added dropwise to a stirred solution of NJ-di- methylaniline (5.0 ml) in 5.0 ml of CSz at room temperature. After 15 min of stirring of the reaction mixture, 3.8 mmol of nitrocatechol was added and reaction mixture was stirred for 20 hat room temperature. Five milliliters cold 5 M KOH was added to the reaction mixture under stirring in an ice bath. The potassium salt of nitrocatechole sulfate was precipitated by addition of 10 vol of ethanol. The precipitate thus obtained was washed twice with ethanol and dried under vacuum. The crude nitrocatechol sulfate was dissolved in minimum volume of water and free sulfate was precipitated by addition of barium acetate (the amount of barium acetate used was approx 10% of the total weight of crude potassium salt of nitrocatechol sulfate present). The barium sulfate which formed was centrifuged off. The remaining solution was adjusted to ethanol concentration of 90% (V/V). The resulting precipitate of nitrocateehol sulfate was recovered and dried. The nitrocatechol sulfate preparation was twice crystallized from 90% ethanol. The purity of the labeled nitrocatechol sulfate was established by spectrophotometric analysis and by elemental analysis of unlabeled nitrocatechol sulfate synthesized by the same procedure. The specific activity of the resulting nitrocateehol sulfate was 2.264 x lo5 dpmf~mol(l.519 x lo5 cpm/pmol). This material was diluted with cold substrate and recrystallized. The resulting specific activity was 1.735 x lo* dpm/Fmol (1.1643 x lo4 cpm!pmol). The latter preparation was used in the present experiments.

Protein concentration determination. The protein concentration of highly purified or homogeneous enzyme was determined using an extinction coefficient, E 1% 1 cm of aryl sulfa&e A determined in sodium acetate buffer pH 4.5, Z = 0.1 or in Tris-HCI buffer pH 7.5, Z = 0.1 at room temperature. Absorption specta were recorded on a Beckman Acta V spectro- photometer using 1 cm cells. Absorbances at 280 nm were used to calculate the extinction coefficient of homogeneous enzyme using the protein concentration data determined from amino acid analysis results. The . . extmction coefficients E :7m at 280 nm and pH 7.5 or pH 4.5 were 10.9 and 10.0, respectively. Lowry’s procedure (20) was used for routine protein concentration determination.

Enzyme assay and kinetics. Enzyme activity was determined using 5 mM nitrocatechol sulfate in 0.5 M sodium acetate pH 5.5 at 37°C. The assay mixture (2.0 ml) containing a known amount of enzyme was incubated for 3-5 min and the reaction was quenched with 1 ml of 0.2 M NaOH. The reaction product was measured at 515 nm on a Gilford Model 2000 spectro- photometer. A molar extinction coefficient of nitro-

ANOMALOUS KINETICS OF RABBIT LIVER ARYL SULFATASE A 13

catechol, EM = 12,600 was used in calculations of the turnover number of the enzyme reaction.

The rate of hydrolysis of nitrocatechol sulfate was determined by a discontinuous method. The reaction mixture and the enzyme solutions were equilibrated at the required temperature and mixture together. At different time intervals 0.2 ml-aliquots were removed and added to 2 or 2.5 ml of 0.2 M NaOH in order to quench the reaction. The absorbance of free nitroeatechol was measured at 515 nm using an ap- propriate blank.

Preparation of turnover-modifid aryl sulfatase A. The reaction mixture contained 35.0 @g/ml aryl sulfatase A, 40 mg/ml nitrocatechol sulfate and 3 mg/ml BaCl, all in 2-4 ml of 0.1 M sodium acetate buffer pH 6.0 plus 0.4 M NaCl and was incubated at 50°C for 5 min. (However, during the preparation of the turnover-modified aryl sulfatase A at 37°C the reaction mixture was incubated for 30 min). Barium sulfate which formed during the course of the reaction was removed by centrifugation. The remaining supernatant was applied on a Sephadex G-25 column (2.5 x 110 cm) equilibrated in sodium acetate buffer pH 5.6, I = 0.2 and the protein was eluted in 6.0-ml fractions. The turnover-modified aryl sulfatase A was located in elution profile by determining the residual enzyme activity in the presence of 3 mM SO;*. Fractions showing activity were pooled and concentrated at 4°C in a dialysis bag using Aquacide. The concentrated enzyme preparation was extensively dialyzed at 4°C against sodium acetate pH 5.5, I = 0.1.

In a similar manner, turnover-modified enzyme was isolated by application of the reaction mixture to a 2.5 x 110 cm column equilibrated with an otherwise identical buffer mixture which was also 10 mM in sodium sulfate. All subsequent chromatographic operations were carried out with thii sulfate-containing buffer, and the resulting protein preparation was utilized directly for spectral studies.

Isolation of sulfate ion-induced reactivated aryl sulfatase A. The 2-ml reaction mixture contained 36-50 pg of turnover-modified enzyme, 100 mM nitrocatechol sulfate, and 5 mM N&SO, in 0.1 M sodium acetate buffer pH 6.0 + 0.4 M NaCl. The reaction mixture was incubated at 37°C for 3-6 h and the reactivated aryl sulfatase A was purified by passage through a Sephadex G-25 column (2.5 x 100 cm) equilibrated with sodium acetate buffer pH 5.6, I = 0.2. The reactivated aryl sulfatase A concentrated in a dialysis bag using Aquacide. The concentrated enzyme preparation was dialyzed against sodium acetate buffer pH 5.5, I = 0.1 at 4°C.

Samples of this protein were also isolated by chromatography in buffers containing sulfate as described for the turnover-modified enzyme itself.

