Many halogenated xenobiotics are detoxified by conversion to the
glutathione adduct, which is hydrolyzed to the
cysteine-S-conjugate and N-acetylated to the
corresponding mercapturate(1) . However, in some cases,
reactions through part of this pathway lead to bioactivation. For
example, trichloroethylene and dichloroacetylene (DA)
are
metabolized to the glutathione adduct (S-(1,2-dichlorovinyl)glutathione) and cysteine adduct (S-(1,2-dichlorovinyl)-L-cysteine, DCVC), both of
which are nephrotoxic to experimental animals(2) . DA is also
toxic to rabbit brain (3) and to human brain (4) (presumably via DCVC), resulting in degeneration of the
brainstem sensory nucleus of the trigeminal nerve and axons within its
tract(3, 4) . In the kidney, mitochondria are
especially vulnerable(5) . DCVC is a substrate of
cysteine-S-conjugate
-lyase(s) and is converted to
pyruvate, ammonia, and a fragment containing a reactive sulfhydryl (a retro-Michael elimination reaction). The reactive fragment is
a thioacetylating agent and binds to
macromolecules(6, 7) . The sulfhydryl-containing
breakdown product of DCVC apparently destroys renal epithelial cells by
a combination of covalent binding to proteins and nucleic acids,
depletion of non-protein thiols, and lipid peroxidation(8) .
Stevens previously showed that a major cysteine-S-conjugate
-lyase of the rat liver is kynureninase(9) . Somewhat
later, Stevens et al.(10) showed that a major
cysteine-S-conjugate
-lyase of rat kidney (DCVC as
substrate) is cytosolic glutamine transaminase K (cytGTK), an enzyme
previously characterized in this laboratory(11, 12) .
In the rat, DCVC-induced damage to the kidney is confined largely to
the S3 region. GTK is localized, however, to the S1(5) ,
S2(5) , and S3(5, 13) regions of the rat
nephron. (
)In addition, GTK in the rat brain is most
abundant in the choroid plexus with smaller concentrations in other
regions(14) . It seems reasonable to assume that cytGTK (in its
capacity as a cysteine-S-conjugate
-lyase) contributes to
the nephrotoxicity and neurotoxicity of DA. However, the lack of
correlation between the regional distribution of this enzyme in kidney
and brain and the regional susceptibility to DA-induced damage suggests
that other factors must be involved (for reviews, see (15) and (16) ). Some authors have suspected that rat kidney may contain
multiple cysteine-S-conjugate
-lyases(16) . To
investigate this possibility, we devised an activity stain that detects
DCVC
-lyase in tissue homogenates subjected to nondenaturing
polyacrylamide gel electrophoresis (ND-PAGE)(17) . Two major
bands of cysteine-S-conjugate
-lyase activity in rat
kidney homogenates are detected by this procedure(17) . The
lower staining band has an apparent M
of
90,000 and corresponds to GTK. The upper staining band has an
apparent M
of
330,000. The high M
species is present in relatively low
concentrations in rat liver homogenates(17) , but is not
detectable in brain(14) . Activity staining revealed that the
high M
species has some weak GTK-type
activity(17) . However, the relationship of the 330-kDa species
to GTK was not apparent. We have now partially purified the 330-kDa
species and have begun to characterize it. The enzyme is not closely
related to GTK and appears to be a previously unrecognized pyridoxal
5`-phosphate (PLP)-containing enzyme. Details are presented below.
EXPERIMENTAL PROCEDURES
Materials
All compounds were of the highest
quality available. L-Phenylalanine, pyridoxal 5`-phosphate
(PLP), dithiothreitol (DTT), sodium salts of
-keto-
-methiolbutyrate (
KMB) and other
-keto acids,
aminooxyacetate, Coomassie Blue, phenazine methosulfate (PMS), and
nitro blue tetrazolium (NBT), Crotalus adamanteusL-amino acid oxidase (8.9 units/mg) and beef liver
catalase (36,000 units/mg) were purchased from Sigma. Leukotriene
E
(LTE
) was obtained from Cayman Chemical Co.
(Ann Arbor, MI). 2,4-Dinitrophenylhydrazine (DNP) was obtained from
Eastman Kodak and crystallized from ethanol before use. S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFEC) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) were generous
gifts from Dr. James L. Stevens (W. Alton Jones Cell Science Center,
Lake Placid, NY). 5`-S-Cysteinyldopamine was kindly provided
by Dr. Jeffrey Tong (University of Toronto). Frozen rat (Wistar)
kidneys were obtained from Pel-Freez. Sodium cyclohexylpyruvate was
made essentially as described(18) . Bacillus subtilisL-alanine dehydrogenase (20 units/mg) was purchased from
Boehringer Mannheim.Protein determinations were done essentially by
the procedure of Lowry et al.(19) .
Enzyme Assays
Cytosolic glutamine transaminase K
(cytGTK) was purified from rat kidneys to homogeneity as described
previously (18, 20) . The specific activity
(transaminase assay; pH 9.0; 37 °C) was 8.9 units/mg of protein.
GTK was routinely assayed by measuring phenylpyruvate formed in
reaction mixtures (0.1 ml) containing 100 mM ammediol-HCl
buffer (pH 9.0), 20 mM phenylalanine, 5 mM
-keto-
-methiolbutyrate (
KMB), and
enzyme(20) . After incubation for 15-60 min at 37 °C,
0.9 ml of 3.3 M KOH is added, and the absorbance at 322 nm is
read (
= 24,000). In some
experiments, phenylalanine-
KMB transaminase activities of cytGTK
and high M
lyase were determined in the presence
of 100 mM potassium phosphate buffer (pH 7.2) or Tris-HCl
buffer (pH 8.0). One unit of GTK activity is defined as the amount of
enzyme that catalyzes the formation of 1 µmol of phenylpyruvate per
min at 37 °C (pH 9.0). DCVC lyase activity was assayed by two
procedures. In the first method, the reaction mixture contains 2 mM DCVC, 0.1 mM sodium phenylpyruvate, 100 mM ammediol-HCl buffer (pH 9.0), and enzyme in a volume of 0.1 ml.
After incubation at 37 °C, 0.4 ml of water is added, and the
phenylpyruvate is removed by addition of 5 mg of activated charcoal.
(Stevens et al.(10) previously reported that with
DCVC as substrate, the products of the GTK reaction are approximately
50% ``keto''DCVC (formed by transamination) and 50% pyruvate
(formed by lyase activity). In the present work, it was found that
``keto''DCVC is also absorbed by the charcoal treatment.)
