Activities of affinity-isolated glutathione S-transferase (GST) from channel catfish whole intestine.
A glutathione S-transferase (GST) fraction was isolated from cytosol prepared from catfish intestinal mucosa by GSH-agarose affinity chromatography and its molecular weight, isoelectric points, substrate specificities and immunochemical cross-reactivity were examined. Intestinal GSTs were purified 100-fold with respect to cytosolic activity with 1-chloro-2, 4-dinitrobenzene and had high activity with ethacrynic acid, (+/-)benzo(a)pyrene-4,5-oxide, and (+/-)anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, but a low activity with 1,2-dichloro-4-nitrobenzene. SDS-polyacrylamide gel electrophoresis revealed the presence of a single band with relative molecular mass of 26700. Gel isoelectric focusing showed a major band with a pI of 8.2. A polyclonal antibody prepared against a GST pi-protein isolated from catfish proximal intestine cross-reacted well with the affinity isolated GST fraction. The catfish antibody also cross-reacted with GST from human placenta which contains predominantly pi-class GST (Mannervik, B., Guthenberg, C., 1981. Glutathione transferase (human placenta). In: Jakoby, W.B. (Ed.), Methods in Enzymology, 77. Academic Press, New York, pp. 231-235; Polidoro, G., Dillio, C., Arduini, A., Frederici, G., 1981. Molecular and catalytic properties of purified glutathione transferase from human placenta. Biochem. Med. 22, 247-259; Dao, D.D., Partridge, C.A., Kurosky, A., Awasthi, Y.C., 1982. Subunit structure of glutathione-S-transferase of human liver and placenta. IRSC Med. Sci, Lib. Compend. 10, 175; Dao, D.D., Partridge, C.A., Kurosky, A., Awasthi, Y.C., 1984. Human glutathione transferase. Characterization of the anionic forms from lung and placenta. Biochem. J. 221, 33-41), but poorly with human liver cytosol. The affinity-isolated protein fraction from whole intestine contained proteins that were immunologically related to all four major classes of human GSTs tested. N-terminal sequence analysis of the predominant band obtained by 2D electrophoresis indicated a marked homology (63-70% identical) to mammalian pi form GST isozymes and very strong similarity (80%) to a salmon hepatic GST that was designated a pi form (Dominey, R.J., Nimmo, I.A., Cronshaw, A.D., Hayes, J.D., 1991. The major glutathione S-transferase in salmonid fish livers is homologous to the mammalian pi class GST. Comp. Biochem. Physiol. (B) 100 (1), 93-98). Other bands contained insufficient protein for N-terminal analysis. Taken together, these results indicate that the predominant intestinal GST isoform is related to the pi-class enzymes, but minor GSTs related to other families are also present.
Gadagbui
BK
Department of Medicinal Chemistry, College of Pharmacy, University of Florida, PO Box 100485, Gainesville, FL, USA
James
MO