Evidence for the presence of a latent active site in the large subunit of rat kidney γ-glutamyl transpeptidase

Abstract

γ-Glutamyl transpeptidase (EC 2.3.2.2) of rat kidney is composed of two nonidentical polypeptide chains, the small and large subunits. The active site of this enzyme has previously been shown to be located in the small subunit [Inoue, M., Horiuchi, S. & Morino, Y. (1977) Eur, J. Biochem. 73 , 335–342; Tate, S. S. & Meister, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74 , 931–935] The denaturation of this oligomeric enzyme in 6 M urea, followed by chromatography on a Sephadex G-150, resulted in the separation of the large and small subunits. The removal of urea gave rise to an enzymatically active preparation from the denatured large subunit. Under several renaturation conditions, the small subunit polypeptide chain did not exhibit the enzymatic activity. Upon incubation with 6-diazo-5-oxo-L-[1,2,3,4,5- 14C]norleucine, an affinity label for γ-glutamyl transpeptidase, the renatured preparation of the large subunit was covalently labeled with the affinity label with concomitant loss of the enzymatic activity. When the native enzyme was inactivated by the 14C-affinity label, radioactivity was selectively incorporated into the small subunit. These findings indicate that the isolated large subunit possesses an active site which is masked in the native state of the enzyme.