Pyruvate is not absorbed by the charcoal. After centrifugation (12,000
g for 10 min), 0.4 ml of the supernatant is removed
and added to 50 µl of 0.2% (w/v) DNP in 2 N HCl. After 10
min at room temperature, 0.55 ml of 1 M KOH is added, and the
absorbance is read against a blank (lacking enzyme) carried through the
same procedure. The extinction coefficient of pyruvate DNPone is
17,000 under these conditions. Since 0.1 mM phenylpyruvate provides little stimulation of the high M
lyase-catalyzed reaction in the absence of PLP
(see ``Results''), this assay procedure provides a method for
distinguishing
-lyase activity due to cytGTK from that due to the
high M
lyase. In the second assay procedure for
-lyase activity, the reaction mixture contains 5 mM DCVC,
5 mM DTT, 5 mM
KMB, 100 µM PLP, 100
mM potassium phosphate buffer (pH 7.2), and enzyme in a final
volume of 0.1 ml. After incubation at 37 °C, pyruvate formation is
quantitated with alanine dehydrogenase as described(17) . (
)One unit of lyase activity is defined as the amount that
catalyzes the formation of 1 µmol of pyruvate from DCVC per min at
37 °C.Both low (cytGTK) and high M
lyases
catalyze the formation of pyruvate when incubated with the second
-lyase assay mixture. Thus, in order to estimate the lyase
activity due to the high M
enzyme in kidney
homogenates containing both enzymes, it is necessary to separate the
two in a small aliquot. This separation is accomplished by adding 1 ml
of the supernatant of the kidney homogenate to a hydroxylapatite column
(1
4 cm). The low M
lyase (cytGTK) is
eluted from the column with 20 ml of 50 mM potassium phosphate
buffer (pH 7.2). The column is then eluted with 3 ml of 1 M potassium phosphate buffer (pH 7.2). The high M
lyase is eluted between 1.5 and 3 ml.
In some experiments, the
purified high M
lyase (14 µg) was incubated at
37 °C for 1 h with 0.1 ml of a reaction mixture containing 10
mM of an amino acid (other than a cysteine conjugate), 5
mM DTT, 10 µM PLP, and 100 mM potassium
phosphate buffer (pH 7.2) to determine whether an
-keto acid was
produced. The reaction mixture was then treated with 50 µl of 0.2%
(w/v) DNP in 2 N HCl. After incubation at room temperature for
10 min, 0.85 ml of 1 M KOH was added, and the absorbance was
read at 430 nm against a blank lacking enzyme. Amino acids tested
included serine, cysteine, threonine, homoserine, homocysteine, and
cystathionine.
-Elimination from serine, cysteine, or
cystathionine will yield pyruvate;
-elimination from threonine and
-elimination from homoserine, homocysteine, and cystathionine will
give rise to
-ketobutyrate. Both pyruvate and
-ketobutyrate
readily form DNPones which strongly absorb at 430 nm in base (
= 13,000-19,000). In addition, if the high M
lyase has threonine aldolase (= serine
hydroxymethyltransferase), acetaldehyde will be generated from
threonine; acetaldehyde also reacts with DPN to form a DPNone.
Paper Chromatography
Typically, 0.2-ml reaction
mixtures were treated with 50 µl of 0.2% (w/v) DNP in 2 N HCl. After 20 min at room temperature, 20 µl of ethyl acetate
was added. After vigorous shaking, the ethyl acetate layer was spotted
5 µl at a time onto Whatman No. 20 chromatography paper. After
development by the ascending technique in a solvent consisting of sec-butyl alcohol:water (4:1, v/v), the DNPones were
visualized as yellow spots (21) . To increase the sensitivity
of detection and to cut down on background, the paper was sprayed with
50% ethanol:50% 1 M KOH (v/v). The pyruvate, phenylpyruvate,
and
KMB DNPones each migrate as two spots; DNP migrates as a
single spot close to the solvent front (R
0.94).
The R
values of the DNPones are as follows:
pyruvate (0.50, 0.65), phenylpyruvate (0.72, 0.85), and
KMB (0.68,
0.77). In each case, the more rapidly migrating spot remains yellow,
whereas the less rapidly migrating spot turns from yellow to an intense
brown color upon spraying with ethanol-KOH. The brown color then slowly
fades back to yellow. The more slowly migrating, base-sensitive spot is
assumed to be the trans-isomer, and the more rapidly
migrating, base-insensitive spot is assumed to be the cis-isomer(22) . One nanomole of
-keto acid
DNPone is easily visualized by this technique.
Electrophoresis Procedures
Nondenaturing
polyacrylamide gel electrophoresis (ND-PAGE) was carried out as
described previously (14) except that 1) the electrophoresis
was carried out at 50 V for 3 h, followed by 120 V for 12 h and 200 V
for 4 h, and 2) the gels contained a linear gradient (4-25%) of
polyacrylamide. Protein bands were visualized by staining with
Coomassie Blue (23) or with silver nitrate (24) . In
other cases, the gels were stained for glutamine transaminase K-type
activity (transamination between L-phenylalanine and
KMB)
or for lyase activity with DCVC (or with TFEC)(17) . Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
carried out by the procedure of Laemmli (25) except that the
gels were constructed from a linear gradient of 4-25%
polyacrylamide. Antibodies to cytosolic glutamine transaminase K were
prepared in rabbits as described previously(14) . Western blots
to cytGTK were carried out by a modification of the previously
described procedure (14) . Kidney homogenates, purified cytGTK,
or purified high M
lyases were subjected to
ND-PAGE. The position on the gel of the high M
lyase and the low M
lyase (cytGTK) was
determined by activity staining. The stained bands were then cut from
the gel and washed with water. The protein was extracted from these gel
fragments electrophoretically by using a unidirectional electroeluter
(IBI, New Haven, CT) as described in the manufacturer's
instructions. Alternatively, the protein was eluted passively from the
gel slices as described by Ward and Simpson(26) . The excised
gel pieces were placed in 1 ml of elution buffer containing 20 mM Tris-HCl (pH 7.4) and 0.02% (v/v) Tween 20 in a 1.5-ml
microcentrifuge tube and gently shaken at 25 °C for 48 h in the
dark. The tubes were then centrifuged at 12,000
g for
15 min. The supernatant was removed and dialyzed against 1 liter of 5
mM potassium phosphate buffer (pH 7.2) and concentrated by
using a Centricon 3 microconcentrator (Amicon, Beverly, MA). The
proteins thus obtained from the ND-PAGE corresponding to the high M
region and in the low M
region were then separately subjected to SDS-PAGE on a
4-15% linear polyacrylamide gradient and electrophoretically
transferred to a nitrocellulose membrane. After transfer, the
nitrocellulose paper was washed in Tris-buffered saline (TBS, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 3 mM KCl)
and blocked for 1 h with 2% (w/v) powdered nonfat ALBA milk dissolved
in the same buffer. Affinity-purified rabbit anti-rat kidney cytGTK (14) was added to the filter at a dilution of 1:200 and
incubated at room temperature for 2 h. The filter was washed twice with
TBS, once with TBS containing 0.05% (v/v) Tween 20, and finally with
TBS. The filter was then incubated with
I-labeled protein
A (30 µCi/µg; Amersham) for 2 h at room temperature and washed
as before. The filter was air-dried, exposed to Kodak X-Omat film (at
70 °C), and developed.
