Toxicology Letters, 53 (1990) 253-255
253
Elsevier
TOXLET
02432
Human renal C-S lyases: two cytosolic isoenzymes
Lorraine D. Buckberry, Ian S. Blagbrough, Barrie W. Bycroft and P. Nicholas Shaw Department of Pharmaceutical Sciences, School of Pharmacy. University of Nottingham, Nottingham (U.K.)
Key words: Cysteine transaminase;
conjugate
P-lyase; C-S lyase; Cytosolic
Dichlorovinyl-L-cysteine;
isozymes;
Human
renal tissue; Glutamine
DCVC
INTRODUCTION
The C-S lyase enzymes are responsible for the generation of mutagenic and cytotoxic metabolites via aberrant drug-metabolizing pathways in mammalian tissues [1,2]. We have examined human renal cytosolic and mitochondrial fractions for evidence of C-S lyase and transaminase activity, as described elsewhere [3]. The enzyme cysteine conjugate b-lyase (C-S lyase, EC 4.4.1.13) is one member of a family of enzymes which effect transamination reactions on amino acid substrates. We have recently established that human hepatic cytosolic C-S lyase co-purifies as an aminotransferase: kynurenine aminotransferase [4]. In this article, we discuss the purification of human C-S lyase from the cytosolic subcellular fraction of female renal tissue. We report the purification of two isoenzymes of the cytosolic C-S lyase, which display physicochemical and biochemical properties consistent with glutamine transaminase K [5]. METHODS
AND RESULTS
Substrates [S-(E- 1,2-dichlorovinyl-L-cysteine (DCVC) and S-benzothiazolyl-L-cysteine (BTC)] were synthesised by standard literature methods [6]. The substrates were incubated with individual enzyme fractions which were then assayed for pyruvic acid content using the enzyme lactic acid dehydrogenase and quantifying the change in absorbance at 340 nm which accompanies the turnover of the NADH co-factor. Glutamine transaminase K (GTK) activity was determined by monitoring the proAddress for correspondence: Dr. Ian S. Blagbrough, Department of Pharmaceutical Sciences, Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
037%4274/90/$3.50
@ 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
School
of
254 TABLE I ENZYME ACTIVITY IN HUMAN RENAL SUBCELLULAR
FRACTIONS
Cortex
Specific activity (nmol/min/mg)
Medulla
Specific activity (nmol/min/mg)
Cytosol Mitochondria Microsomes
2.00 f 0.01 2.08kO.28 6.07kO.57
Cytosol Mitochondria Microsomes
1.6OkO.57 3.18kO.25 5.25ki.75
duction of phenylpyruvic acid at 322 nm, from L-phenylalanine co-incubated with sodium cl-keto-y-methiolbutyrate (KMB) (Sigma) [7]. A kidney from a 90-year-old female was identified as pathologically normal and unsuitable for transplantation. Death had followed multiple fractures. The tissue was frozen at -80°C within 4 h of death. A kidney from a 30-year-old pregnant female was identified as normal, as determined at autopsy, and unsuitable for transplantation. Death had followed a severe brain haemorrhage. The tissue was frozen at - 80°C within 4 h of death. The purification of the cortical enzyme from the 30-year-old subject followed the preparation by differential cent~fugation of a 100 000 x g supernatant. The supernatant was saturated with ammonium sulphate (to 40%) and then centrifuged at 36000 x g (45 min, 4°C). The pellet was resuspended and then further purified by Fast Protein Liquid Chromatography over Fast Flow Q Sepharose (anionic exchange), Mono P (chromatofocusing), and Superose 12 (size exclusion). The cytosolic, mitochondrial and microsomal fractions obtained from the medulla were tested for activity, without further purification, together with those subcellular fractions from the cortex and using DCVC (7.5 mmol) as the substrate of choice. The results are shown in Tables I and II. The results shown in these Tables were reproducible in similar experiments with tissue obtained from the 90-year-old subject. TABLE II ENZYME PURIFICATION
PROFILE OF HUMAN RENAL CYTOSOLIC C-S LYASE
Fraction
DCVC
DCVC + KMB
Total activity
Cytosol Pellet
2.00 to.01 3.88kO.11 n.d. 8.79hO.05 0.54kO.15 3.67 i 2.77
_ _
2080 1185 53
FFQ Mono P Superose 12a Superose l2b
14.64+0.00 17.33+0.4 89.91+0.45 75.02+5.13
27 1.5
BTC+KMB _ 0.18+0.03 9.33kO.25 6.21 kO.8 5.31kO.5 47.57+0.00
GTK _ 3.34 19.00 14.74 2.33 20.64
Enzyme activity (nmol/min/mg), totat activity ~nmol/~n/total protein). n.d. = not detectabie; Superose I2a was fractions 1 and 2 (1 ml each) combined; Superose l2b was fraction 30.