Circular dichvism measurements. Circular dichroism (CD)3 experiments were performed on a Cary 60

3 Abbreviations used: CD, circular dichroism.

spectropolarimeter using 0.5-1.0 cm light path jacketed cells at 15, 25, 37, and 50°C. The path length of the cells was chosen to give the highest possible ellipticity at a photomultiplier voltage not exceeding 700 V. All samples were filtered through a Millipore filter before measurement. The mean residue ellipticities, [0] in deg cm* dmol-1 were calculated from the measured ellipticities 0 by the equation [0], = [(e/10) x (Mollc)] where f3 is the observed ellipticity in degrees, 1 is the optical path length in centimeters, c is the protein concentration in grams per milliliter, and MO is the mean residue weight (molecular weight divided by the number of amino acid residues). The secondary structures of the aryl sulfatase A preparations were computed from CD data using the procedure of Chen et al. (21).

Fluoresence measurements. Fluorescence spectra were measured using a Perkin-Elmer MPF-44A fluorescence spectrometer equipped with a thermo- statted cell holder. The ratio mode of operation was used in all measurements to minimize the effect of variation in xenon lamp intensity. The excitation and emission slit widths were kept constant at 6 nm. Fluo- rescence measurements were done at 37°C except where noted. Fluoresence intensities were standard- ized using protein solutions of approx 1 mg/ml and are presented as relative values. Standard solutions of tyrosine and tryptophan were scanned each day before experimental measurements. If there was any difference in the spectra, the fluorescence spectra of the aryl sulfa&e A preparations were corrected accordingly.

Thermal stabilities of the native and turnover- modified enzyme were examined in the temperature range of 20-60°C. The protein sample consisting of 6-18 kg/ml protein in 3 ml was placed in a cuvette and equilibrated at the required temperature for 10 min before fluorescence measurements. The protein samples were excited at 230 nm and the emission spectra were recorded in the wavelength region of 290- 360 nm. Under similar experimental conditions, fluorescence emission spectra of tryptophan solutions were recorded at different temperatures. The relative fluorescence of the enzyme preparations at 330 nm was plotted against temperature.

Preparation of 35S-labeled aryl sulfatase A. Rabbit liver aryl sulfatase A, 50 pglml, was incubated with 40 mg/ml [35S]nitrocatechol sulfate in 0.1 M sodium acetate buffer pH 6.0-0.4 M NaCl at 50°C for 5 min in presence of 4 mg/ml barium chloride. Barium sulfate formed during the reaction was removed by centrifugation at 5000 rpm for 2.0 min and the remaining supernatant was applied to a Sephadex G-25 column (2.5 X 100 cm) equilibrated with sodium acetate buffer pH 5.6, Z = 0.2. The turnover-modified enzyme was eluted in 3 to 6-ml fractions immediately following the void volume of the column. Fractions of 3 ml were eluted directly in counting vials. The enzyme activity


g 0.10 5 m 0.08 5 ; 0.06 a

0.04

0 40 60 120 160 200 TIME,min

FIG. 1. Progress curve for the hydrolysis of 4 mM

nitrocatechol sulfate by aryl sulfatase A. Reactions were carried out at 37°C in sodium acetate pH 5.5, I = 0.1 as follows: control (A); 3 mM Na2S04 was added after 30 min of incubation (0); 3 mM Na,SO, was present in the reaction mixture (0); (-) indicates the differential curve between the reactivated enzyme (0) and the control (A).

in each fraction was determined using 4 mM nitrocatechol sulfate + 3 mM K,SO, in 0.5 M sodium acetate buffer pH 5.5 at room temperature. A 100~~1 aliquot was removed from each fraction and added to 1 ml of 4 mM nitrocatechol sulfate + 3 mM K2S04. The reaction mixture was incubated for 30 min and the reaction was quenched with 1 ml of 0.2 M NaOH. Absorbance was recorded at 515 nm on Spectronic 20. Fractions showing enzyme activity were counted in a Packard Tri-Carb Model 3320 liquid scintillation spectrometer using Bray’s scintillation liquid (22).

For quantitative radioactivity measurements, the protein fractions showing enzyme activity were pooled and concentrated at room temperature in a dialysis bag using Aquacide. The concentrated enzyme samples were extensively dialyzed against sodium acetate buffer pH 5.6, I = 0.2. The protein concentration and radioactivity were determined. For radioactivity determinations each vial was counted for 100 min and counting was repeated at least eight times in order to minimize counting errors (23).

RESULTS

Anomalous kinetics. Typical experiments illustrating the anomalous kinetic behavior of rabbit liver aryl sulfatase A are shown in Fig. 1. The enzyme is sub- stantially inactivated within 20 min during the hydrolysis of 4 mM nitrocatechol sulfate

in sodium acetate buffer pH 5.5, I = 0.1 at 37°C. The addition of 3 IIIM Na,S04 after 30 min partially reactivates the inactive enzyme (Fig. 1). The reactivation of the inactive enzyme by sulfate is not apparent during the first 15 min of incubation time. The time course of the enzyme reactivation shows a distinct lag phase. Since the inactive enzyme had some residual activity, the extent of nitrocatechol sulfate hydrolysis by the reactivated enzyme is determined by subtracting the absorbance due to the inactivated enzyme sample from the absorbance of the reactivated enzyme sample at different time intervals. The sigmoidal nature of the curve for reactivation process is clearly evident (Fig. 1, solid line).

The presence of sulfate in the reaction mixture prevents the complete inactivation of rabbit liver aryl sulfatase A (Fig. 1). Moreover, the initial rate of nitrocatechol sulfate hydrolysis is lowered because of the competitive inhibition of the enzyme activity by sulfate (5).