Purification of Cytosolic High M
Cysteine-S-Conjugate
-Lyase from Rat Kidneys
Unless
otherwise stated, all procedures were carried out at 0-4 °C.
Fifty rat kidneys (38 g) were homogenized in 200 ml of 0.25 M sucrose, 50 mM Tris-HCl (pH 7.3), 150 mM KCl, 50
MgCl
, and 1 mM EDTA. The crude homogenate was
centrifuged for 30 min at 3,000
g, followed by another
centrifugation at 14,000
g for 30 min, and a final
centrifugation at 100,000
g for 90 min. The
supernatant was then heated at 45 °C for 30 min. The resulting
coagulate was removed by centrifugation at 14,000
g for 30 min. The supernatant was then extensively dialyzed against
5 liters of 5 mM potassium phosphate buffer (pH 7.2). A
precipitate formed during the dialysis and was removed by
centrifugation. The supernatant was then applied to a column of DE52 (8
2.5 cm; Whatman) that had previously been equilibrated with 5
mM potassium phosphate buffer (pH 7.2). The column was washed
with 500 ml of the same buffer. Glutamine transaminase K and high M
lyase were eluted coincidentally from the column
by application of a linear gradient of potassium phosphate buffer
(5-200 mM, pH 7.2; 500 ml). At this point, it was
convenient to determine the elution position of both enzymes by
activity staining. The active fractions were combined, extensively
dialyzed against 5 mM potassium phosphate buffer (pH 7.2), and
applied to a column of hydroxylapatite (8
2.5 cm; Bio-Rad)
equilibrated with 5 mM potassium phosphate buffer (pH 7.2).
The column was then successively washed with 250 ml of 5 mM,
300 ml of 50 mM, and 150 ml of 100 mM potassium
phosphate buffer (pH 7.2). cytGTK was eluted in the 50 mM potassium phosphate wash, whereas the high M
lyase was not eluted by the 50 mM buffer or by the 100
mM buffer. The high M
lyase was eluted
from the column by application of a linear gradient of potassium
phosphate buffer (100-1000 mM, pH 7.2; 500 ml). Active
fractions, identified by activity staining, were combined and
extensively dialyzed against 5 mM potassium phosphate buffer
(pH 7.2). The enzyme was then concentrated by passing the dialysate
over a small column of DE52 (1
0.5 cm). The enzyme was then
eluted in
2 ml of 1 M potassium phosphate buffer (pH
7.2). The concentrated enzyme was applied to the top of a Sephadex
G-200 column (110
1.5 cm) equilibrated with 5 mM potassium phosphate buffer (pH 7.2) and eluted with the same
buffer. Active fractions detected by activity staining were pooled and
concentrated on a small DE52 column as described above. A summary of
the purification is presented in Table 1. The procedure has been
successfully repeated three times. Activity stains of enzyme at various
stages of the purification are presented in Fig. 1.
Figure 1:
DCVC
lyase activity staining on ND-PAGE at different stages of the
purification of high M
-lyase from
rat kidney homogenates. Lane 1, purified high M
lyase (30 µg of protein from the Sephadex G-22 step); lane 2, activity eluted from the hydroxylapatite column at
high potassium phosphate concentration (50 µg of protein); lane
3, activity eluted from the hydroxylapatite column at low
phosphate concentration and which contains cytGTK (20 µg of
protein); lane 4, DE52 eluate (100 µg of protein); lane 5, supernatant after heat treatment and dialysis (100
µg of protein); lane 6, 14,000
g supernatant (100 µg of protein); lane 7, 3,000
g supernatant; lane 8, crude kidney
homogenate (100 µg of protein). Note the co-purification of cytGTK
and high M
lyase on DE52 (lane 4) but
complete separation on hydroxylapatite (lanes 2 and 3). The gels were incubated in the dark at 25 °C in the
presence of 100 mM potassium phosphate buffer (pH 7.2), 1
mM DCVC, 0.6 mM
KMB, 0.1 mM PMS, and
0.1 mM NBT. After 1 h, the gel was photographed. The
polyacrylamide gradient is from 4-25% and the gel dimensions are
16
15
1.5 cm. The numbers on the left-hand
side refer to the positions of molecular weight protein standards
(thyroglobulin monomer (670,000), ferritin (440,000), catalase
(232,000), lactate dehydrogenase (140,000), and bovine serum albumin
(67,000)). Occasionally, on shallower gradients (5-15%), it was
noted that the purified high M
lyase exhibited an
apparent M
that was slightly lower than that
observed in crude homogenates (data not
shown).
Other Procedures
Alanine was measured as its o-phthaldialdehyde derivative by HPLC as
described(27) . Stock solutions of ``keto''DCVC were
made by incubating 5 mM DCVC with 0.6 unit of L-amino
acid oxidase and 10 units of catalase in 10 µl of 100 mM potassium phosphate buffer (pH 7.2) for 2 h at room temperature.
Frozen kidneys were homogenized and fractionated into crude
mitochondrial and cytosolic fractions as described(14) .