255 CONCLUSIONS
These enzymes display properties which are comparable to those isolated from other tissue sources [6]. Two proteins which displayed both C-S lyase and transaminase activity were purified from kidney cytosol, isolated from the organs of a 30-yearold female. These proteins were also isolated, in separate experiments, from the kidneys of a 90-year-old female subject. The isoelectric points were the same for both proteins (from the two kidneys). The two proteins have different relative molecular weights. Both the proteins exhibited GTK activity; however, the protein in fractions 1 and 2 (when separated by size exclusion chromatography) had a lower specific activity with L-phenylalanine in comparison with that of the enzyme in fraction 30 (see Table II). The proteins were essentially homogeneous as judged (data not shown) by gelelectrophoresis (PAGE). In contrast to recently published results [5], the requirement for KMB in the incubation medium increased as the enzyme was purified. That the co-factor vitamin B6 is tightly bound was confirmed by this requirement of KMB for lyase activity, together with a lack of dependence on exogenous pyridoxal phosphate. In agreement with Lash et al. [5], we conclude that this enzyme activity is similar to that displayed by glutamine transaminase K. This is the first report, to the best of our knowledge, of isoenzymes of human renal cytosolic C-S lyase. ACKNOWLEDGEMENTS
We wish to acknowledge the generous financial support of the Health and Safety Executive, and thank Dr. E.M. Gibby for her interest in these studies. REFERENCES 1 Green, T. and Odum, J. (1985) Structure/activity studies on the nephrotoxic and mutagenic action of cysteine conjugates of chloro-pd fluoroalkenes. Chem.-Biol. Interact. 54, 15-3 1. 2 Green, T., Odum, J., Nash, J.A. and Foster, J.R. (1990) Perchloroethylene-induced rat kidney tumors: an investigation of the mechanisms involved and their relevance to humans. Toxicol. Appl. Pharmacol. 103,77-90. 3 Blagbrough, I.S., Buckberry, L.D., Bycroft, B.W. and Shaw, P.N. (1990) Human renal C-S lyase: structure-activity relationships of cytosolic and mitochondrial enzymes. Toxicol. Lett. 53,255-257. 4 Buckberry, L.D., Blagbrough, I.S., Bycroft, B.W. and Shaw, P.N. (1990) Human hepatic C-S lyase: copurification with kynurenine amino transferase. J. Pharm. Pharmacol. 41, Suppl. 30P (in press). 5 Lash, L.H., Nelson, R.M., Van Dyke, R.A. and Anders, M.W. (1990) Purification and characterization of human kidney cytosolic cysteine conjugate p-lyase activity. Drug Metab. Disposit. 18,5&54. 6 Shaw, P.N. and Blagbrough, IS. (1989) Cysteine conjugate /I-lyase. II. Isolation, properties and structure-activity relationships. In: L.A. Damani (Ed.), Sulphur-Containing Drugs and Related Organic Compounds, Vol. 2, Part B. Ellis Horwood, Chichester, pp. 135155. 7 Cooper, A.J.L. and Meister, A. (1985) Glutamine transaminase-K from rat kidney. In: A. Meister (Ed.), Methods in Enzymology, Vol. 113. Academic Press, London-New York, pp. 344-349.