Effect of temperature on the anomalous kinetics. Progress curves for the hydrolysis at different temperatures of 4 mM nitrocatechol sulfate in sodium acetate buffer pH 5.5, I = 0.1 containing added 3 IIIM BaCl, are shown in Fig. 2. The kinetic parameters were calculated using the data of Fig. 3 by Stinshoff’s procedure (3), whose

0.24

0.20

e 0.16

5 p 0.12

a

2 O.OS

0.04

TIME, min

FIG. 2. Progress curve for the hydrolysis of 4 mM nitrocatechol sulfate by aryl sulfatase A. The enzyme reaction was carried out in sodium acetate buffer pH 5.5, I = 0.1 + 3 mM BaGI, at 15 (X); 25 (0); 37 (0); 42 (A); and 50°C (0).


theoretical basis has been considered in detail by Roy (24). The results are tabulated in Table I. The initial velocity v0 is lower at reduced temperatures. The value of &, which is a measure of the rate of inactivation of the enzyme, is also greater at lower temperature values, suggesting a possible correlation between the initial velocity of substrate hydrolysis and the rate of inactivation of aryl sulfatase A. Aryl sulfatase A which had been inactivated at 50°C could be reactivated by sulfate ions in a manner similar to inactive enzyme prepared at 37°C.

Effect of organic solvents on the anomalous kinetics. Rabbit liver aryl sulfatase A showed the anomalous kinetic behavior in the presence of lo-40% ethanol or dioxane and in 20% (3.3 M) urea in sodium acetate buffer pH 5.5, Z = 0.5 at 3’7°C. The resulting inactive aryl sulfatase A (turnover- modified enzyme) was not reactivated by addition of sulfate to the dioxane, ethanol, or urea solutions. Kinetic parameters calculated according to the procedure of Stin- shoff (3) are given in Table II. Typical protein denaturants (25) lower the initial velocity of the aryl sulfatase A and cause a corresponding increase in t 1,2 (Table II).

Reactivation of turnover-modi,fied aql

I /= I

2.0

t z 1.6 a g 1.2 0 z 0.6 a

0.4

no _._ 0 20 40 60 80 100 120

TIME, min

FIG. 3. Progress curve for the hydrolysis of 4 mM nitrocatechol sulfate by turnover-modified aryl sulfatase A. The enzyme reaction was carried out in sodium acetate buffer pH 5.5, I = 0.1 M at 37°C (0) and in the presence of 1 X 10m3 mM sulfite (0); 0.025 mM phosphate (0); 0.15 mM pyrophosphate (A); 2.5 mM sulfate (A); and 1.0 mM arsenate (W).

TABLE I

EFFECTOFTEMPERATURE ONTHE KINETIC PARAMETERS OF ARYL SULFATASE A

Temper- ature (“C)

15 25 37 42 50

Initial velocity, zlo (woY minimg)

79 130 400 450

1000

Half-life, t 112

(min)

276.0 146.5

5.5 3.7 0.8

Maximum turnover,

U max (mmopmg)

53.0 46.0

6.0 4.0 2.0

sulfatase A. Turnover-modified rabbit liver aryl sulfatase A can be reactivated by different ions such as sulfate, phosphate, pyrophosphate, and arsenate. In order to determine the optimum concentration of these ions, we carried out the reactivation at different concentrations of the ions using 4 mM nitrocatechol sulfate in sodium acetate buffer pH 5.5, Z = 0.5 at 37°C. The concentrations of ions needed for half-maximal reactivation (apparent dissociation constant) parallel the Ki values. The K.., values obtained for sulfate, phosphate, pyrophosphate, and arsenate were 2.50, 0.025, 0.12, and 1.0 mM, respectively.

Once it was established that the apparent dissociation constants K Bpp for the ions were similar to their Ki values, we studied the effects these inhibitors had on the reactivation of inactive enzyme. The reactivation of the turnover-modified enzyme was carried out with concentrations of inhibitors near their Ki. The results are shown in Fig. 3. The maximum reactivation was observed with arsenate and the reactivation effects of the ions were in the order arsenate > sulfate > pyrophoshate > phosphate. Sulfite did not reactivate the turnover-modified enzyme to any measurable extent.

Incorporation of Y5’ in turnover-mod$ed aryl sulfatase A. A typical chromatographic profile for the preparation of turnover- modified aryl sulfatase A is shown in Fig. 4. The chromatographic profile showed a good coincidence between the enzyme activity peak and the radioactivity peak although a small shoulder near the trailing end of the elution profile was observed which may be

WAHEED AND VAN ETTEN

TABLE II

EFFECT OF DIOXANE, ETHANOL, AND UREA ON THE KINETIC PARAMETERS OF ARYL SULFATASE Aa

Solvent

None 10% dioxane 20% dioxane 40% dioxane 10% ethanol 20% ethanol 40% ethanol 20% urea

Initial Maximum velocity, Half-life, turnover, v. (cLmoV t 112 u max min/mg) (min) (mmopmg)

319 5.2 4.0 305 5.3 4.0 146 11.0 4.0 45 18.4 2.0

273 6.1 4.0 207 7.9 4.0 96 8.6 2.0 29 14.3 1.0

a All reaction mixtures contained 0.5 M sodium acetate buffer, pH 5.5 and data were obtained at 3’7°C.

due to presence of small amounts of labeled protein having no remaining enzyme activity. The major radioactivity fraction was due to free sulfate and unreacted substrate and there was a good separation from the turnover-modified enzyme. Radio- activity remained associated with the turnover-modified enzyme upon rechromatog- raphy on Sephadex, upon dialysis, and upon concentration using Diaflo filtration.

