Isolation and expression of a cDNA coding for rat kidney cytosolic cysteine conjugate beta-lyase.
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.
Perry
S J
SJ
Molecular Toxicology Research Group, School of Biological Sciences, University of Surrey, Guildford, UK.
Schofield
M A
MA
MacFarlane
M
M
Lock
E A
EA
King
L J
LJ
Gibson
G G
GG
Goldfarb
P S
PS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000201:enzymology*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D007668:Kidney / Q000201:enzymology*; D008190:Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D000637:Transaminases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D012333:RNA, Messenger; D011732:Pyridoxal Phosphate; D004247:DNA; D000637:Transaminases; C427605:glutamine - phenylpyruvate transaminase; C021481:kynurenine-oxoglutarate transaminase; D008190:Lyases