Purification and characterization of human kidney cytosolic cysteine conjugate beta-lyase activity.
The central role of cysteine conjugate beta-lyase (beta-lyase) in the bioactivation of nephrotoxic halogenated hydrocarbons and the possibility of human exposure to these chemicals has focused interest on the beta-lyase from human kidney. Human kidney tissue was collected as surgical waste material, and subcellular fractions were isolated by differential centrifugation. Human beta-lyase activity, determined with S-(2-benzothiazolyl)-L-cysteine (BTC) as the substrate, was present in the cytosolic, mitochondrial, and microsomal fractions, but was highest in the cytosolic fraction. Activity in human kidney cytosol was about 10% of that present in rat kidney cytosol. Human kidney cytosolic beta-lyase activity, with BTC as the substrate, was not stimulated by pyridoxal phosphate or by exogenous 2-keto acids. Cytosolic beta-lyase activity was purified 280-fold with a yield of 12% from human kidneys unsuitable for transplantation. The beta-lyase activity copurified with cytosolic glutamine transaminase K and exhibited a molecular mass of 85 kDa on a Sephacryl 5300 column and a subunit molecular mass of 45 kDa by gel electrophoresis. Both BTC and its homocysteine analogue S-(2-benzothiazolyl)-L-homocysteine were excellent substrates, exhibiting Km and kcat values of 0.97 mM and 2.78 mM and 9.35 min-1 and 6.90 min-1, respectively. beta-Lyase activity was inhibited by aminooxyacetic acid, indicating that the human cytosolic enzyme contains pyridoxal phosphate, and by the nephrotoxins S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)-L-homocysteine, which serve as alternative substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
Lash
L H
LH
Department of Pharmacology, University of Rochester School of Medicine and Dentistry, NY.
Nelson
R M
RM
Van Dyke
R A
RA
Anders
M W
MW
D000645:Ammonium Sulfate; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology*; D006801:Humans; D006882:Hydroxyapatites; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008190:Lyases / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D000637:Transaminases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism
D006882:Hydroxyapatites; D000637:Transaminases; C427605:glutamine - phenylpyruvate transaminase; C021481:kynurenine-oxoglutarate transaminase; D008190:Lyases; D000645:Ammonium Sulfate