Intranephron distribution of cysteine S-conjugate beta-lyase activity and its implication for hexachloro-1,3-butadiene-induced nephrotoxicity in rats

The intranephron distribution of two major cysteine S-conjugate beta-lyases was determined in order to clarify the role of these enzymes in promoting the nephrotoxicity associated with certain halogenated xenobiotics. Various nephron segments [i.e., glomerulus, early, middle, and terminal portions of the proximal tubule (S-1, S-2, and S-3 respectively), the thick ascending limb, the distal tubule, and the collecting tubule] were isolated by microdissection from collagenase-treated rat kidneys. Each segment was dissected in Hanks' solution, solubilized with Triton X-100, and applied to a micropolyacrylamide gel constructed with a continuous gradient. The gels were subjected to electrophoresis and then incubated in the dark in a solution containing S(1,2 dichlorovinyl)-L-cysteine (DCVC), sodium alpha-keto-gamma-methiolbutyrate, phenazine methosulfate, and nitroblue tetrazolium. The position of cysteine S-conjugate beta-lyase- and L-amino acid oxidase activities in the gels was revealed by the presence of blue formazen dye bands. The relative intensities of the bands were determined by optical scanning with a microdensitometer. Three bands were detected: band I (M(r) similar to 330 000) corresponds to a recently described high M, cysteine S-conjugate beta-lyase whereas band III (M(r) similar to 90 000) corresponds to a lower M(r) cysteine S-conjugate beta-lyase (identical to cytosolic glutamine transaminase K). Band II (M(r) similar to 240000) corresponds to L-amino acid oxidase (a unique activity of the B isoform of rat kidney L-hydroxy acid oxidase).