Analysis of the two fractions for glutamate dehydrogenase
(mitochondrial marker) and lactate dehydrogenase (cytosolic marker)
activities showed that, despite the freeze-thawing of the kidneys, the
crude cytosolic fraction contained <10% contamination with
mitochondrial matrix proteins and that the crude mitochondrial fraction
contained <15% contamination with cytosolic proteins. In some
experiments, ammonia generated in the the
-lyase reaction was
measured by a modification of the indophenol method of
Chaykin(28) . The method is quite sensitive but has the
disadvantage that it must be standardized for each application. The
following procedure was empirically found to give good results for the
-lyase assay. The reaction mixture contains 100 mM potassium phosphate buffer (pH 7.2), 5 mM DCVC, 100
µM PLP, 10 mM DTT, and enzyme in a final volume
of 0.5 ml. After incubation at 37 °C, the reaction is stopped by
the addition of 0.74 ml of 0.14 M phenol. The reaction mixture
is vigorously shaken. As the mixture is being shaken, 10 µl of 100
mM sodium nitroprusside is added and, immediately,
200 µl of alkaline bleach (1 ml of 0.75 M NaOH and 0.02 ml
of Clorox) is also added. After 20 min, the absorbance of the mixture
is read against a blank (assay mixture to which enzyme is added just
prior to addition of the phenol reagent) at 625 nm. Under these
conditions, 2 nmol of ammonia is easily detected.Data are expressed
as mean ± S.E. Statistical comparisons of control enzyme
activities versus activities in the presence of additions
(DTT, PLP,
-keto acids) was carried out using the Mann-Whitney U test. p values of <0.05 were considered to
indicate a significant difference.
RESULTS
Transaminase and Cysteine-S-Conjugate
-Lyase
Activities of Purified Rat Kidney cytGTK
These activities were
determined to provide a basis for comparison of low M
lyase (cytGTK) with the purified cytosolic rat kidney high M
lyase. The
phenylalanine-
-keto-
-methiolbutyrate transaminase activities
(37 °C) of purified cytGTK at pH 9.0 and at pH 7.2 are 8.9 ±
0.6 and 5.3 ± 0.3 µmol/min/mg of protein, respectively. The
transaminase activity is not stimulated by the addition of 10 mM DTT or of 0.1 mM PLP as was noted
previously(11, 12) . The
-lyase activities in the
presence of 5 mM
KMB (4 mM DCVC as substrate) at
pH 9.0 and pH 7.2 are 0.78 ± 0.08 and 0.47 ± 0.06
µmol/min/mg of protein, respectively. The corresponding values for
the DCVC lyase reaction in the presence of 0.1 mM phenylpyruvate are 0.76 ± 0.06 and 0.42 ± 0.02,
respectively. TFEC is a somewhat better substrate for the lyase
reaction than is DCVC. Thus,
-lyase activities with 4 mM TFEC in the presence of 5 mM
KMB are 2.90 ±
0.20 and 1.30 ± 0.04 µmol/min/mg of protein at pH 9.0 and
7.2, respectively. Evidently, although cytGTK has a high pH optimum,
considerable transaminase and
-lyase activities (
45-60%
of maximum) are manifest at physiological pH values. Interestingly, 5
mM DTT inhibits the
KMB-supported
-lyase activities
with DCVC and TFEC at pH 9.0 by 27% and 73%, respectively. However, 5
mM DTT has no effect on the
-lyase reaction with DCVC and
TFEC at pH 7.2. Ammonia was also measured in separate experiments. The
reaction mixture contained 100 mM potassium phosphate buffer
(pH 7.2), 5 mM DCVC, and 5 mM
KMB in a final
volume of 0.05 ml. In some cases, the reaction mixture also contained 5
mM DTT or 100 µM PLP. The rate of ammonia
production was 0.56 (no addition), 0.60 (+DTT), 1.26 (+PLP),
and 1.06 (+PLP + DTT) µmol/min/mg of protein; 37 °C
(average of two determinations). In addition, rates of ammonia
production when cytGTK was incubated with 100 mM potassium
phosphate buffer (pH 7.2), 5 mM sodium pyruvate (or
-ketoglutarate), and 5 mM DCVC (
0.02 µmol/min/mg
of protein) were not significantly different from rates obtained in the
absence of added
-keto acid. Although the transaminase activity of
cytGTK is not stimulated by the addition of PLP, the
-lyase
activity of this enzyme is stimulated by addition of this cofactor (Table 2).
General Properties of the Purified Cytosolic High M
Lyase
The current purification procedure provides a
preparation devoid of cytGTK but which is not homogeneous. Two bands
are visible when the purified enzyme preparation is subjected to
ND-PAGE and stained with Coomassie Blue (data not shown). The upper
band corresponds to a band which stains positively for
-lyase
activity with DCVC. The lower protein band on ND-PAGE (M
280,000) does not stain for
-lyase activity and is
presumably an impurity. The lower band is estimated to contribute at
least 40% to the protein content of the purified high M
lyase. The purified enzyme is stable at 4 °C in 20% glycerol,
5 mM potassium phosphate buffer (pH 7.2) for at least 3
months.Activity staining showed that the purified enzyme has an
apparent M
of approximately 330,000 on ND-PAGE (Fig. 2). The subunit composition was determined as follows. The
band corresponding to high M
lyase in the purified
enzyme preparation (delineated by activity staining) was excised from
the nondenaturing gel, passively eluted with 1 ml of 20 mM Tris-HCl (pH 7.4), 0.02% (v/v) Tween 20 for 48 h in the dark; 25
°C(26) . The eluted protein was dialyzed extensively
against 5 mM potassium phosphate buffer (pH 7.2) and
lyophilized. The residue was taken up in 100 µl of gel-loading
buffer and subjected to SDS-PAGE. Two bands were revealed by silver
staining of the denaturing gel. The two proteins have apparent M
values of
50,000 and
70,000 (data not
shown). The enzyme does not generate a product with an active carbonyl
when incubated with serine, cysteine, threonine, homoserine,
homocysteine, or cystathionine in the presence of PLP and DTT. (
)
Figure 2:
-Lyase activity staining of crude
cytosolic and mitochondrial homogenates of rat kidney. Rat kidney
cytosol (lanes 1, 3, 5, and 7; 150
µg of protein (
1.7 transaminase milliunits of cytGTK) in each
lane) and mitochondria (lanes 2, 4, 6, and 8; 150 µg of protein (
2.4 transaminase milliunits of
mitGTK) in each lane) were subjected to ND-PAGE and stained for
-lyase activity. The staining solution contained 100 mM potassium phosphate buffer (pH 7.2), 1 mM DCVC (or 1
mM TFEC), 0.6 mM
KMB (or 0.6 mM
-ketoglutarate (
KG)), 0.1 mM PMS, and
0.1 mM NBT. The gels were incubated in the dark for 2 h at 25
°C and then photographed. The numbers on the left-hand
side refer to the positions of molecular mass protein standards
(as in Fig. 1).
