Purification and characterization of gamma-glutamyl transpeptidase from Ascaris suum.
gamma-Glutamyl transpeptidase, which is of central importance in the degradation of glutathione, was purified from Ascaris suum to apparent homogeneity. The enzyme was found to be a predominantly membrane-bound protein and was solubilized by Triton X-100. The purified enzyme, which exhibits a specific activity of 1009 U (mg protein)-1, showed a molecular mass of 70 kDa and was found to be composed of two non-identical subunits of molecular mass 43 and 30 kDa. Concerning the kinetic properties of the enzyme, the data presented in this study showed that various amino acids and dipeptides with L-configuration served as acceptors for the gamma-glutamyl moieties of the enzyme reaction products and showed Km-values in the mM range. The apparent Km-value for the gamma-glutamyl donor L-glutamyl-gamma-7-amido-4-methylcoumarin of the enzyme was found to be 0.03 mM. L- and D-serine in combination with borate ions were competitive inhibitors of the enzyme activity with Ki-values of 0.30 and 0.61 mM, respectively. Acivicin was an irreversible inhibitor of the enzyme with a Ki-value of 0.42 mM and with a pseudo-first-order kinetics (kinact) of 0.18 min-1. In vitro treatment of the adult A. suum with acivicin resulted in a dose-dependent inhibition of the enzyme activity and an increase of the glutathione levels. These findings indicate the physiological role of the gamma-glutamyl transpeptidase of this parasitic nematode in the catabolism of glutathione.
Hussein
A S
AS
Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Walter
R D
RD
D000596:Amino Acids; D000818:Animals; D017165:Ascaris suum / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004151:Dipeptides; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007555:Isoxazoles / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D017830:Octoxynol; D013379:Substrate Specificity; D005723:gamma-Glutamyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D000596:Amino Acids; D004151:Dipeptides; D004791:Enzyme Inhibitors; D007555:Isoxazoles; D017830:Octoxynol; D005723:gamma-Glutamyltransferase; C006303:acivicin