Table III shows the quantitative results from the several labeling experiments. The results are comparable with those obtained in earlier preliminary experiments (17). The number of gram atoms of sulfur incorporated per mole of molecular weight 140,000 was in the range of 1.8-2.3 at different protein concentrations. However, only 0.2 g atoms of sulfur were incorporated per mole of monomer in reaction mixtures where sulfate was initially present and the anomalous kinetics could not be observed. In order to demonstrate that the incorporation of 35S was not due to a nonspecific adsorption of SOi ions on the turnover-modified aryl sulfatase A molecule, we carried out an experiment where the enzyme was exposed to [35Sl- SO:2 ions under otherwise comparable experimental conditions. The results indi- cated an incorporation of, at most, 0.2 g atom of sulfur per mole of the aryl sulfatase A monomer (Table III).

Circular dichroism of the native enzyme

in the far ultraviolet region. Rabbit liver aryl sulfatase A showed a maximum near 275 nm and a shoulder near 265 nm at pH 4.5, Z = 0.1 37°C; however, at pH 7.5, Z = 0.1 only one maximum was observed at 270 nm. The optical ellipticity was positive at both pH values. The mean residue weight optical density at pH 4.5 was about 23% higher than at pH 7.5 (Fig. 5).

Circular dichroism of native and turnover-modi$ed aryl sulfatase A in the ultraviolet region. The CD spectra of native aryl sulfatase A at pH 7.5, 5.5 and 4.5, Z = 0.1 37°C are shown in Fig. 6. It can be seen that the CD spectra of aryl sulfatase A at all pH values between 250 and 204 nm had minima near 218 nm and a shoulder near 208 nm. The average optical ellipticity of aryl sulfatase A near 220 nm was - 11,000 deg cm2 dmol-’ at all pH values, the small variation in the values being within experimental error.

The CD spectra of turnover-modified enzyme and sulfate ion-induced, reactivated aryl sulfatase A are shown in Fig. 6. Turn- over-modified enzyme showed a minimum near 220 nm. The optical ellipticity of the modified enzyme decreased to -2500 deg cm2 dmol-’ from - 11,000 deg cm2 dmol-’ observed for native enzyme at pH 5.5, Z = 0.1 37°C. Samples at 50°C had similar CD spectra indicating that the enzyme preparations

“”

28 32

FIG. 4. Separation of YS-labeled aryl sulfatase A from substrate and products on Sephadex G-25 column. The turnover-modified enzyme was prepared using [YSlnitrocatechol sulfate as described under Experimental Procedures. The radioactivity was counted as cpm per vial (3 ml), (the vertical bar indicates average deviation). The enzyme activity was measured using an assay for nitrocatechol release at 515 nm.


TABLE III

INC~RP~RATI~NOF~~SFROM~-HYDROXY-~-NITROPHENYLSULFATEINTORABBITLIVERARYLSULFATASE A

Sample

Total counts” W Incorporated Protein per mole of

(/a) Observed Calculated* Net counts” protein monomer

Aryl sulfa&e A + [%]nitrocatechol 7.5 2541 2 17 2553 107 lr 31 1.3 sulfate, pH 6.0, I = 0.5, 50°C 10.5 2614 + 21 2594 190 k 33 2.3

13.0 2647 + 42 2630 228 -t 49 2.2 24.0 2790 r+ 45 2784 391 ? 52 2.0 25.0 2787 2 28 2799 388 2 38 1.9 50.0 3182 k 49 3150 838 + 55 2.1

Aryl sulfatase A + [35S]nitrocatechol sulfate, pH 6.0, I = 0.5, 50 mM sop, 50°C 35.5 2498 k 48 2948 58 + 55 0.2

Aryl sulfatase A + [%]nitrocatechol sulfate, pH 4.5, I = 0.5, 50°C 30.0 2484 + 40 2869 52 f 48 0.2

Aryl sulfatase A + 0.1 PCi [35S]SO~2, pH 6.0, I = 0.5, 50°C 30.0 2493 t 42 2869 52 + 49 0.2

a The background count was 2447 ? 26 counts/100 min; all count totals are per 100 min. * Calculated assuming that 2 g atoms W are incorporated per molecule of aryl sulfatase A monomer

of M, 140,000.

were not detectably different. The CD spectrum of sulfate ion-induced, reactivated aryl sulfa&e A showed no negative ellipticity; the optical ellipticity near 220 nm was positive (see Fig. 6).

The turnover-modified aryl sulfatase isolated in the presence of 10 mM Na,SO, showed an optical ellipticity of -1814 deg cm2 dmol-’ at 220 nm, at pH 5.5, 37”C, which is similar, within experimental error, to the optical ellipticity of the turnover- modified enzyme isolated in the absence of sulfate ions (Fig. 6). The “reactivated” turnover-modified aryl sulfatase A, which was isolated in the presence of 10 lllM Na,SO,, had a CD spectrum which was identical to that of turnover-modified aryl sulfatase A and the optical ellipticity at 200 nm at pH 5.5, 3’i’“C, was -1800 deg cm2 dmol-‘.

The amounts of secondary structure, a-helix, p-structure, and remaining structure were calculated from CD data obtained for preparations of aryl sulfatase A using a representative algorithm (21) and the results are tabulated in Table IV.

Fluorescence measurements of native, turnover-modi,fied, and “reactivated” turnover-modifid aryl sulfatase A. Native aryl sulfatase A solutions were excited at 295,

282, or 275 nm and the corresponding emission were recorded. Typical fluorescence spectra are shown in Fig. 7. Native aryl sulfatase A showed emission maxima near 330 nm at pH 7.5, 5.5, and 4.5. The relative fluorescence intensity near 330 nm of aryl sulfatase A at pH 7.5 was only slightly higher than at pH 4.5 This fluorescence quenching behavior of aryl sulfatase A at pH 4.5 was seen at all wavelengths of excitation.