Transamination Catalyzed by the High M
Species
Activity staining experiments previously suggested
that the high M
lyase in crude kidney homogenates
possesses weak phenylalanine-
KMB transaminase
activity(17) . In the present work, we showed directly that the
purified high M
species does indeed possess this
activity ( Table 1and Table 3). In addition to
KMB,
other
-keto acids (chosen to represent a range of sizes, shapes,
and charges) were investigated as potential substrates of the high M
lyase-supported transaminase reaction. These
included glyoxylate, pyruvate, oxaloacetate,
-ketoglutarate,
-ketobutyrate,
-ketononanoate, cyclohexylpyruvate, and
branched-chain
-keto acids. Each (at a concentration of 5
mM) is active as an amine acceptor in the high M
-lyase-catalyzed transamination of
phenylalanine in the presence of 100 mM ammediol buffer (pH
9.0), 100 µM PLP, and 5 mM DTT. Activities
relative to that noted with 5 mM
KMB are
20-40%, except in the case of cyclohexylpyruvate where the
relative activity is
60%. More precise studies were not carried
out because of the low transaminase activity of the high M
-lyase. No increase in transaminase
activity was noted upon raising the concentration of
KMB from 5
mM to 40 mM, suggesting that the K
for
KMB is <5 mM. Phenylpyruvate was not tested
directly as a transaminase substrate of the high M
-lyase, but since the reaction between phenylalanine and
KMB is fully reversible, the enzyme is expected to utilize
phenylpyruvate as an amine acceptor. The pH profile of the
phenylalanine-
KMB transaminase reaction catalyzed by the high M
-lyase is quite broad; roughly equivalent
activity was noted in the presence of 100 mM potassium
phosphate (pH 7.2), 100 mM Tris-HCl (pH 8.0), and 100 mM ammediol (pH 9.0) buffers (Table 3). The
phenylalanine-
KMB transaminase activity of the high M
-lyase is stimulated by the addition of 10
mM DTT and by 100 µM PLP. The transaminase and
-lyase reactions are inhibited by 1 mM aminooxyacetate (Table 3).
Cysteine-S-Conjugate
-Lyase Reaction of the
Cytosolic High M
Species
The
-lyase activity of
the high M
species was routinely measured by
quantitating pyruvate formed from DCVC with alanine dehydrogenase. The
formation of pyruvate was also demonstrated by the following procedure.
A reaction mixture (0.1 ml) containing 5 mM DCVC, 5 mM
KMB (or 5 mM phenylpyruvate), 5 mM DTT, 100
µM PLP, 100 mM potassium phosphate (pH 7.2), and
14 µg of purified enzyme was incubated for 2 h at 37 °C. Acidic
DNP reagent was then added, and the resulting DNPones were separated by
paper chromatography as described under ``Experimental
Procedures.'' The cis- and trans-pyruvate
DNPones (which migrate with different R
values
from those of
KMB and phenylpyruvate) were readily detected. In
addition, the
-lyase reaction with DCVC as substrate is expected
to yield ammonia and 1-mercapto-1,2-dichloroethylene; however, the
latter compound is exceedingly unstable and has not been previously
isolated (see for example, (6) ). Thus, no attempt was made to
quantitate this fragment. The production of a species with a free
sulfhydryl was, however, indirectly demonstrated. The reaction mixture
contained 100 mM potassium phosphate buffer (pH 7.2), 0.2
mM DCVC, 0.5 mM
KMB, purified high M
-lyase (25 µg), 0.1 mM NBT,
and 0.1 mM PMS in a final volume of 100 µl. (The reaction
mixture lacked PLP or DTT as these interfere with the color
development.) Upon incubation at room temperature in the dark, the
solution rapidly (within a few mins) turned blue, indicating reduction
of the dye to a blue formazan. No color development occurred within 2 h
in the absence of enzyme; in the absence of
KMB, color development
was very slow. Other
-keto acids (0.5 mM) including
glyoxylate, pyruvate,
-ketoglutarate, cyclohexylpyruvate, and
-ketoisocaproate also stimulated the high M
-lyase-catalyzed color development, but more slowly than
with
KMB. The reaction was carried out at pH 7.2 because at higher
pH values a blue color forms in the absence of enzyme. Phenylpyruvate
slowly reacts with NBT/PMS precluding its inclusion in reaction
mixtures containing this dye system.In another experiment, ammonia
generated in the lyase reaction was also determined. The reaction
mixture (0.1 ml) contained 5 mM DCVC, 100 µM PLP,
5 mM DTT, and 100 mM potassium phosphate buffer (pH
7.2), and 14 µg of purified high M
lyase.
After incubation at 37 °C for 2 h, pyruvate (by the alanine
dehydrogenase reaction) and ammonia (by the indophenol method) were
separately quantitated. Pyruvate and ammonia formation were 0.11
± 0.02 and 0.13 ± 0.02 µmol, respectively. In a
separate experiment, it was shown that the
-lyase reaction with
DCVC under the above conditions is linear for at least 2 h.
Several
-keto acids also stimulate the
-lyase reaction. Thus,
pyruvate formation from 5 mM DCVC in 100 mM potassium
phosphate buffer (pH 7.2) in the presence of various
-keto acids
was <0.01 (no addition of
-keto acid), 0.05 ± 0.01
(+5 mM
KMB), 0.04 ± 0.02 (+5 mM
-ketoglutarate), 0.04 ± 0.01 (+5 mM phenylpyruvate), and <0.01 (+0.1 mM phenylpyruvate) µmol/min/mg of protein; 37 °C. Ammonia
formation from DCVC in the presence of 5 mM pyruvate was 0.03
± 0.01 µmol/min/mg of protein. PLP and DTT also stimulate
the
-lyase reaction (Table 3). PLP stimulates the lyase
activity of the high M
enzyme to an even greater
extent than do
-keto acids. Inclusion of 5 mM
-keto
acid (
KMB, pyruvate,
-ketoglutarate, phenylpyruvate) in a
reaction mixture containing 5 mM DCVC, 100 µM PLP, and 5 mM DTT did not result in any further
significant stimulation of
-lyase activity (data shown only for
KMB in Table 3).
Interaction of High M
Lyase with Leukotriene
E
(LTE
)
When 14 µg of purified high M
lyase was incubated in the dark at 25 °C in
a reaction mixture (200 µl) containing 22 µM LTE
, 2.5 mM
KMB, 0.1 mM NBT,
0.1 mM PMS, and 100 mM potassium phosphate buffer (pH
7.2) in a plastic tube, development of a blue formazan was rapid,
indicating release of a compound with a free sulfhydryl. No color
development was noted in the absence of enzyme or of
KMB. In
contrast, when purified high M
lyase in the above
reaction mixture was replaced by cytGTK, no color development was
observed. Because of the small quantity of substrate (
4 nmol), it
was not possible to quantitate pyruvate formation by measuring the
decrease of absorption at 340 nm upon addition of NADH (
= 2.23
10
), ammonium acetate and
alanine dehydrogenase. However, pyruvate formation was demonstrated
directly by other methods. Purified high M
lyase
(14 µg) was incubated in a reaction mixture (200 µl) containing
22 µM LTE
, 2.5 mM
KMB, 5 mM DTT, and 100 mM potassium phosphate (pH 7.2) under helium
in the dark for 24 h at 25 °C. The reaction mixture was then
treated with acidic DNP, and the
-keto acid DNPones were analyzed
by paper chromatography (see ``Experimental Procedures'').
