Purification of Glutamine Transaminase K/Cysteine Conjugate β-Lyase from Rat Renal Cytosol Based on Hydrophobic Interaction HPLC and Gel Permeation FPLC

doi:10.1006/prep.1993.1073

Protein Expression and Purification

Volume 4, Issue 6, December 1993, Pages 552-562

https://doi.org/10.1006/prep.1993.1073 Get rights and content

Abstract

Cysteine conjugate β-lyase (β-lyase, EC 4.4.1.13) was purified to homogeneity from rat renal cytosol using a new and highly efficient method, based on C₃-hydrophobic interaction (HI) high-performance liquid chromatography (HPLC) in combination with gel permeation fast protein liquid chromatography. The purity of the enzyme was judged from SDS-PAGE and C₁₈-reversed-phase HPLC. The β-lyase was estimated to be a homodimer consisting of a 47,400-Da subunit with absorption maxima at 280 and 420-430 am. The specific activity of the purified β-lyase toward S-(1,2-dichloro-vinyl)-L-cysteine (1,2-DCVC) in the presence of α-keto-γ-methiolbutyric acid (KMB) was 6.4 μmol/min/mg protein, which is by far the highest value so far reported. Kinetic analysis of 1,2-DCVC metabolism by the enzyme in the presence of KMB gave K_m and V_max of 0.33 mM and 8.4 μmol/min/mg protein, respectively. No significant activity of the purified enzyme was detectable with S-2-benzothiazolyl-L-cysteine up to 2 mM. The purified enzyme also had glutamine transaminase K activity (EC 2.6.1.64) as assayed with phenylalanine and KMB as substrates. This specific activity was 16.0 μmol/min/mg. Amino acid analysis of the purified β-lyase was carried out and was found to be closely similar to the amino acid composition of five other pyridoxal phosphate (PLP)-containing amino acid aminotransferases. This suggests that glutamine transaminase K/cysteine conjugate β-lyase is a typical member of the PLP-containing aminotransferase group.

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Cited by (25)

