AFFINITY LABELING OF RAT-KIDNEY GAMMA-GLUTAMYL TRANSPEPTIDASE BY 6-DIAZO-5-OXO-D-NORLEUCINE

Reaction of rat kidney γ‐glutamyl transpeptidase with 6‐diazo‐5‐oxo‐D‐norleucine, a D‐glutamine analog, resulted in irreversible inactivation of the enzyme. Double reciprocal plots of the inactivation rate versus the inactivator concentration yielded an apparent Michaelis constant of 1.1 mM for the reagent at pH 7.4. The inactivation was prevented by the presence of S‐methyl glutathione, a well known substrate of this enzyme. The rate of inactivation was greatly accelerated by the presence of maleate, which was believed to enhance the inherent glutaminase activity of this enzyme. Inactivation of the enzyme with 6‐diazo‐5‐oxo‐D‐[6‐14C]norleucine was accompanied by covalent incorporation of an amount of radioactivity corresponding to approximately 1 mole of the 14C‐label per 69000 g of the enzyme. Upon sodium dodecylsulfate/polyacrylamide disc gel electrophoresis, only the small subunit of this oligomeric enzyme was found to contain radioactivity. All these findings clearly indicate that the D‐form of the norleucine derivative acts as an affinity label for γ‐glutamyl transpeptidase. Thus, most aspects of its reaction with the enzyme were quite similar to those described previously for 6‐diazo‐5‐oxo‐L‐norleucine [Inoue, M., Horiuchi, S., and Morino, Y. (1977) Eur. J. Biochem. 73, 335–342; Tate, S. S. and Meister, A. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 931–935] except that the inactivation rate was 4–5‐fold greater with the L‐form derivative than with D‐isomer. Both of the covalent bonds formed upon inactivation with D‐form and L‐form of [6‐14C]norleucine derivatives were similarly resistant to mild hydrolytic treatment with acid or alkali and performic acid oxidation. A high degree of similarity thus observed in chemical stability suggests that these two enantiomeric norleucine derivatives reacted with an indentical amino acid residue or alternatively, with two different residues having chemically similar reactivity. The L‐form reagent was actively transported into the ascites tumour cell AH‐130, while no significant amount of the D‐form isomer was taken up by the cells. However, the D‐form of norleucine derivative also served as an affinity label for the enzyme in the intact cells as was the case with the L‐form isomer. The potential usefulness of this D‐form reagent for the study of the physiological function of γ‐glutamyl transpeptidase was discussed. Copyright © 1979, Wiley Blackwell. All rights reserved