©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation from Rat Kidney of a Cytosolic High Molecular Weight Cysteine-S-Conjugate -Lyase with Activity toward Leukotriene E(*)

(Received for publication, August 18, 1994; and in revised form, October 3, 1994)

Dicky G. Abraham (1)(§) Pulin P. Patel (1) Arthur J. L. Cooper (1) (2)(¶)

From the  (1)Departments of Biochemistry and (2)Neurology and Neuroscience, Cornell University Medical College, New York, New York 10021

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A cytosolic high M(r) cysteine-S-conjugate beta-lyase (apparent M(r) of 330,000) has been partially purified from rat kidneys. The high M(r) lyase is also present in the mitochondria. The purified enzyme contains at least two proteins with apparent M(r) values of 50,000 and 70,000. Activity is stimulated by dithiothreitol, alpha-keto acids, and pyridoxal 5`-phosphate; aminooxyacetate is an inhibitor. The enzyme catalyzes a competing (half) transamination reaction between pyridoxal 5`-phosphate cofactor and cysteine-S-conjugate substrate; added alpha-keto acids promote conversion of active site pyridoxamine 5`-phosphate to pyridoxal 5`-phosphate. The enzyme also catalyzes a full (but weak) transamination between L-phenylalanine and alpha-keto--methiolbutyrate. The purified enzyme is not recognized by polyclonal rabbit antibodies to cytosolic rat kidney glutamine transaminase K (another cysteine-S-conjugate beta-lyase of rat kidney) and has no obvious similarities to other pyridoxal 5`-phosphate-containing enzymes. In addition to catalyzing elimination reactions with S-(1,2-dichlorovinyl)-L-cysteine and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, the enzyme reacts with leukotriene E(4) and 5`-S-cysteinyldopamine. Finally, the cytosolic and mitochondrial enzymes are activated by alpha-ketoglutarate. Thus, the possibility must be considered that, in kidneys of animals exposed to various cysteine conjugates, the high M(r) lyase contributes to the generation of pyruvate, ammonia, and reactive fragments in vivo. Many cysteine conjugates are nephrotoxic, and the high M(r) lyase(s) may be involved.


INTRODUCTION

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)^1 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 beta-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 beta-lyase of the rat liver is kynureninase(9) . Somewhat later, Stevens et al.(10) showed that a major cysteine-S-conjugate beta-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. (^2)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 beta-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 beta-lyases(16) . To investigate this possibility, we devised an activity stain that detects DCVC beta-lyase in tissue homogenates subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE)(17) . Two major bands of cysteine-S-conjugate beta-lyase activity in rat kidney homogenates are detected by this procedure(17) . The lower staining band has an apparent M(r) of 90,000 and corresponds to GTK. The upper staining band has an apparent M(r) of 330,000. The high M(r) 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(r) 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 alpha-keto--methiolbutyrate (alphaKMB) and other alpha-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(4) (LTE(4)) 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 alpha-keto--methiolbutyrate (alphaKMB), 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-alphaKMB transaminase activities of cytGTK and high M(r) 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 times 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(r) lyase-catalyzed reaction in the absence of PLP (see ``Results''), this assay procedure provides a method for distinguishing beta-lyase activity due to cytGTK from that due to the high M(r) lyase. In the second assay procedure for beta-lyase activity, the reaction mixture contains 5 mM DCVC, 5 mM DTT, 5 mM alphaKMB, 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) . (^3)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(r) lyases catalyze the formation of pyruvate when incubated with the second beta-lyase assay mixture. Thus, in order to estimate the lyase activity due to the high M(r) 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 times 4 cm). The low M(r) 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(r) lyase is eluted between 1.5 and 3 ml.