WAVELENGTH, nm

FIG. 5. The circular dichroism spectra of rabbit liver aryl sulfatase A in the near ultraviolet region at 37°C in sodium acetate buffer pH 4.5, Z = 0.1 (- - -) and in Tris-HCl buffer pH 7.5, Z = 0.1 (-).


200 210 220 230 240 WAVELENGTH, nm

FIG. 6. The circular dichroism spectra of rabbit liver aryl sulfatase A in the ultraviolet region at 37°C. Native aryl sulfatase A in sodium acetate pH 4.5, Z = 0.1 (. . .); sodium acetate pH 5.5, Z = 0.1 (-); and Tris-HCl pH 7.5, Z = 0.1 (- - -). Turn- over-modified aryl sulfa&e A prepared at 37°C (-.-) and 50°C (0) in sodium acetate pH 5.5, Z = 0.1; “reactivated” turnover-modified aryl sulfatase (-. .-).

The fluorescence spectra of turnover- modified aryl sulfatase A is shown in Fig. 8. It can be seen that the relative fluorescence intensity of the turnover-modified aryl sulfatase A decreased from 2.76 to 0.89 for native aryl sulfatase at pH 5.5, Z = 0.1 M. The fluorescence spectra of turnover- modified aryl sulfatase A preparation which had been concentrated in a Diaflo apparatus

or in a dialysis bag using Aquacide were virtually identical.

The relative fluorescence of the enzyme molecule at 330 nm was used as a measure of possible structural changes occurring under various experimental conditions used to study the anomalous kinetic behavior (Table V). When aryl sulfatase A was incubated in sodium acetate buffer pH 4.5, Z = 0.1 and pH 6.0, Z = 0.5 at 50 and 3’7°C respectively, for 10 min, there was no loss in the relative fluorescence of the enzyme molecule. Aryl sulfatase A isolated from a reaction mixture containing nitrocatechol sulfate (10 mti) in sodium acetate buffer pH 4.5, Z = 0.1 [where the enzyme does not show the anomalous kinetics (16, lS>] revealed no change in the relative fluorescence of the enzyme. Similarly, aryl sulfatase A obtained from a reaction mixture containing sulfate (5 InM) and nitrocatechol sulfate (10 InM) in sodium acetate buffer pH 6.0, Z = 0.5 at 5O”C, where the anomalous kinetics of the enzyme is prevented by sulfate ions, exhibited a relative fluorescence similar to that of the native enzyme. Therefore, these results indicate that the isolation procedures themselves do not bring about a decrease in the relative fluorescence of the enzyme.

Effect of sulfate and of temperature on the structure of the enzyme. Since sulfate ions induce activity in inactive turnover-modified enzyme in the presence of nitrocatechol sulfate and also prevent the native enzyme from being inactivated during substrate hydrolysis, the effect of sulfate ions on the CD spectra of native and turnover-modified

TABLE IV

SECONDARY STRUCTURE OF RABBIT LIVERARYLSULFATASE A AT 3’7YP

Sample

Random a-Helix &Structure Structure

(%) (%I 6)

Aryl sulfatase A in Tris-HCl, pH 7.5, Z = 0.1 32 30 38 Aryl sulfatase A in sodium acetate, pH 5.5, Z = 0.1 31 31 38 Aryl sulfatase A in sodium acetate, pH 4.5, Z = 0.1 33 30 37 Turnover-modified aryl sulfatase A in sodium acetate, pH 5.5, Z = 0.1 8 8 84 Reactivated aryl sulfa&se A in sodium acetate, pH 5.5, Z = 0.1 0 4 96

a Calculated according to (21) using circular dichroism data obtained in the present study.


enzyme was studied. The results are shown in Fig. 9. There was no detectable difference between the CD spectra of the enzyme preparations in the presence or absence of 10 mM Na,S04. Similarly, over the range of 25-60°C temperature did not influence the CD spectra of the native enzyme. A small decrease in the optical ellipticity of the native enzyme was observed at 60°C.

Structural stability of native and turnover- modified aryl sulfatase A. The relative fluorescence yields of native and of turnover- modified aryl sulfatase A at 330 nm at different temperatures are shown in Fig. 10. The relative fluorescence of a solution of L-tryptophan under identical experimental conditions (sodium acetate buffer pH 5.5, Z = 0.1) is also given in Fig. 10. The relative fluorescence of the native enzyme decreased with increasing temperature. A similar decrease in the fluorescence of tryptophan was observed but the extent of decrease in the relative fluorescence of tryptophan was greater than that for native aryl sulfatase A. Up to 37”C, the relative fluorescence of the turnover-modified enzyme decreased with increasing temperature. However, heating of the turnover-modified enzyme sample above 37°C caused a transition between 37 and 52°C which was consistent with an increased exposure of tryptophan residues accompanying a loss of remaining structure from turnover-modified aryl sulfatase A.

I3

WAVELENGTH, nm

FIG. 7. The fluorescence emission spectra of rabbit liver aryl sulfatase A at 37°C. The fluorescence measurements were made in (A) Tris-HCl pH 7.5, I = 0.1; (B) sodium acetate pH 4.5, Z = 0.1. Num- bers indicate the excitation wavelength in nanometers.

2 ::;I , , , , J

290 350 370 310 330

WAVELENGTH,nm

FIG. 8. The fluorescence emission spectra of turnover-modified rabbit liver aryl sulfatase A in sodium acetate buffer pH 5.5, Z = 0.1 and at 37°C. The excitation wavelengths are 275 nm (0); 272 nm (A); and 295 nm (0).