Pyruvate DNPone was detected in the complete reaction mixture
containing high M
lyase, but not in mixtures
lacking
KMB or enzyme. In another experiment, purified high M
lyase (14 µg) was incubated with 200 µl
of 22 µM LTE
, 100 µM PLP, and 5
mM DTT in 100 mM potassium phosphate buffer (pH 7.2)
under helium. After 24 h at 25 °C,
3 nmol of ammonia
(determined by the indophenol method) was produced. No ammonia was
detected in a control lacking enzyme. In a separate experiment, the
above reaction mixture (100 µl) was treated with 100 µl of 100
µM NADH and 1 µl of alanine dehydrogenase (2.2 units
in 2.4 M ammonium sulfate). After incubation for 5 h, alanine
was measured by HPLC of the o-phthaldialdehyde derivative.
Approximately 1.0 nmol of alanine was detected in the complete reaction
mixture, but no alanine was detected in a control lacking enzyme.
Interaction of Purified Cytosolic High M
Lyase with 5`-S-Cysteinyldopamine
When the purified enzyme
(14 µg) was incubated at 25 °C in the dark in a reaction
mixture (200 µl) containing 2 mM 5`-S-cysteinyldopamine, 2.5 mM
KMB, 0.1
mM NBT, and 0.1 mM PMS in 100 mM potassium
phosphate buffer (pH 7.2), rapid color development was observed. When
enzyme or
KMB was omitted, color development was very slow. In
contrast, when high M
lyase was replaced by 1 unit
of purified cytGTK in the above reaction mixture, color development was
no different from that of a control lacking enzyme. Pyruvate and
ammonia formation in a reaction mixture (200 µl) containing 2.5
mM 5`-S-cysteinyldopamine, 5 mM DTT, 100
µM PLP, 100 mM potassium phosphate (pH 7.2), and
14 µg of enzyme were also determined separately by the alanine
dehydrogenase and indophenol methods, respectively. After 12 h at 25
°C, pyruvate and ammonia production were both
20 nmol. A
similar figure was obtained when the reaction mixture was treated with
NADH and alanine dehydrogenase followed by analysis for alanine
formation by HPLC.Of interest was the finding that when
5`-S-cysteinyldopamine is incubated with phenylpyruvate, a
nonenzymatic reaction occurs in which the free carbonyl of
phenylpyruvate is lost. The reaction involved is not clear.
Subcellular Distribution of High and Low M
Lyases in Rat Kidney Homogenates
Activity staining of rat
kidney homogenates shows that both the cytosolic and mitochondrial
compartments possess cysteine-S-conjugate
-lyase activity
(with both DCVC and TFEC) that migrates with an apparent M
of 330,000 (Fig. 2). Since (a)
approximately equal amounts of protein were added to each lane and (b) the intensity of the band in the mitochondrial fraction is
similar to that in the corresponding cytosolic fraction, the specific
activity of the high M
lyase in the mitochondria
must be similar to that in the cytosol. In contrast, the apparent
specific activity of
KMB-stimulated DCVC (and TFEC) lyase
migrating with an apparent M
of
90,000 is
lower in the mitochondrial fractions (lanes 2 and 4)
than that in the cytosolic fractions (lanes 1 and 3; Fig. 2) despite the fact that a greater activity of mitGTK than
of cytGTK (in terms of transaminase units) was applied to the gel
lanes.
-Ketoglutarate is a poor substrate for the transaminase
activity of rat kidney cytGTK(11) , and, as expected, this
-keto acid does not significantly support the
-lyase activity
of purified cytGTK (i.e. no staining is visible in lanes
5-8 in the region of the gel corresponding to a M
of 90,000; Fig. 2). In contrast to the
low M
lyase (cytGTK), the high M
lyase in the cytosolic and mitochondrial fractions is active with both
KMB and
-ketoglutarate.
Lack of Immunological Relatedness between High and Low
M
-Lyases
Western blot analysis showed that
affinity-purified rabbit antibodies to rat kidney cytGTK fail to react
with the high M
-lyase in rat kidney
homogenates (Fig. 3).
Figure 3:
Immunoblot analyses of high and low M
-lyases. Rat kidney homogenates
(containing 500 µg of protein;
8 transaminase milliunits of
GTK) were subjected to ND-PAGE followed by lyase activity staining. The
positions on the gel corresponding to the high and low M
-lyases were delineated by activity staining with DCVC, and
these regions were excised. The proteins in the excised gel pieces (see Fig. 1) were separately extracted either by electroelution (lanes 2 and 3) or by passive elution (lanes 4 and 5). The eluted proteins were then subjected to
SDS-PAGE and immunoblotted (see ``Experimental Procedures''). Lane 1, purified cytGTK standard (0.5 µg;
4.5
transaminase milliunits of GTK) subjected directly to SDS-PAGE and
immunoblotted; lane 2, electroeluted high M
lyase; lane 3, electroeluted low M
lyase (cytGTK); lane 4, passively eluted high M
lyase; lane 5, passively eluted low M
lyase. Lane 1 was exposed to film for
12 h, lanes 3 and 5 were exposed to film for 48 h,
and lanes 2 and 4 were exposed for 7 days. The numbers on the left-hand side refer to the positions
of molecular mass protein standards (fumarase (57,800), ovalbumin
(40,800), and triose-phosphate isomerase
(34,100)).
DISCUSSION
Comments on the Catalytic Properties of Purified Rat
Kidney cytGTK
Relatively large noncharged amino acids are
preferred substrates(11, 12, 31) , including
DCVC(10) . However, when DCVC binds at the active site,
-elimination competes with transamination(10) . For the
-elimination (
-lyase) reaction to proceed efficiently,
KMB (or other suitable
-keto acid) or PLP must be included in
the reaction mixture. The PMP form of the enzyme, resulting from
transamination between PLP and cysteine conjugate, cannot catalyze a
-elimination reaction. Addition of an
-keto acid to the
reaction mixture ensures that the PMP at the active site is
transaminated back to PLP; the PLP form of the enzyme can then catalyze
a further round of
-elimination(10, 15) . The
transaminase activity of purified rat kidney cytGTK is not stimulated
by addition of PLP, suggesting that the cofactor in the aldehyde form
is tightly bound (via Schiff base linkage to the
-nitrogen of an
active site lysine). The PMP at the active site, however, must be less
tightly bound as it is easily displaced by PLP.