In some experiments, the purified high M(r) 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 alpha-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. beta-Elimination from serine, cysteine, or cystathionine will yield pyruvate; beta-elimination from threonine and -elimination from homoserine, homocysteine, and cystathionine will give rise to alpha-ketobutyrate. Both pyruvate and alpha-ketobutyrate readily form DNPones which strongly absorb at 430 nm in base ( = 13,000-19,000). In addition, if the high M(r) 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 alphaKMB DNPones each migrate as two spots; DNP migrates as a single spot close to the solvent front (R(F) 0.94). The R(F) values of the DNPones are as follows: pyruvate (0.50, 0.65), phenylpyruvate (0.72, 0.85), and alphaKMB (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 alpha-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 alphaKMB) 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(r) lyases were subjected to ND-PAGE. The position on the gel of the high M(r) lyase and the low M(r) 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 times 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(r) region and in the low M(r) 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(r) Cysteine-S-Conjugate beta-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(2), and 1 mM EDTA. The crude homogenate was centrifuged for 30 min at 3,000 times g, followed by another centrifugation at 14,000 times g for 30 min, and a final centrifugation at 100,000 times 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 times 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 times 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(r) 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 times 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(r) lyase was not eluted by the 50 mM buffer or by the 100 mM buffer. The high M(r) 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 times 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 times 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 beta-lyase from rat kidney homogenates. Lane 1, purified high M(r) 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 times g supernatant (100 µg of protein); lane 7, 3,000 times g supernatant; lane 8, crude kidney homogenate (100 µg of protein). Note the co-purification of cytGTK and high M(r) 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 alphaKMB, 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 times 15 times 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(r) lyase exhibited an apparent M(r) 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 beta-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 beta-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, alpha-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 beta-Lyase Activities of Purified Rat Kidney cytGTK

These activities were determined to provide a basis for comparison of low M(r) lyase (cytGTK) with the purified cytosolic rat kidney high M(r) lyase. The phenylalanine-alpha-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 beta-lyase activities in the presence of 5 mM alphaKMB (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, beta-lyase activities with 4 mM TFEC in the presence of 5 mM alphaKMB 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 beta-lyase activities (45-60% of maximum) are manifest at physiological pH values. Interestingly, 5 mM DTT inhibits the alphaKMB-supported beta-lyase activities with DCVC and TFEC at pH 9.0 by 27% and 73%, respectively. However, 5 mM DTT has no effect on the beta-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 alphaKMB 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 alpha-ketoglutarate), and 5 mM DCVC (0.02 µmol/min/mg of protein) were not significantly different from rates obtained in the absence of added alpha-keto acid. Although the transaminase activity of cytGTK is not stimulated by the addition of PLP, the beta-lyase activity of this enzyme is stimulated by addition of this cofactor (Table 2).



General Properties of the Purified Cytosolic High M(r) 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 beta-lyase activity with DCVC. The lower protein band on ND-PAGE (M(r) 280,000) does not stain for beta-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(r) 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(r) of approximately 330,000 on ND-PAGE (Fig. 2). The subunit composition was determined as follows. The band corresponding to high M(r) 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(r) 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. (^4)


Figure 2: beta-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 beta-lyase activity. The staining solution contained 100 mM potassium phosphate buffer (pH 7.2), 1 mM DCVC (or 1 mM TFEC), 0.6 mM alphaKMB (or 0.6 mM alpha-ketoglutarate (alphaKG)), 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(r) Species

Activity staining experiments previously suggested that the high M(r) lyase in crude kidney homogenates possesses weak phenylalanine-alphaKMB transaminase activity(17) . In the present work, we showed directly that the purified high M(r) species does indeed possess this activity ( Table 1and Table 3). In addition to alphaKMB, other alpha-keto acids (chosen to represent a range of sizes, shapes, and charges) were investigated as potential substrates of the high M(r) lyase-supported transaminase reaction. These included glyoxylate, pyruvate, oxaloacetate, alpha-ketoglutarate, alpha-ketobutyrate, alpha-ketononanoate, cyclohexylpyruvate, and branched-chain alpha-keto acids. Each (at a concentration of 5 mM) is active as an amine acceptor in the high M(r) beta-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 alphaKMB 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(r) beta-lyase. No increase in transaminase activity was noted upon raising the concentration of alphaKMB from 5 mM to 40 mM, suggesting that the K(m) for alphaKMB is <5 mM. Phenylpyruvate was not tested directly as a transaminase substrate of the high M(r) beta-lyase, but since the reaction between phenylalanine and alphaKMB is fully reversible, the enzyme is expected to utilize phenylpyruvate as an amine acceptor. The pH profile of the phenylalanine-alphaKMB transaminase reaction catalyzed by the high M(r) beta-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-alphaKMB transaminase activity of the high M(r) beta-lyase is stimulated by the addition of 10 mM DTT and by 100 µM PLP. The transaminase and beta-lyase reactions are inhibited by 1 mM aminooxyacetate (Table 3).



