938 Chem. Res. Toxicol., Vol. 12, No. 10, 1999
Uttamsingh and Anders
substrate. The fractions that had activity with N-acetyl-L-
cysteine and N-acetyl-L-methionine as substrates were purified
as described previously (22). Fractions with activity with
NADCVC as the substrate were combined and concentrated in
an Amicon ultrafiltration cell, and 30 mL of the concentrated
fraction was applied to a Sephacryl S-200 HR (5.0 cm × 40 cm,
Pharmacia, Piscataway, NJ ) column equilibrated with 10 mM
potassium phosphate buffer (pH 7.4); the column was eluted
with the same buffer. Eluate fractions (4 mL) were collected
and assayed for protein concentrations and enzyme activities.
Fractions with the highest activities were combined, concen-
trated in an Amicon ultrafiltration cell, and applied to a
hydroxyapatite column (1.5 cm × 12 cm, Macro-Prep Ceramic
Hydroxyapatite TYPE I, 40 µm, Bio-Rad Laboratories, Hercules,
CA) equilibrated with 10 mM potassium phosphate buffer (pH
7.4). Proteins were eluted with the column equilibration buffer,
and eluate fractions (2 mL) were collected and assayed for
protein concentrations and enzyme activities. Fractions with the
highest activity were combined and applied to a Mono-Q HR5/5
column (0.5 cm × 5 cm, Pharmacia), which was eluted with a
linear gradient of 10 to 200 mM (∼40 mL) potassium phosphate
buffer (pH 7.4) with a FPLC system (Pharmacia, Piscataway,
NJ ). Eluate fractions (1 mL) were collected and assayed for
protein concentrations and enzyme activities. Fractions with the
highest activities were analyzed by SDS-PAGE (10% acryla-
mide). Proteins were visualized by Coomassie Blue staining. The
purified rat kidney acylase was stored at 4 °C for 3 months
without a significant loss of acylase activity.
lase is associated with Canavan disease, an autosomal
recessive leukodystrophy characterized by spongy degen-
eration of white matter; hence, aspartoacylase is thought
to play a role in brain biology (16, 17). cDNAs encoding
the amino acid sequence of bovine and human aspartoa-
cylase have been reported (18). Acylase III has been
partially purified from rat kidney (19), and a rat liver
acylase with substrate selectivities similar to those of rat
kidney acylase III has been identified (20).
The enzymes responsible for the deacetylation of
xenobiotic-derived mercapturates have not been fully
identified. Recent studies show that acylase I catalyzes
the deacetylation of S-alkyl-N-acetyl-L-cysteines, includ-
ing N-acetyl-L-methionine and S-ethyl-N-acetyl-L-cys-
teine, and some haloalkene-derived mercapturates, in-
cluding S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-cysteine,
S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, and
S-(2-bromo-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine (21).
We report herein the results of studies in which a rat
kidney acylase that catalyzed the hydrolysis of the
haloalkene-derived mercapturates (NADCVC), S-(1,2,3,4,4-
pentachloro-1,3-butadienyl)-N-acetyl-L-cysteine, and S-(2,2-
dibromo-1,1-difluoroethyl)-N-acetyl-L-cysteine was iden-
tified. The acylase was purified from rat kidney cytosol
by column chromatography and partially characterized.
The data indicate that the substrate selectivity of the
purified rat kidney acylase is similar to that of the
previously identified rat kidney and liver acylase III.
Protein concentrations were determined by the method of
Bradford (25) with bovine serum albumin as the standard.
Acyla se Activity Assa ys. Acylase activity with NADCVC
as the substrate was determined by measuring the amount of
deacetylated product formed by reaction with fluorescamine (26).
The reaction mixture (1 mL) contained 0.1-0.5 mL of enzyme
solution and 2 µmol of substrate in 50 mM potassium phosphate
buffer (pH 7.4). The reaction mixture was incubated at 37 °C
for 1 h, and the reaction was stopped by addition of 0.2 mL of
20% trichloroacetic acid. The mixture was allowed to stand for
10 min in an ice bath and was then centrifuged at 500g for 10
min. A sample (40 µL) of the supernatant was added to 3.6 mL
of 50 mM potassium phosphate buffer (pH 7.4), and the volume
was increased to 4 mL by the addition of water. Fluorescamine
(300 µL of a solution containing 10 mg of fluorescamine dissolved
in 33 mL of acetone) was added to the sample, and the
fluorescence intensity (390 nm excitation, 475 nm emission) was
measured with a Perkin-Elmer LS-5 fluorescence spectropho-
tometer (Norwalk, CT). A standard curve was prepared with
L-methionine as the analyte.