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2018, Comprehensive Toxicology: Third Edition
The kidney possess most of the common drug-metabolising enzymes, and thus is able to make an important contribution to the body’s metabolism of drugs and other xenobiotics. The catalytic activity of the various enzymes is lower than in the liver, however enzymes involved in the processing of GSH conjugates to their mercapturic acids primarily involves the kidney. Xenobiotic-metabolising enzymes are not evenly distributed along the nephron: cytochromes P450 and enzymes involved in conjugation, glucuronidation or sulfation are present in higher concentrations in proximal tubular cells then other regions. There are however exception where some isoenzymes of P450 and GST’s are selectively localized in cells of the thick ascending limb. Most of the major form of cytochrome P450 are detected in the kidneys of experimental animals and humans. However, members of the CYP1 family, CYP2D6 and CYP2E1 appear to be, absent in human kidney. GST’s have been most extensively characterised in rat and humans kidney. The renal enzyme cysteine conjugate β-lyase involved in the bioactivation of haloalkane and haloalkene S-conjugates have been cloned and sequenced from both rat and human kidney and shown to be identical to glutamine transaminase K and KAT. Cloning approaches have also demonstrated the presence in the kidney of multiple forms of UGT, flavin monooxygenases, CES and SULTS. Bioactivation of certain chemicals by SULTS can generate reactive metabolites that modify DNA and leads to renal cancer. All of the enzymes discussed in addition to detoxification of foreign chemicals, and in a few case causing bioactivation have endogenous functions in the body. Such as regulation of salt and water balance, the control of blood pressure and synthesis of vitamin D and there are many more physiological roles for these enzymes awaiting discovery.
Halogenated Hydrocarbons
2018, Comprehensive Toxicology: Third Edition
Halogenated hydrocarbons (HHCs) encompass a broad group of aliphatic and aromatic chemicals. Many are environmental contaminants or industrial solvents. This article focuses on select HHCs that are known to exhibit nephrotoxicity in animals and/or humans. Chemicals that are reviewed include select examples from haloalkanes, haloacids, haloalkenes, halogenated aromatics, halogenated ethers (which include some commonly used anesthetic agents), and ifosfamide and cyclophosphamide. The article is divided into processes involved in nephrotoxicity. The first major section summarizes metabolic pathways for bioactivation and/or detoxification. Metabolism comprises two major pathways: cytochrome P450 (CYP)-dependent oxidation, which can result in bioactivation or generate substrates for Phase II conjugation reactions; and glutathione-dependent bioactivation pathways, which involve subsequent metabolism by cysteine conjugate beta-lyase or flavin-containing monooxygenase. The next major section summarizes key features of acute and chronic nephrotoxicity, which typically involve single dose or short-term, high-dose exposures for acute and long-term, low-dose exposures for chronic. The final section summarizes select human health risk assessments for HHCs.
Trichloroethylene biotransformation and its role in mutagenicity, carcinogenicity and target organ toxicity
2014, Mutation Research - Reviews in Mutation Research
Citation Excerpt :
DCVC metabolism by enzymes that possess CCBL activity can occur in either the cytoplasm or mitochondria. Studies of the CCBL-catalyzed reaction in vitro in kidney preparations, with DCVC as a substrate, show that in the rat this reaction is 3- to as much as 10-fold higher than that in analogous human kidney models [97,110,111,122–128]. Rates of CCBL-dependent metabolism of DCVC in rats are considerably slower than those for the initial GSH conjugation step that yields DCVG, suggesting that the CCBL-mediated bioactivation reaction is rate-limiting for the conversion of TCE to nephrotoxic and/or mutagenic metabolites.
Metabolism is critical for the mutagenicity, carcinogenicity, and other adverse health effects of trichloroethylene (TCE). Despite the relatively small size and simple chemical structure of TCE, its metabolism is quite complex, yielding multiple intermediates and end-products. Experimental animal and human data indicate that TCE metabolism occurs through two major pathways: cytochrome P450 (CYP)-dependent oxidation and glutathione (GSH) conjugation catalyzed by GSH S-transferases (GSTs). Herein we review recent data characterizing TCE processing and flux through these pathways. We describe the catalytic enzymes, their regulation and tissue localization, as well as the evidence for transport and inter-organ processing of metabolites. We address the chemical reactivity of TCE metabolites, highlighting data on mutagenicity of these end-products. Identification in urine of key metabolites, particularly trichloroacetate (TCA), dichloroacetate (DCA), trichloroethanol and its glucuronide (TCOH and TCOG), and N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (NAcDCVC), in exposed humans and other species (mostly rats and mice) demonstrates function of the two metabolic pathways in vivo. The CYP pathway primarily yields chemically stable end-products. However, the GST pathway conjugate S-(1,2-dichlorovinyl)glutathione (DCVG) is further processed to multiple highly reactive species that are known to be mutagenic, especially in kidney where in situ metabolism occurs. TCE metabolism is highly variable across sexes, species, tissues and individuals. Genetic polymorphisms in several of the key enzymes metabolizing TCE and its intermediates contribute to variability in metabolic profiles and rates. In all, the evidence characterizing the complex metabolism of TCE can inform predictions of adverse responses including mutagenesis, carcinogenesis, and acute and chronic organ-specific toxicity.
Renal Xenobiotic Metabolism
2010, Comprehensive Toxicology, Second Edition
All of the common drug-metabolizing enzymes involved in phase 1 and phase 2 metabolism of xenobiotics are present in the kidney, although the catalytic activity of the various enzymes in the whole kidney is lower than that in the liver. Renal drug-metabolizing enzymes are, however, not evenly distributed along the nephron; cytochrome’s P450 and enzymes involved in conjugation with glutathione (GSH), glucuronic acid, or sulfate are present at much higher concentrations in cells of the proximal tubule than in other parts of the nephron, and thus comparison on a whole organ basis underestimates renal cell activity. Hence, the proximal tubule is the principal site of xenobiotic biotransformation and is particularly susceptible to chemical insult, while the localization of prostaglandin H-synthase (PHS) to the inner medulla and papilla may be a contributory factor to the toxicity produced by chemicals in this part of the nephron.