Cysteine-S-Conjugate beta-Lyase Reaction of the Cytosolic High M(r) Species

The beta-lyase activity of the high M(r) 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 alphaKMB (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(F) values from those of alphaKMB and phenylpyruvate) were readily detected. In addition, the beta-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 alphaKMB, purified high M(r) beta-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 alphaKMB, color development was very slow. Other alpha-keto acids (0.5 mM) including glyoxylate, pyruvate, alpha-ketoglutarate, cyclohexylpyruvate, and alpha-ketoisocaproate also stimulated the high M(r) beta-lyase-catalyzed color development, but more slowly than with alphaKMB. 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(r) 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 beta-lyase reaction with DCVC under the above conditions is linear for at least 2 h.

Several alpha-keto acids also stimulate the beta-lyase reaction. Thus, pyruvate formation from 5 mM DCVC in 100 mM potassium phosphate buffer (pH 7.2) in the presence of various alpha-keto acids was <0.01 (no addition of alpha-keto acid), 0.05 ± 0.01 (+5 mM alphaKMB), 0.04 ± 0.02 (+5 mM alpha-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 beta-lyase reaction (Table 3). PLP stimulates the lyase activity of the high M(r) enzyme to an even greater extent than do alpha-keto acids. Inclusion of 5 mM alpha-keto acid (alphaKMB, pyruvate, alpha-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 beta-lyase activity (data shown only for alphaKMB in Table 3).

Interaction of High M(r) Lyase with Leukotriene E(4) (LTE(4))

When 14 µg of purified high M(r) lyase was incubated in the dark at 25 °C in a reaction mixture (200 µl) containing 22 µM LTE(4), 2.5 mM alphaKMB, 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 alphaKMB. In contrast, when purified high M(r) 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 times 10^3), ammonium acetate and alanine dehydrogenase. However, pyruvate formation was demonstrated directly by other methods. Purified high M(r) lyase (14 µg) was incubated in a reaction mixture (200 µl) containing 22 µM LTE(4), 2.5 mM alphaKMB, 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 alpha-keto acid DNPones were analyzed by paper chromatography (see ``Experimental Procedures''). Pyruvate DNPone was detected in the complete reaction mixture containing high M(r) lyase, but not in mixtures lacking alphaKMB or enzyme. In another experiment, purified high M(r) lyase (14 µg) was incubated with 200 µl of 22 µM LTE(4), 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(r) 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 alphaKMB, 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 alphaKMB was omitted, color development was very slow. In contrast, when high M(r) 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(r) Lyases in Rat Kidney Homogenates

Activity staining of rat kidney homogenates shows that both the cytosolic and mitochondrial compartments possess cysteine-S-conjugate beta-lyase activity (with both DCVC and TFEC) that migrates with an apparent M(r) 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(r) lyase in the mitochondria must be similar to that in the cytosol. In contrast, the apparent specific activity of alphaKMB-stimulated DCVC (and TFEC) lyase migrating with an apparent M(r) 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.

alpha-Ketoglutarate is a poor substrate for the transaminase activity of rat kidney cytGTK(11) , and, as expected, this alpha-keto acid does not significantly support the beta-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(r) of 90,000; Fig. 2). In contrast to the low M(r) lyase (cytGTK), the high M(r) lyase in the cytosolic and mitochondrial fractions is active with both alphaKMB and alpha-ketoglutarate.

Lack of Immunological Relatedness between High and Low M(r) beta-Lyases

Western blot analysis showed that affinity-purified rabbit antibodies to rat kidney cytGTK fail to react with the high M(r) beta-lyase in rat kidney homogenates (Fig. 3).


Figure 3: Immunoblot analyses of high and low M beta-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(r) beta-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(r) lyase; lane 3, electroeluted low M(r) lyase (cytGTK); lane 4, passively eluted high M(r) lyase; lane 5, passively eluted low M(r) 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, beta-elimination competes with transamination(10) . For the beta-elimination (beta-lyase) reaction to proceed efficiently, alphaKMB (or other suitable alpha-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 beta-elimination reaction. Addition of an alpha-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 beta-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. beta-Lyase activity is somewhat greater in the presence of PLP than of saturating levels of alphaKMB (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 alphaKMB.