DISCUSSION

Rabbit liver aryl sulfatase A is 80% inactivated within 20 min during the course of hydrolysis of 4 InM nitrocatechol sulfate in sodium acetate pH 5.5, Z = 0.1, at 3’7°C (Fig. 1). Qualitatively similar results have been reported for other mammalian aryl sulfatase A enzymes (l-3). Inactivation of rabbit liver aryl sulfatase A during substrate hydrolysis seems to depend on the initial velocity of the reaction (Table I). Also consistent with this interpretation is the fact that the enzyme is more rapidly inactivated at higher temperatures where initial velocities are higher. Baum and Dodgson (2) and Roy (12) have also observed the more rapid inactivation of human liver and ox liver aryl sulfatase A at higher temperatures. Since the initial velocity of the reaction is an important factor in the formation of the turnover-modified aryl sulfatase A, one may expect that substrates exhibiting lower turnover rates (such as cerebroside sulfate, L-tyrosine sulfate, as- corbate sulfate, methyl umbelliferone sulfate, and several steroid sulfates) will not readily exhibit anomalous kinetics. Con- sistent with this, the ox liver enzyme (15, 26), chicken brain enzyme (14), and human liver enzyme (6) are apparently not inactivated during the hydrolysis of cerebroside sulfate substrate. In contrast, a recent

20 WAHEED ANEbV&N E-i’TEN

TABLE V

RELATIVE FLUORESCENCE OFRABBITLIVERARYLSULFATASE Aa

Samples

Native aryl sulfa&e A in: Tris-HCl, pH 7.5 Sodium acetate, pH 5.5 Sodium acetate, pH 4.5 Sodium acetate, pH 4.5, 50°C for 10 min Sodium acetate, pH 6.0, 37°C for 10 min

Native aryl sulfatase A exposed to:

Relative fluorescence ( Aem, 330 nm)

2.67 2.76 2.67 2.84 2.84

Sodium acetate, pH 4.5, 40 mM nitrocatechol sulfate Sodium acetate, pH 6.0, 40 mM nitrocatechol sulfate plus 5 mM SO;*, 37°C

Turnover-modified aryl sulfatase A prepared in sodium acetate, pH 5.5, 40 mM nitrocatechol sulfate

“Reactivated” turnover-modified aryl sulfatase A in sodium acetate, pH 5.5

’ All measurements were conducted at 37°C and Z = 0.1 M.

2.73 2.70

0.89

0.85

report (12) has suggested that the inactivation rate of ox liver aryl sulfatase A may not depend on the initial velocity of the substrate hydrolysis.

Rabbit liver aryl sulfatase A shows the anomalous kinetics in the presence of 40%

1 1 1 I I

200 210 220 230 240

WAVELENGTH, nm

FIG. 9. The circular dichroism spectra of rabbit liver aryl sulfa&e A in the ultraviolet region in sodium acetate pH 5.5, Z = 0.1. Symbols: native aryl sulfa&se A at 37°C (-); 50°C (- - -); 37°C with added 10 mM Na+!SO, (-.-); turnover-modified aryl sulfatase A at 37°C in presence and absence of 10 mMN%SO,(O).

ethanol and dioxane. The initial velocity of the reaction is decreased from 319 pmoll min/mg as a control to 95 and 45 pmol/min/ mg in 40% ethanol and 40% dioxane, respectively (Table II). Since the initial velocities of the enzyme at 15°C in sodium acetate buffer pH 5.5 (Table I) and in 40% ethanol and 40% dioxane at 37°C (Table II) are not much different, the values of tllz (a measure of the rate of inactivation) might be expected to be similar. However, the value of t l/2 at 15°C in buffer is 276 min while in 40% ethanol and 40% dioxane at 37°C the values are 8.6 and 18.4 min, respectively. These results suggest that the inactivation of the enzyme during hydrolysis of the substrate seems to depend on the nature of the solvent and that nonpolar organic solvents enhance the anomalous kinetics of the enzyme.

Turnover-modified aryl sulfatase A can be partially reactivated by sulfate ions (Fig. 1) as well as several other ions including arsenate, phosphate, and pyrophosphate. The sigmoidal shape of the reactivation curve suggests that SOi ions may induce a structural rearrangement in the turnover- modified enzyme in order to restore the biological activity of the enzyme (Fig. 1). Aryl sulfatase A is also inactivated during substrate hydrolysis in the presence of

ANOMALOUS KINETICS OF RABBIT LIVER ARYL SULFA’I’ASE A 21

ethanol and dioxane but the inactive enzyme is not detectably reactivated by SOa2. Either a SOh2 ion-induced conformational change is prevented in the presence of organic solvents or else organic solvents prevent the necessary initial interaction of SOi ions with the inactive aryl sulfatase A.

In most cases, the apparent dissociation constants of several ions in promoting activation of the inactive enzyme are similar to the Ki values of the ions. The extent of activation of the inactive enzymes by activators present at concentrations near their Ki values is different for different ions (Fig. 3). Strikingly, sulfite (SOTS), which is a strong (Ki = 1 PM) competitive inhibitor of rabbit liver aryl sulfatase A (5), neither activates the turnover-modified enzyme nor inhibits the residual activity of the turnover-modified enzyme. Thus, not all inhibitors are capable of acting as activators but, so far, all known activators are competitive inhibitors.

The results of 35S incorporation indicate that the inactivation of aryl sulfatase A is due to the covalent modification of the enzyme molecule. The presence of inorganic sulfate, which prevents the anomalous kinetic behavior, also prevents the incorporation of 35S from labeled substrate into the enzyme molecule (Table III). Incubation of [35S]SO;* ions with the enzyme molecule, under otherwise identical experimental conditions, does not result in the incorporation of 35S in aryl sulfatase A. These results establish that the incorporation of 35S is a specific reaction dependent upon the catalytic process. The stoichiometry of labeling corresponds to 2.0 + 0.1 g atom per mole of enzyme monomer ofM, 140,000 (Table III). The monomer of M, 140,000 consists of two identical protein subunits (5). Thus, the stoichiometry of incorporation is consistent with two reactive sites per monomer, or one per peptide chain.