253
Elsevier
TOXLET
02432
Human renal C-S lyases: two cytosolic isoenzymes
Lorraine D. Buckberry, Ian S. Blagbrough, Barrie W. Bycroft and P. Nicholas Shaw Department of Pharmaceutical Sciences, School of Pharmacy. University of Nottingham, Nottingham (U.K.)
Key words: Cysteine transaminase;
conjugate
P-lyase; C-S lyase; Cytosolic
Dichlorovinyl-L-cysteine;
isozymes;
Human
renal tissue; Glutamine
DCVC
INTRODUCTION
The C-S lyase enzymes are responsible for the generation of mutagenic and cytotoxic metabolites via aberrant drug-metabolizing pathways in mammalian tissues [1,2]. We have examined human renal cytosolic and mitochondrial fractions for evidence of C-S lyase and transaminase activity, as described elsewhere [3]. The enzyme cysteine conjugate b-lyase (C-S lyase, EC 4.4.1.13) is one member of a family of enzymes which effect transamination reactions on amino acid substrates. We have recently established that human hepatic cytosolic C-S lyase co-purifies as an aminotransferase: kynurenine aminotransferase [4]. In this article, we discuss the purification of human C-S lyase from the cytosolic subcellular fraction of female renal tissue. We report the purification of two isoenzymes of the cytosolic C-S lyase, which display physicochemical and biochemical properties consistent with glutamine transaminase K [5]. METHODS
AND RESULTS
Substrates [S-(E- 1,2-dichlorovinyl-L-cysteine (DCVC) and S-benzothiazolyl-L-cysteine (BTC)] were synthesised by standard literature methods [6]. The substrates were incubated with individual enzyme fractions which were then assayed for pyruvic acid content using the enzyme lactic acid dehydrogenase and quantifying the change in absorbance at 340 nm which accompanies the turnover of the NADH co-factor. Glutamine transaminase K (GTK) activity was determined by monitoring the proAddress for correspondence: Dr. Ian S. Blagbrough, Department of Pharmaceutical Sciences, Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
037%4274/90/$3.50
@ 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
School
of
254 TABLE I ENZYME ACTIVITY IN HUMAN RENAL SUBCELLULAR
FRACTIONS
Cortex
Specific activity (nmol/min/mg)
Medulla
Specific activity (nmol/min/mg)
Cytosol Mitochondria Microsomes
2.00 f 0.01 2.08kO.28 6.07kO.57
Cytosol Mitochondria Microsomes
1.6OkO.57 3.18kO.25 5.25ki.75
duction of phenylpyruvic acid at 322 nm, from L-phenylalanine co-incubated with sodium cl-keto-y-methiolbutyrate (KMB) (Sigma) [7]. A kidney from a 90-year-old female was identified as pathologically normal and unsuitable for transplantation. Death had followed multiple fractures. The tissue was frozen at -80°C within 4 h of death. A kidney from a 30-year-old pregnant female was identified as normal, as determined at autopsy, and unsuitable for transplantation. Death had followed a severe brain haemorrhage. The tissue was frozen at - 80°C within 4 h of death. The purification of the cortical enzyme from the 30-year-old subject followed the preparation by differential cent~fugation of a 100 000 x g supernatant. The supernatant was saturated with ammonium sulphate (to 40%) and then centrifuged at 36000 x g (45 min, 4°C). The pellet was resuspended and then further purified by Fast Protein Liquid Chromatography over Fast Flow Q Sepharose (anionic exchange), Mono P (chromatofocusing), and Superose 12 (size exclusion). The cytosolic, mitochondrial and microsomal fractions obtained from the medulla were tested for activity, without further purification, together with those subcellular fractions from the cortex and using DCVC (7.5 mmol) as the substrate of choice. The results are shown in Tables I and II. The results shown in these Tables were reproducible in similar experiments with tissue obtained from the 90-year-old subject. TABLE II ENZYME PURIFICATION
PROFILE OF HUMAN RENAL CYTOSOLIC C-S LYASE
Fraction
DCVC
DCVC + KMB
Total activity
Cytosol Pellet
2.00 to.01 3.88kO.11 n.d. 8.79hO.05 0.54kO.15 3.67 i 2.77
_ _
2080 1185 53
FFQ Mono P Superose 12a Superose l2b
14.64+0.00 17.33+0.4 89.91+0.45 75.02+5.13
27 1.5
BTC+KMB _ 0.18+0.03 9.33kO.25 6.21 kO.8 5.31kO.5 47.57+0.00
GTK _ 3.34 19.00 14.74 2.33 20.64
Enzyme activity (nmol/min/mg), totat activity ~nmol/~n/total protein). n.d. = not detectabie; Superose I2a was fractions 1 and 2 (1 ml each) combined; Superose l2b was fraction 30.