In our hands, for cytGTK the ratio of phenylalanine-alphaKMB 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-alphaKMB 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 beta-lyase activity. The present findings that TFEC is a more effective substrate than is DCVC for the beta-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 beta-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 beta-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 beta-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 beta-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 beta-lyase reaction in the presence of alphaKMB 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 beta-substituted amino acids appears to involve aminoacrylate release by hydrolysis of the PLP-eneamine intermediate and rotation in the active site; the beta 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(r) Cysteine-S-Conjugate beta-Lyase of Rat Kidney Cytosol

The enzyme has an apparent native M(r) of 330,000 and is composed of at least two types of subunit with apparent M(r) values of 50,000 and 70,000. Like the low M(r) enzyme (cytGTK), the high M(r) enzyme contains PLP, its beta-lyase activity is stimulated by PLP (or by alpha-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(r) beta-lyase is <1% that of the rate catalyzed by the low M(r) lyase (cytGTK) (calculated from the data in Table 1). The ratio of maximal beta-lyase activity to transaminase activity catalyzed by the high M(r) beta-lyase is 10 (Table 3). This value is much greater than that of cytGTK where the ratio is leq0.3. Nevertheless, because some transamination does occur, PLP or alpha-keto acid must be present for stimulation of the beta-lyase reaction. As is the case with cytGTK (Table 2), the beta-lyase reaction of the high M(r) enzyme is more strongly stimulated by PLP than by addition of alpha-keto acids (Table 3). Finally, the high M(r) species is active with large cysteine conjugates such as cysteinyldopamine and LTE(4), whereas cytGTK is not. The data suggest that cytGTK and high M(r) lyase are catalytically distinct enzymes. Moreover, antibodies to rat kidney cytGTK do not react with high M(r) 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 beta-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(r) species may be a PLP enzyme that does not normally catalyze a beta-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(r) lyase is a PLP enzyme with a physiologically important beta-lyase function. Serine and threonine can undergo beta-elimination reactions to generate pyruvate and alpha-ketobutyrate, respectively. -Cystathionase catalyzes a -elimination reaction with cystathionine (or homoserine), but can also catalyze beta-elimination reactions with cysteine (or cystine) to yield pyruvate, ammonia, and H(2)S (or sulfocysteine) (cf. (38) ). The high M(r) 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(r) 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 beta-elimination reactions (see discussion in (40) ), but clearly the activity of the high M(r) lyase cannot be due to these PLP enzymes. Other possible PLP enzymes ruled out as being identical with the high M(r) beta-lyase on the basis of size and substrate specificity include cystathionine beta-synthase (rat liver enzyme, 96,000(41) ) and serine hydroxymethyltransferase (rabbit liver, 230,000(42) ).

Possible in Vivo Reactions Catalyzed by the High M(r) Lyase

LTE(4) (along with LTC(4) and LTD(4)) is the slowly reacting substance of anaphylaxis. LTE(4) 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(4) (glutathione conjugate) and LTD(4) (-glutamylcysteine conjugate) are rapidly converted to LTE(4) (cysteine conjugate) in vivo(48) . LTE(4) 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(4) 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(4), but gave no details. In confirmation of this statement, we found that highly purified cytGTK is inactive with LTE(4). However, we were able to show that LTE(4) is a substrate of the rat kidney cytosolic high M(r) beta-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(4) in humans have not so far revealed a fragment derived from a lyase reaction on LTE(4)(51) . Moreover, balance studies with labeled leukotrienes (49) would seem to suggest that the conversion of LTE(4) 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(4) and of the high M(r) beta-lyase occur within the kidneys, it is possible that one of the natural functions of the high M(r) beta-lyase is to convert LTE(4) 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)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 alphaKMB and high M(r) beta-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). (^6)Evidently, the chemistry of this interesting cysteinyl conjugate in the presence of alpha-keto acids and high M(r) beta-lyase needs to be further elucidated.

Some evidence suggests that multiple forms of cysteine-S-conjugate beta-lyase may exist in the rat kidney. Thus, S-2-benzothiazolyl-L-cysteine is known to undergo beta-elimination in the rat kidney, but this cysteine conjugate is not a substrate of highly purified cytGTK(32) . beta-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 beta-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. (^7)On the other hand, the high M(r) species is present in the S3 region of the nephron^2 and in both the mitochondrial and cytosolic fractions of rat kidney (Fig. 2). (Whether the high M(r) species exists in two isoforms has not yet been determined.) The beta-lyase activities of both the high and low M(r) enzymes in vitro are strongly dependent on the presence of PLP or alpha-keto acids. Whether endogenous levels of PLP are adequate to maintain the beta-lyase activities in vivo is not known, but some data suggest that endogenous alpha-keto acid levels are sufficient to promote beta-lyase activity in the kidney in vivo. The DCVC lyase activities of both the low and high M(r) species are stimulated by 5 mM alphaKMB, whereas only the low M(r) species is significantly stimulated by 0.1 mM phenylpyruvate. The concentrations of alphaKMB acid and phenylpyruvate in the rat kidney in vivo are leq1 µM(^8)and 2 µM(56) , respectively. Since the apparent K(m) exhibited by cytGTK for phenylpyruvate is leq10 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(r) species (but not the low M(r) species) is activated by alpha-ketoglutarate (Fig. 2). In the experiments reported in Fig. 2, alpha-ketoglutarate was present at a concentration of 0.6 mM. The concentrations of alpha-ketoglutarate in rat kidney is 0.11-0.32 mM(57) . Thus, it is possible that the high M(r) beta-lyase contributes to the turnover of cysteine conjugates in both the cytosolic and mitochondrial compartments of renal cells. Therefore, the high M(r) beta-lyase may contribute to the nephrotoxicity of DCVC (and similar cysteine conjugates) and may exacerbate mitochondrial damage.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 16739. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Merck Research Laboratories, West Point, PA 19486.