The circular dichroism spectra of aryl sulfatase A in the near ultraviolet region (250-310 nm) (Fig. 5) and the fluorescence spectra of the enzyme (Fig. 7) suggest that the environments of chromophores in the enzyme molecule are changed when aryl sulfatase A dimerizes at pH 4.5 (18). The quenching of the relative fluorescence at pH 4.5 and the increase in the ellipticities of

FIG.

I 8 1.2 -

El 2 1.0 -

g 0.8 -

3 L 0.6 -

w 2 0.4- l- B

20 30 40 50 60 TEMPERATURE, OC

10. The intensity of fluorescence at the wavelength of maximum emission of rabbit liver aryl sulfataae A in sodium acetate pH 5.5, Z = 0.1 as a function of temperature. The wavelength of excitation was 282 nm; native aryl sulfatase A (A); turnover-modified aryl sulfatase A (0); and L- tryptophan (0).

the protein molecule at pH 4.5 indicate that tyrosine and tryptophan residues are buried during the dimerization of the enzyme monomer. However, the CD spectra in the ultraviolet region (200-250 nm) (Fig. 6) indicate that there is no difference in the secondary structure of the enzyme molecule at pH 7.5 and 4.5. This is more clearly seen in the results of Table IV. The average content of a-helix and p-structure was 32 and 30%, respectively, at all pH values studied. The low value of the helical content of rabbit liver aryl sulfatase A, estimated from circular dichroism data, may be due in part to carbohydrate moieties (27) since rabbit liver aryl sulfatase A is a glycopro- tein containing approx 5% carbohydrate (28). However, the possible importance of the high amount of proline residues, a strong helix breaker (29), in rabbit liver aryl sulfatase A (28) should be kept in mind. Nichol and Roy (30) found a nonlinear plot of the optical rotatory dispersion for the ox liver enzyme and concluded that it was due to a disruption of the helical structure by the high content of proline residues in that enzyme.

Circular dichroism measurements on turnover-modified rabbit liver aryl sulfatase


TABLE VI

THE ENZYMATICACTIVITYOFDIFFERENTPREPARATIONSOFARYL SULFATASE AAT~%,I = 0.1, PH 5.5

Sample

Native aryl sulfatase A Turnover-modified aryl sulfa&e A prepared at 37°C Turnover-modified aryl sulfa&e A prepared at 37”C, incubated with 3 mM Na,SO, for 30 min Turnover-modified aryl sulfatase A prepared at 50°C “Reactivated” aryl sulfatase A; reactivation carried out for 3 h “Reactivated” aryl sulfatase A; reactivation carried out for 3 h; incubated

with 3 mM Na,SO, for 200 min “Reactivated” aryl sulfatase A; reactivation carried out for 6 h

Specific activity (pmol/min/mg)

340” 63”

173 60” 0.700

3.63 0.52”

n The specific activity of the enzyme preparation was determined using 4 mM nitrocatechol sulfate at 37°C and incubating for 3 min.

A (Fig. 6) suggest that the substrate- induced inactivation of the enzyme results in a loss of secondary structure (Table IV). There is an approximately 76% decrease in a-helical content of the enzyme molecule upon inactivation. The turnover-modified enzymes prepared at 37 and 50°C have similar circular dichroism spectra (Fig. 6) indicating that the loss of secondary structure, due to substrate-induced inactivation, is not influenced by the temperature. The fluorescence spectra of the turnover-modified enzyme at different excitation wavelengths (Fig. 8) also supports the above eontention that the turnover-modified has lost about 67% of its structure. A reduction of fluorescence has been found as a result of loss of secondary structure (31). It is significant that the specific activity of the enzyme is also decreased by 82% (Table VI). These results establish that there is a parallelism between the structure and the functional activity of the turnover-modified aryl sulfatase A.

Interestingly enough, the results of CD measurements on “reactivated” aryl sulfatase A isolated in the presence of 10 mM sulfate ions show that sulfate prevents the loss of the small amount of remaining secondary structure from the turnover-modified, “reactivated” enzyme. However, the loss of secondary structure compared to the native enzyme is still dramatic.

Turnover-modified aryl sulfatase A can be partially reactivated by the presence of substrate and sulfate ions (2, 5, 9). Since

high concentrations of substrate (nitrocatechol sulfate) interfere with spectral measurements of the protein molecule in the uv region, the effect of sulfate ions on the circular dichroism spectra of native aryl sulfatase A and turnover-modified enzyme must be studied in the absence of substrate (Fig. 9). In the presence and absence of 10 InM Na,SO,, the native and turnover- modified enzymes exhibit identical CD spectra. Thus sulfate apparently does not induce any conformational change in the absence of substrate. However, the turnover-modified enzyme having 18% of the activity of native enzyme was reactivated after 30 min in the presence of 2 mM Na,S04 and 4 mM nitrocatechol sulfate to a form having 50% of the activity of the native enzyme (Table VI). Thus, this preparation of turnover-modified enzyme evidently has sufficient structural integrity remaining in at least a portion of the protein molecules to permit a partial reactivation. Whether the loss in structure is also temporarily reversed by sulfate ion plus substrate is not yet known with complete certainty because of the impossibility of making CD measurements on solutions having high concentrations of nitrocatechol and nitrocatechol sulfate. However, when a sample of “reactivated” aryl sulfatase A was isolated from a reactivation mixture where turnover- modified enzyme has been partially reactivated with respect to activity both the circular dichroism (Fig. 6 and Table IV) and fluorescence measurements (Table V) of the


“reactivated” aryl sulfatase A show that it has little or no remaining secondary structure. The enzymatic activity is only 0.2% of native aryl sulfatase A (Table VI). When the isolated “reactivated” enzyme is incubated with 4 lllM nitrocatechol sulfate and 3 mM N&SO, for extended periods, a small increase in specific activity is observed (Table VI). This result suggests that isolated “reactivated” rabbit liver aryl sulfatase A has lost that part of the structure required for the SO72 ion-induced reactivation.

