Abstract
Selenocysteine Se-conjugates have recently been proposed as potential prodrugs to target pharmacologically active selenol compounds to the kidney. Although rat renal cytosol displayed a high activity of β-elimination activity toward these substrates, the enzymes involved in this activation pathway as yet have not been identified. In the present study, the possible involvement of cysteine conjugate β-lyase/glutamine transaminase K (β-lyase/GTK) in cytosolic activity was investigated. To this end, the enzyme kinetics of 15 differentially substituted selenocysteine Se-conjugates and 11 cysteineS-conjugates was determined using highly purified rat renal β-lyase/GTK. The results demonstrate that most selenocysteine Se-conjugates are β-eliminated at a very high activity by purified β-lyase/GTK, implicating an important role of this protein in the previously reported β-elimination reactions in rat renal cytosol. As indicated by the rapid consumption of α-keto-γ-methiolbutyric acid, purified β-lyase/GTK also catalyzed transamination reactions, which appeared to even exceed that of β-elimination. The corresponding sulfur analogs also showed significant transamination but were β-eliminated at an extremely low rate. Comparison of the obtained enzyme kinetic data of purified β-lyase/GTK with previously obtained data from rat renal cytosol showed a poor correlation. By determining the activity profiles of cytosolic fractions applied to anion exchange fast protein liquid chromatography and gel filtration chromatography, the involvement of multiple enzymes in the β-elimination of selenocysteine Se-conjugates in rat renal cytosol was demonstrated. The identity and characteristics of these alternative selenocysteine conjugate β-lyases, however, remain to be established.
Footnotes
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Send reprint requests to: Dr. Jan N. M. Commandeur, Leiden/Amsterdam Center for Drug Research (LACDR), Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, the Netherlands. E-mail: command{at}chem.vu.nl
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↵1 Financial support was provided by the European Science Foundation.
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↵2 I.A. was a visiting scientist from School of Pharmacy of the Aristotelian University of Thessaloniki, Greece.
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↵3 Due to a typing error, Abraham and Cooper (1996)originally reported a mass of Mr 45,800 for this protein. The correct mass of the protein, based on its cDNA sequence, is Mr 48,500 (D. G. Abraham, personnel communication).
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↵4 MALDI-TOF mass spectrometry measurements were carried out on a VISION 2000 (Finnigan MAT, Bremen, Germany) (for details, see Jespersen et al., 1995). Samples containing 125 fmol of β-lyase/GTK in 2,5-dihydroxybenzoic acid as matrix were irradiated with a nitrogen laser at 337 nm. Mass spectra were accumulated for 25 laser shots fired at the same spot on the sample surface.
- Abbreviations:
- GSH
- glutathione
- β-lyase
- cysteineS-conjugate β-lyase
- GTK
- glutamine transaminase K
- KMB
- α-keto-γ-methiolbutyric acid
- OPD
- o-phenylene diamine
- FPLC
- fast protein liquid chromatography
- BTC
- S-(benzothiazolyl)-l-cysteine
- 1,2-DCV-Cys
- S-(1,2-dichlorovinyl)-l-cysteine
- TFE-Cys
- S-(1,1,2,2-tetrafluoroethyl)-l-cysteine
- PMSF
- phenylmethylsulfonyl fluoride
- CTFE-Cys
- S-(2-chloro-1,1,2-trifluoroethyl)-l-cysteine
- DCDFE-Cys
- S-(2,2-dichloro-1,1-difluoroethyl)-l-cysteine
- MALDI-TOF
- matrix-assisted laser desorption time-of-flight
- Received January 6, 2000.
- Accepted April 19, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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