-Lyase activity is
somewhat greater in the presence of PLP than of saturating levels of
KMB (Table 3). This is presumably due to more effective
conversion of the PMP form of the enzyme to the PLP form in the
presence of exogenous PLP than in the presence of
KMB.In our
hands, for cytGTK the ratio of phenylalanine-
KMB transaminase
activity to DCVC lyase activity is
7-11. The ratio may
depend on conditions of assay; others report a ratio of 5.5 (10) and 2.5(32) . The ratio for phenylalanine-
KMB
transaminase activity to TFEC lyase activity is
2-3.0. The
accumulated data suggest that the inherent maximal transaminase
activity of purified rat kidney cytGTK is somewhat greater than that of
-lyase activity. The present findings that TFEC is a more
effective substrate than is DCVC for the
-lyase reaction of cytGTK
is in accord with the previous finding of Hayden and Stevens (7) .
The production of pyruvate from DCVC and TFEC is
significantly diminished in the presence of 5 mM DTT at pH
9.0, but not at pH 7.2. The finding is in accord with the postulated
aminoacrylate intermediate (cf.(10) and (15) ) in the cytGTK-catalyzed
-lyase reaction. At pH 9.0,
a considerable portion of the DTT sulfhydryls is expected to be ionized
to -S
(thiolate) but not at pH 7.2. DTT (in the
thiolate form but not in the un-ionized form) is expected to add to the
double bond of aminoacrylate reducing the amount of pyruvate formed. On
the other hand, DTT has no effect on the transamination of
phenylalanine at either pH 7.2 or 9.0, suggesting that the transaminase
activity of cytGTK is not dependent on the presence of a reducing
agent.
Cytosolic aspartate aminotransferase converts
-chloroalanine, L-serine-O-sulfate, and L-cysteine sulfinate to pyruvate and ammonia. However, the
enzyme is slowly inactivated in the process (33, 34, 35) via attack of aminoacrylate on
the PLP Schiff base at the active site. Escherichia coliL-glutamate decarboxylase also catalyzes
-elimination with L-serine-O-sulfate and is
similarly inactivated(36) . On the other hand, inactivation of
bacterial aspartate decarboxylase by chloroalanine involves attack on a
susceptible group at the active site(37) . Interestingly, due
to nucleophilic attack on the aminoacrylate product, thiosulfate
protects cytosolic aspartate aminotransferase against L-serine-O-sulfate-induced inactivation(33) .
As noted in the introduction, kynureninase catalyzes a
-elimination reaction with DCVC(9) . The enzyme is
inactivated in the process(9) , presumably via aminoacrylate.
Interestingly, cytGTK is not inactivated by DCVC or by TFEC, and the
-lyase reaction in the presence of
KMB is linear for at least
2 h at pH 7.2 and 37 °C. Inactivation of cytosolic aspartate
aminotransferase and E. coli glutamate decarboxylase by
-substituted amino acids appears to involve aminoacrylate release
by hydrolysis of the PLP-eneamine intermediate and rotation in the
active site; the
carbon of aminoacrylate is positioned for
nucleophilic attack on carbon 4` of the coenzyme resulting in a
PLP-pyruvate adduct(35, 36) . Evidently, the geometry
at the active site of cytGTK is such that this rotation is hindered and
there are no catalytically essential residues attacked by
aminoacrylate.
Comments on the Catalytic Properties of the High M
Cysteine-S-Conjugate
-Lyase of Rat Kidney Cytosol
The
enzyme has an apparent native M
of
330,000
and is composed of at least two types of subunit with apparent M
values of
50,000 and
70,000. Like the
low M
enzyme (cytGTK), the high M
enzyme contains PLP, its
-lyase activity is stimulated by
PLP (or by
-keto acids), it is not inactivated by aminoacrylate,
and it possesses transaminase activity. However, the rate of the
transaminase reaction catalyzed by the cytosolic high M
-lyase is <1% that of the rate catalyzed by the low M
lyase (cytGTK) (calculated from the data in Table 1). The ratio of maximal
-lyase activity to
transaminase activity catalyzed by the high M
-lyase is
10 (Table 3). This value is much
greater than that of cytGTK where the ratio is
0.3. Nevertheless,
because some transamination does occur, PLP or
-keto acid must be
present for stimulation of the
-lyase reaction. As is the case
with cytGTK (Table 2), the
-lyase reaction of the high M
enzyme is more strongly stimulated by PLP than
by addition of
-keto acids (Table 3). Finally, the high M
species is active with large cysteine conjugates
such as cysteinyldopamine and LTE
, whereas cytGTK is not.
The data suggest that cytGTK and high M
lyase are
catalytically distinct enzymes. Moreover, antibodies to rat kidney
cytGTK do not react with high M
lyase on Western
blots (Fig. 3) suggesting that the protein structures of the two
enzymes are also distinct.As noted above, to date the only well
characterized mammalian cysteine-S-conjugate
-lyases are
kynureninase (9) and cytGTK(10) . Apparently, DCVC and
some other cysteine-S-conjugates are similar enough to the
physiological substrates that they bind effectively to the active
sites. The presence of a strong electron-withdrawing group attached to
the sulfur of the cysteine conjugate appears to promote a lyase
reaction that is not part of the usual reaction
pathway(10, 15) . Similarly, the high M
species may be a PLP enzyme that does not normally catalyze a
-elimination reaction but that is ``coerced'' into
catalyzing such a reaction because of the strong electron-withdrawing
group on the sulfur of the cysteine conjugate. Alternatively, it is
possible that the high M
lyase is a PLP enzyme
with a physiologically important
-lyase function. Serine and
threonine can undergo
-elimination reactions to generate pyruvate
and
-ketobutyrate, respectively.