In contrast to the general belief that reactivation restores the activity of modified aryl sulfatase A to that of native aryl sulfatase A (2, 9) we were surprised to find that the “reactivated” rabbit liver aryl sulfatase A had lost most of its activity as well as structure upon isolation from reaction mixtures where the enzyme activity is higher. This is probably a consequence of the reduced structural stability of the turnover-modified enzyme. A comparison of fluorescence measurements on the native enzyme, turnover-modified aryl sulfatase A and of free tryptophan at different temperatures (Fig. 10) suggests that the native enzyme does not unfold between 20 and 60°C under our experimental conditions. The decrease in fluorescence of the native enzyme was similar to that observed for tryptophan except that the quenching of fluorescence per degrees centrigrade for aryl sulfatase A was lower than for the amino acid. [A similar observation has been reported for ribo- nuclease (32)]. In contrast to the native enzyme, turnover-modified sulfatase exhibited a transition in the temperature range 37 to 52°C. Thus the results indicate that the structure of the turnover-modified rabbit liver enzyme is very unstable and is readily denatured between 37 and 52°C whereas the native enzyme is unaffected in this temperature range. It appears likely that during the reactivation process the turnover-modified aryl sulfatase temporarily assumes a conformation induced by sulfate ions in the presence of substrate and that this conformation is catalytically more active that that of turnover-modified enzyme. However, due to the poor stability of the turnover-modified enzyme, the protein molecule loses its remaining secondary structure

during the isolation of the “reactivated” enzyme and this results in a nearly total loss of enzymatic-activity.

A reasonable chemical model can now be provided to explain the anomalous kinetic behavior of aryl sulfatase A. One mechanism by which sulfate esters are considered to undergo hydrolysis is via a unimolecular process involving the loss of SO3 (33). Such a reaction, involving as it does a dispersal of charge in the transition state, should be facilitated by transfer from an aqueous to a less polar environment, consistent with the present results. Even more im- portantly, such a reaction process would be facilitated by increasingly stable phenolate leaving groups. Thus, substrates such as nitrocatechol sulfate or o-nitrophenyl sulfate, precisely those substrates which result in the anomalous kinetic behavior, may be expected to undergo hydrolysis by a unimolecular type of mechanism. A subsequent reaction of SO, with critical groups on the protein rather than with solvent water could result in an effectively irreversible modification of the protein. Such a reaction evidently occurs in one of every lo3 to lo4 turnover reactions.

If sulfate ion is simultaneously present, we would hypothesize that the rapid non- covalent association of sulfate with free enzyme (or with an E-S intermediate) can protect this particular group on the protein from reaction with SO,. Seen in this way, the fact that many competitive inhibitors can also act to prevent or reduce the anomalous kinetic behavior is easily explained.

Moreover, the present work also provides a possible model for the partial reactivation of the modified enzyme. If the covalently modified enzyme is very fragile with respect to a loss of secondary and tertiary structure, then the apparent reactivation due to high concentrations of sulfate is similar to the effects of competitive inhibitors generally in stabilizing protein structure. Sulfate acts like a kind of weak “glue” which can (while present) temporarily “repair” at least some of the subunits.

The covalent modification reaction of rabbit liver aryl sulfatase A is pH de-


pendent, occurring readily at a pH value of 5.5 or 6.0 but not at pH 4.5 (17). The reaction is also highly specific in that 2 g atom of sulfur are introduced per enzyme monomer, and each enzyme monomer of M, 140,000 consists of two equivalent polypeptide chains of M, 70,000 (5). In connection with the chemical nature of the covalent modification, we consider it significant that the use of a chemical sul- fating agent, pyridine-sulfur trioxide complex, results in a loss of enzymatic activity and in the formation of a protein which has also lost the major portion of its secondary structure (17). It is probable that the stoichiometry of the reaction with pyridine-sulfur trioxide is very different from that for modification by substrate. The chemical nature of the covalently modified protein clearly deserves study. Finally, the fragile nature of the tertiary structure of turnover-modified rabbit liver aryl sulfatase A prepared at the pH optimum is remarkable, and it will be of interest to see how characteristic this is for modified aryl sulfatase A enzymes from other organisms or prepared under other conditions.

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11.

12.

13.

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ROY, A. B. (1978) B&him. Biophys. Acta 526, 489-506.

FLUHARTY, A. L., AND EDMOND, J. (1978) in Complex Carbohydrates, Part C (Ginsburg, V., ed.), Vol. 50, pp. 537-547, Academic Press, New York.

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ACKNOWLEDGMENT

CHEN, Y. H., YANG, J. T., AND CHAU, K. H. (1975) Biochemistry 13,3350-3359.

BRAY, G. A. (1960) Anal. Biochem. 1, 279-285. WANG, C. H., AND WILLIS, D. L. (1965) Radio-

tracer Methodology in Biological Science, pp. 186-209, Prentice-Hall, Englewood Cliffs, N. J.

We thank Ms. Susan Burkhardt for proficient and valued technical assistance.

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276, 4’75-490. 4. FAROOQUI, A. A., AND BACHHAWAT, B. K.

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The structural basis of the anomalous kinetics of rabbit liver aryl sulfatase A

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