Isolation and expression of a cDNA coding for rat kidney cytosolic cysteine conjugate beta-lyase. 1993

S J Perry, and M A Schofield, and M MacFarlane, and E A Lock, and L J King, and G G Gibson, and P S Goldfarb
Molecular Toxicology Research Group, School of Biological Sciences, University of Surrey, Guildford, UK.

The role of rat kidney cysteine conjugate beta-lyase in the production of nephrotoxic thiols from S-cysteine conjugates of xenobiotics has been well established. However, the factors controlling the cellular distribution and substrate specificity of the enzyme have yet to be elucidated. As an approach to this we have isolated a cDNA for cysteine conjugate beta-lyase from a rat kidney cDNA library, using a combination of immunological and hybridization screening. A full length cDNA was sequenced and its identity was confirmed by deduced molecular weight, deduced amino acid composition, the presence of a consensus pyridoxal phosphate (PLP) binding site in the deduced amino acid sequence, kidney-specific expression of the corresponding mRNA, and the expression of beta-lyase and glutamine transaminase K activities in tissue culture cells transfected with the cDNA. The cDNA coded for a protein of 48 kDa containing the sequence Ser-Ala-Gly-Lys-Ser-Phe, which corresponds closely to the PLP binding site in other PLP-containing enzymes. Use of the cDNA to detect beta-lyase mRNA sequences in rat liver and kidney RNA demonstrated that expression was kidney specific and that the mRNA size (2.1 kilobases) was in good agreement with the size of the cDNA. When the cDNA was inserted into the expression vector pUS1000 and transfected into COS-1 tissue culture cells, a 7-10-fold increase in cytosolic beta-lyase and glutamine transaminase K activities could be detected. The use of beta-lyase cDNA for the elucidation of the mechanism of action of this enzyme and for the development of in vitro systems to examine xenobiotic cysteine conjugate toxicity is discussed.

UI MeSH Term Description Entries
D007668 Kidney Body organ that filters blood for the secretion of URINE and that regulates ion concentrations. Kidneys
D008190 Lyases A class of enzymes that catalyze the cleavage of C-C, C-O, and C-N, and other bonds by other means than by hydrolysis or oxidation. (Enzyme Nomenclature, 1992) EC 4. Desmolase,Desmolases,Lyase
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D008970 Molecular Weight The sum of the weight of all the atoms in a molecule. Molecular Weights,Weight, Molecular,Weights, Molecular
D011732 Pyridoxal Phosphate This is the active form of VITAMIN B 6 serving as a coenzyme for synthesis of amino acids, neurotransmitters (serotonin, norepinephrine), sphingolipids, aminolevulinic acid. During transamination of amino acids, pyridoxal phosphate is transiently converted into pyridoxamine phosphate (PYRIDOXAMINE). Pyridoxal 5-Phosphate,Pyridoxal-P,Phosphate, Pyridoxal,Pyridoxal 5 Phosphate,Pyridoxal P
D002461 Cell Line, Transformed Eukaryotic cell line obtained in a quiescent or stationary phase which undergoes conversion to a state of unregulated growth in culture, resembling an in vitro tumor. It occurs spontaneously or through interaction with viruses, oncogenes, radiation, or drugs/chemicals. Transformed Cell Line,Cell Lines, Transformed,Transformed Cell Lines
D002522 Chlorocebus aethiops A species of CERCOPITHECUS containing three subspecies: C. tantalus, C. pygerythrus, and C. sabeus. They are found in the forests and savannah of Africa. The African green monkey is the natural host of SIMIAN IMMUNODEFICIENCY VIRUS and is used in AIDS research. African Green Monkey,Cercopithecus aethiops,Cercopithecus griseoviridis,Cercopithecus griseus,Cercopithecus pygerythrus,Cercopithecus sabeus,Cercopithecus tantalus,Chlorocebus cynosuros,Chlorocebus cynosurus,Chlorocebus pygerythrus,Green Monkey,Grivet Monkey,Lasiopyga weidholzi,Malbrouck,Malbrouck Monkey,Monkey, African Green,Monkey, Green,Monkey, Grivet,Monkey, Vervet,Savanah Monkey,Vervet Monkey,Savannah Monkey,African Green Monkey,Chlorocebus cynosuro,Green Monkey, African,Green Monkeys,Grivet Monkeys,Malbrouck Monkeys,Malbroucks,Monkey, Malbrouck,Monkey, Savanah,Monkey, Savannah,Savannah Monkeys,Vervet Monkeys
D003600 Cytosol Intracellular fluid from the cytoplasm after removal of ORGANELLES and other insoluble cytoplasmic components. Cytosols
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein

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