Ma ter ia ls a n d Meth od s
Ma ter ia ls. N-Acetyl-L-cysteine, N-acetyl-L-methionine, L-
methionine, N-acetyl-L-tyrosine, and fluorescamine were pur-
chased from Sigma Chemical Co. (St. Louis, MO). S-Benzyl-N-
acetyl-L-cysteine was purchased from Schweizerhall, Inc.
(Plainfield, NJ ). N-Acetyl-L-aspartic acid, trichloroethylene, and
hexachloro-1,3-butadiene were purchased from Aldrich Chemi-
cal Co. (Milwaukee, WI), and 1,1-dibromodifluoroethylene was
purchased from PCR Inc. (Gainesville, FL).
Syn th eses. S-(1,2-Dichlorovinyl)-N-acetyl-L-cysteine was syn-
thesized as described by McKinney et al. (22). S-(2,2-Dibromo-
1,1-difluoroethyl)-N-acetyl-L-cysteine was synthesized by a pro-
cedure similar to that described for the synthesis of S-(2,2-
dichloro-1,1-difluoroethyl)-N-acetyl-L-cysteine (23). S-(1,1,2,3,4-
Pentachloro-1,3-butadienyl)-N-acetyl-L-cysteine was prepared as
described by Nash et al. (24). The physical constants of the
synthesized products were identical with literature values.
The deacetylation of S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-
cysteine, S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, S-(2-
bromo-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, S-(2,2-dibromo-
1,1-difluoroethyl)-N-acetyl-L-cysteine, S-(1,1,2,3,4-pentachloro-
1,3-butadienyl)-N-acetyl-L-cysteine, S-benzyl-N-acetyl-L-cysteine,
N-acetyl-L-tyrosine, and N-acetyl-L-aspartic acid with the puri-
fied rat kidney acylase was assessed by the method described
above.
En zym e P u r ifica tion . Kidneys from male, Sprague-Dawley
rats (Pel-freeze, Rogers, AR) were homogenized in 50 mM
potassium phosphate buffer (pH 7.4) at 4 °C. All purification
steps were performed at 4 °C. The homogenate was centrifuged
at 9000g, and the supernatant was centrifuged at 100000g to
obtain the cytosolic fraction. Solid ammonium sulfate was added
to the cytosol to 50% saturation, and the mixture was stirred
Kin etic An a lyses. The kinetics of the deacetylation of
NADCVC, S-(2,2-dibromo-1,1-difluoroethyl)-N-acetyl-L-cysteine,
S-(1,1,2,3,4-pentachloro-1,3-butadienyl)-N-acetyl-L-cysteine, and
S-benzyl-N-acetyl-L-cysteine was studied with the purified rat
kidney acylase. For the determination of the Km and Vmax, the
reaction mixtures contained 5 µg of the purified rat kidney
acylase and 1.0-16.0 mM substrate in a final volume of 1 mL
of 50 mM potassium phosphate buffer (pH 7.4). The reaction
mixtures were incubated at 37 °C for 30 min, and the amounts
of deacetylated products that were formed were measured as
described above. Experiments were performed in triplicate. The
Km and Vmax were computed by fitting the data to the Michaelis-
Menten equation with the EZ-FIT program (Perrella Scientific,
Inc., Conyers, GA).
for
1 h and then centrifuged at 9000g for 25 min. The
precipitated proteins were dissolved in a minimum volume of
10 mM potassium phosphate buffer (pH 7.4) and dialyzed
against the same buffer for 24 h. The dialyzed protein solution
was concentrated to 32 mL in an Amicon ultrafiltration cell and
applied to a DEAE-cellulose column (5.0 cm × 32 cm, Express
Ion-D, Whatman, Hillsboro, OR) equilibrated with 10 mM
potassium phosphate buffer (pH 7.4). The column was eluted
with 700 mL of 10 mM potassium phosphate buffer (pH 7.4)
and then with a linear gradient of 10 to 200 mM (∼650 mL)
potassium phosphate buffer (pH 7.4). Eluate fractions (8.2 mL)
were collected and assayed for protein concentrations and
enzyme activities. Fractions with activity with N-acetyl-L-
cysteine and N-acetyl-L-methionine as substrates were sepa-
rated from the fractions with activity with NADCVC as the
Deter m in a tion of th e Molecu la r Ma ss of th e P u r ified
Ra t Kid n ey Acyla se. The molecular mass of the purified rat