-Cystathionase catalyzes a
-elimination reaction with cystathionine (or homoserine), but can
also catalyze
-elimination reactions with cysteine (or cystine) to
yield pyruvate, ammonia, and H
S (or sulfocysteine) (cf. (38) ). The high M
enzyme
appears to be too large (calculated weight from ND-PAGE, 330,000) to be
serine (=threonine) deaminase (rat liver enzyme,
63,000(39) ) and
-cystathionase (rat liver enzyme,
160,000(38) ), and the purified high M
lyase has no detectable activity with serine, threonine,
homoserine, and cystathionine. As noted above, glutamate decarboxylase
(
100,000 Da) and aspartate aminotransferase (
90,000 Da) can
catalyze
-elimination reactions (see discussion in (40) ),
but clearly the activity of the high M
lyase
cannot be due to these PLP enzymes. Other possible PLP enzymes ruled
out as being identical with the high M
-lyase
on the basis of size and substrate specificity include cystathionine
-synthase (rat liver enzyme, 96,000(41) ) and serine
hydroxymethyltransferase (rabbit liver,
230,000(42) ).
Possible in Vivo Reactions Catalyzed by the High M
Lyase
LTE
(along with LTC
and
LTD
) is the slowly reacting substance of anaphylaxis.
LTE
is a potent modulator of renal function and, when
infused in vivo, stimulates both renal blood flow and
glomerular filtration(43, 44, 45) . Such
effects may be due to vasoconstrictor action and modulation of
mesangial cell contraction (46, 47) . LTC
(glutathione conjugate) and LTD
(
-glutamylcysteine conjugate) are rapidly converted to
LTE
(cysteine conjugate) in vivo(48) .
LTE
is deactivated by N-acetylation in the kidney
tubules, and the N-acetylated compound is excreted (49) . Bernström et al.(50) showed that a gut bacterium (Eubacterium
limosum) is capable of converting LTE
to a fragment
(5-hydroxy-6-mercapto-7,9-trans-11,14-cis-eicosatetraenoic
acid) containing a free sulfhydryl group. The authors stated that the
rat kidney lyase (presumably cytGTK) is not active with
LTE
, but gave no details. In confirmation of this
statement, we found that highly purified cytGTK is inactive with
LTE
. However, we were able to show that LTE
is
a substrate of the rat kidney cytosolic high M
-lyase. The products of the reaction included pyruvate,
ammonia, and a fragment containing a reactive sulfhydryl. The exact
turnover number, the chemical nature of the eliminated fragment, and
kinetic characteristics must await further studies. Detailed metabolic
studies of the fate of LTE
in humans have not so far
revealed a fragment derived from a lyase reaction on
LTE
(51) . Moreover, balance studies with labeled
leukotrienes (49) would seem to suggest that the conversion of
LTE
to a new compound via an elimination reaction, if it
occurs in vivo, would be a quantitatively minor pathway in rat
kidney. Nevertheless, since high concentrations of LTE
and
of the high M
-lyase occur within the
kidneys, it is possible that one of the natural functions of the high M
-lyase is to convert LTE
to low
levels of yet another type of leukotriene within that organ.Hydoxyl
radicals react rapidly with dopamine to form neurotoxic 2-, 5-, and
6-hydroxydopamines(52) , which avidly react with
sulfhydryl-containing compounds in vivo(53) . Reaction
with cysteine yields S-cysteinyldopamines.
6-S-Cysteinyldopamine has been detected in brain(54) .
Cysteinyldopamine conjugates may be substrates for the kidney lyases
and thereby contribute to the neurotoxicity. (
)5`-S-Cysteinyldopamine is not a substrate of rat
kidney cytGTK, but a compound containing a reactive sulfhydryl is
generated when 5`-S-cysteinyldopamine is incubated with
KMB and high M
-lyase. The nature of the
eliminated species and whether the reaction occurs in vivo remain to be determined. Moreover, 5`-S-cysteinyldopamine
reacts with phenylpyruvate nonenzymatically (present work). (
)Evidently, the chemistry of this interesting cysteinyl
conjugate in the presence of
-keto acids and high M
-lyase needs to be further elucidated.
Some evidence
suggests that multiple forms of cysteine-S-conjugate
-lyase may exist in the rat kidney. Thus, S-2-benzothiazolyl-L-cysteine is known to undergo
-elimination in the rat kidney, but this cysteine conjugate is not
a substrate of highly purified cytGTK(32) .
-Lyase
activity with S-2-benzothiazolyl-L-cysteine in rat
kidney cytosol of starved female rats was increased
200% by prior
treatment with a dose of N-acetyl-S-(1,2,3,4,4-pentadienyl)-L-cysteine,
but the activities of DCVC lyase, TFEC lyase, and cytGTK were only
increased by
63, 38, and 39%, respectively(55) . The
present results confirm that the rat kidney contains at least two types
of cysteine-S-conjugate
-lyases.
As noted in the
introduction, DCVC induces damage to the S3 region of the nephron in
experimental animals, in part, by destroying mitochondria. Since (a) cytGTK is present in nephrons, (b) cytGTK is a
DCVC lyase, and (c) GTK activity is present in mitochondria,
it has been assumed that mitGTK may somehow contribute to the damage.
However, purified mitGTK has relatively little DCVC and TFEC lyase
activities. (
)On the other hand, the high M
species is present in the S3 region of the nephron
and in both the mitochondrial and cytosolic fractions of rat
kidney (Fig. 2). (Whether the high M
species exists in two isoforms has not yet been determined.) The
-lyase activities of both the high and low M
enzymes in vitro are strongly dependent on the presence
of PLP or
-keto acids. Whether endogenous levels of PLP are
adequate to maintain the
-lyase activities in vivo is not
known, but some data suggest that endogenous
-keto acid levels are
sufficient to promote
-lyase activity in the kidney in
vivo. The DCVC lyase activities of both the low and high M
species are stimulated by 5 mM
KMB, whereas only the low M
species is
significantly stimulated by 0.1 mM phenylpyruvate. The
concentrations of
KMB acid and phenylpyruvate in the rat kidney in vivo are
1 µM(
)and
2
µM(56) , respectively. Since the apparent K
exhibited by cytGTK for phenylpyruvate is
10
µM(11, 12) , it is possible that enough
of the enzyme can be maintained in a PLP form to significantly
contribute to metabolism of DCVC and some other
cysteine-S-conjugates in the kidney in vivo. On the
other hand, the DCVC lyase activity of the high M
species (but not the low M
species) is
activated by
-ketoglutarate (Fig. 2). In the experiments
reported in Fig. 2,
-ketoglutarate was present at a
concentration of 0.6 mM. The concentrations of
-ketoglutarate in rat kidney is 0.11-0.32
mM(57) . Thus, it is possible that the high M
-lyase contributes to the turnover of
cysteine conjugates in both the cytosolic and mitochondrial
compartments of renal cells. Therefore, the high M
-lyase may contribute to the nephrotoxicity of DCVC (and
similar cysteine conjugates) and may exacerbate mitochondrial damage.