Many xenobiotic-biotransformation enzymes have been cloned and sequenced, and their presence in the kidney detected. Most of the major families of cytochrome P450 involved in xenobiotic metabolism have been detected in the kidneys of experimental animals and humans. Members of the CYP2B subfamily appear to be absent from rat and mouse kidney but present in the rabbit and hamster. The CYP4A subfamily involved in ω and ω–1 oxidation of fatty acids and arachidonic acid (AA) are expressed at particularly high levels in the kidney and are involved in the side chain metabolism of many drugs and xenobiotics. Glutathione S-transferases (GSTs) have been most extensively characterized in rat and human kidney, this tissue having a different subunit profile from that of the liver, with a predominance of A (alpha) and P1-1 (pi) classes. Cloning has identified a number of relatively new forms of GSTs, and although these enzymes are mainly located in the cytosol, some are associated with microsomes and mitochondria. The kidney plays a pivotal role in the processing of GSH conjugates to their cysteine conjugates and then mercapturic acids for excretion in urine. The renal enzyme cysteine conjugate β-lyase involved in the bioactivation of haloalkane and haloalkene S-cysteine conjugates has been cloned and sequenced from both rat and human kidney, and is shown to be identical to glutamine transaminase K and kynurenine aminotransferase. Cloning approaches have also demonstrated the presence in the kidney of multiple forms of UDP-glucuronosyltransferase, flavin monooxygenase, carboxylesterase, and sulfotransferase
The presence and localization of xenobiotic biotransformation enzymes in human kidney are still limited, although information on embryonic and fetal tissue is beginning to emerge. Many of the enzymes discussed, in addition to their ability to metabolize drugs or foreign compounds, have endogenous functions in the kidney, for instance, in the regulation of salt and water balance, the control of blood pressure and inflammation, and the synthesis of vitamin D.
Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate β-lyase
2008, Archives of Biochemistry and Biophysics
Citation Excerpt :
Thus, despite the ability of rhGTK to bind large amino acids there are subtle constraints in the geometry of amino acids that can bind most effectively at the active site. As was previously noted for the rat enzyme [6,9], our data show that rhGTK catalyzes β-elimination more effectively than transamination when TFEC and DCVC are substrates (compare Table 1 and 3). Yamauchi et al. [9] obtained a preparation of rat kidney GTK of exceptionally high specific activity both as an aminotransferase and as a β-lyase.
Rat kidney glutamine transaminase K (GTK) exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. The β-lyase reaction products are pyruvate, ammonium and a sulfhydryl-containing fragment. We show here that recombinant human GTK (rhGTK) also exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. S-(1,1,2,2-Tetrafluoroethyl)-l-cysteine is an excellent aminotransferase and β-lyase substrate of rhGTK. Moderate aminotransferase and β-lyase activities occur with the chemopreventive agent Se-methyl-l-selenocysteine. l-3-(2-Naphthyl)alanine, l-3-(1-naphthyl)alanine, 5-S-l-cysteinyldopamine and 5-S-l-cysteinyl-l-DOPA are measurable aminotransferase substrates, indicating that the active site can accommodate large aromatic amino acids. The α-keto acids generated by transamination/l-amino acid oxidase activity of the two catechol cysteine S-conjugates are unstable. A slow rhGTK-catalyzed β-elimination reaction, as measured by pyruvate formation, was demonstrated with 5-S-l-cysteinyldopamine, but not with 5-S-l-cysteinyl-l-DOPA. The importance of transamination, oxidation and β-elimination reactions involving 5-S-l-cysteinyldopamine, 5-S-l-cysteinyl-l-DOPA and Se-methyl-l-selenocysteine in human tissues and their biological relevance are discussed.
Modeling and molecular dynamics of glutamine transaminase K/cysteine conjugate β-lyase
2003, Journal of Molecular Graphics and Modelling
The homodimeric, pyridoxal 5′-phosphate (PLP)-dependent enzyme glutamine transaminase K/cysteine conjugate β-lyase (GTK/β-lyase) has been implicated in the bioactivation of chemopreventive compounds. This paper describes the first homology model of rat renal GTK/β-lyase and its active site residues, deduced from molecular dynamics (MD) simulations of the binding mode of 13 structurally diverse cysteine S-conjugates and amino acids after Amber-parametrization of PLP. Comparison with Thermus thermophilus aspartate aminotransferase (tAAT) and Trypanosoma cruzi tyrosine aminotransferase (tTAT), used as templates for modeling GTK/β-lyase, showed that the PLP-binding site of GTK/β-lyase is highly conserved. Binding of the ligand α-carboxylate-group occurred via the conserved residues Arg⁴³² and Asn²¹⁹, and Asn⁵⁰ and Gly⁷⁰. Two pockets accommodated the various ligand side chains. A small pocket, located directly above PLP, was of a highly hydrophobic and aromatic character. A larger pocket, formed partly by the substrate access channel, was more hydrophilic and notably involved the salt bridge partners Glu⁵⁴ and Arg^99∗ (∗ denotes the other subunit). Ligand-binding residues included Leu⁵¹, Phe⁷¹, Tyr¹³⁵, Phe³⁷³ and Phe^312∗, and π-stacking interactions were often observed. Tyr¹³⁵ and Asn⁵⁰ were prominent in hydrogen bonding with the sulfur-atom of cysteine S-conjugates.
The observed binding mode of the ligands corresponded well with their experimentally determined inhibitory potency toward GTK/β-lyase. The current homology model thus provides a starting point for further validation of the role of active site residues in ligand-binding by means of mutagenesis studies. Ultimately, insight in the binding of ligands to GTK/β-lyase may result in the rational design of new ligands and selective inhibitors.

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Regular ArticlePurification of Glutamine Transaminase K/Cysteine Conjugate β-Lyase from Rat Renal Cytosol Based on Hydrophobic Interaction HPLC and Gel Permeation FPLC

Abstract

Regular Article
Purification of Glutamine Transaminase K/Cysteine Conjugate β-Lyase from Rat Renal Cytosol Based on Hydrophobic Interaction HPLC and Gel Permeation FPLC