255 CONCLUSIONS
These enzymes display properties which are comparable to those isolated from other tissue sources [6]. Two proteins which displayed both C-S lyase and transaminase activity were purified from kidney cytosol, isolated from the organs of a 30-yearold female. These proteins were also isolated, in separate experiments, from the kidneys of a 90-year-old female subject. The isoelectric points were the same for both proteins (from the two kidneys). The two proteins have different relative molecular weights. Both the proteins exhibited GTK activity; however, the protein in fractions 1 and 2 (when separated by size exclusion chromatography) had a lower specific activity with L-phenylalanine in comparison with that of the enzyme in fraction 30 (see Table II). The proteins were essentially homogeneous as judged (data not shown) by gelelectrophoresis (PAGE). In contrast to recently published results [5], the requirement for KMB in the incubation medium increased as the enzyme was purified. That the co-factor vitamin B6 is tightly bound was confirmed by this requirement of KMB for lyase activity, together with a lack of dependence on exogenous pyridoxal phosphate. In agreement with Lash et al. [5], we conclude that this enzyme activity is similar to that displayed by glutamine transaminase K. This is the first report, to the best of our knowledge, of isoenzymes of human renal cytosolic C-S lyase. ACKNOWLEDGEMENTS
We wish to acknowledge the generous financial support of the Health and Safety Executive, and thank Dr. E.M. Gibby for her interest in these studies. REFERENCES 1 Green, T. and Odum, J. (1985) Structure/activity studies on the nephrotoxic and mutagenic action of cysteine conjugates of chloro-pd fluoroalkenes. Chem.-Biol. Interact. 54, 15-3 1. 2 Green, T., Odum, J., Nash, J.A. and Foster, J.R. (1990) Perchloroethylene-induced rat kidney tumors: an investigation of the mechanisms involved and their relevance to humans. Toxicol. Appl. Pharmacol. 103,77-90. 3 Blagbrough, I.S., Buckberry, L.D., Bycroft, B.W. and Shaw, P.N. (1990) Human renal C-S lyase: structure-activity relationships of cytosolic and mitochondrial enzymes. Toxicol. Lett. 53,255-257. 4 Buckberry, L.D., Blagbrough, I.S., Bycroft, B.W. and Shaw, P.N. (1990) Human hepatic C-S lyase: copurification with kynurenine amino transferase. J. Pharm. Pharmacol. 41, Suppl. 30P (in press). 5 Lash, L.H., Nelson, R.M., Van Dyke, R.A. and Anders, M.W. (1990) Purification and characterization of human kidney cytosolic cysteine conjugate p-lyase activity. Drug Metab. Disposit. 18,5&54. 6 Shaw, P.N. and Blagbrough, IS. (1989) Cysteine conjugate /I-lyase. II. Isolation, properties and structure-activity relationships. In: L.A. Damani (Ed.), Sulphur-Containing Drugs and Related Organic Compounds, Vol. 2, Part B. Ellis Horwood, Chichester, pp. 135155. 7 Cooper, A.J.L. and Meister, A. (1985) Glutamine transaminase-K from rat kidney. In: A. Meister (Ed.), Methods in Enzymology, Vol. 113. Academic Press, London-New York, pp. 344-349.