To whom correspondence and reprint requests should be addressed. Tel.: 212-746-6427; Fax: 212-746-8875. ajlc{at}cumc.cornell.edu.

(^1)
The abbreviations used are: TE, trichloroethylene; alphaKMB, alpha-keto--methiolbutyrate; ammediol, 2-amino-2-methyl-1,3-propanediol; Coomassie Blue, Coomassie Brilliant Blue R-250; cyt, cytosolic; DA, dichloroacetylene; DCVC, S-(1,2-dichlorovinyl)-L-cysteine; DNP, 2,4-dinitrophenylhydrazine; DNPone, 2,4-dinitrophenylhydrazone; DTT, dithiothreitol; K, kidney type; ``keto''DCVC, alpha-keto-(S-1,2-dichlorovinyl)--mercaptobutyrate; GTK, glutamine transaminase K; HPLC, high performance liquid chromatography; LTE(4), leukotriene E(4); lyase, cysteine-S-conjugate beta-lyase; mit, mitochondrial; NBT, nitro blue tetrazolium; ND-PAGE, nondenaturing polyacrylamide gel electrophoresis; PLP, pyridoxal 5`-phosphate; PMP, pyridoxamine 5`-phosphate; PMS, phenazine methosulfate; TBS, Tris-buffered saline; TFEC, S-(1,1,2,2-tetrafluoroethyl)-L-cysteine.

(^2)
H. Endou and A. J. L. Cooper, unpublished observations.

(^3)
The pyruvate formed in the lyase reaction is reductively aminated to alanine with subsequent loss of absorbance at 340 nm due to conversion of NADH to NAD. The reaction with alanine dehydrogenase is complete in 10 min, but if there is considerable lyase present, turnover of DCVC to pyruvate may continue in the alanine dehydrogenase reaction mixture resulting in a slow downward drift in the absorbance at 340 nm. This can be avoided by heating the sample at 90 °C for 2 min to inactivate the lyase before assaying for pyruvate with alanine dehydrogenase.

(^4)
alpha-Keto acids could also possibly arise from the action of L-amino acid oxidase. However, the M(r) of the rat kidney L-amino acid oxidase (240,000 ((29) )) is considerably less than that of the high M(r) species, and L-amino acid oxidase does not contain PLP. Moreover, the weak L-amino acid oxidase activity (actually a property of the B form of L-hydroxy acid oxidase(29) ) has a pH optimum of 9.6 and exhibits relatively little activity at pH 7.2(30) . Nevertheless, the purified high M(r) beta-lyase was checked to determine whether it contains L-amino acid oxidase activity. Purified enzyme (14 µg) was incubated at 37 °C for 2 h in a reaction mixture (0.1 ml) containing 10 mM L-leucine (a preferred substrate of rat kidney L-amino acid oxidase (30) ), 100 units of catalase, and 100 mM potassium phosphate buffer, pH 7.2 (or ammediol-HCl buffer, pH 9.0). No alpha-keto acid was detected at either pH with the DNP reagent.

(^5)
J. Tong and A. D. Baines, personal communication.

(^6)
J. Tong, personal communication.

(^7)
D. G. Abraham and A. J. L. Cooper, unpublished results.

(^8)
A. J. L. Cooper, unpublished results.


ACKNOWLEDGEMENTS

We thank Karin Rimpel-Lamhaouar, Randi Thomas, Maumita Makar, Marta Kirsis, and Lena Verkhovsky for expert technical help.


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