Structural studies and two-dimensional gel electrophoresis of gamma-glutamyl transpeptidase

The heterodimeric enzyme gamma-glutamyl transpeptidase (EC 2.3.2.2) was isolated from adult rat kidney and purified to homogeneity for structural studies using papain solubilization and multiple chromatographies. Two-dimensional gel electrophoresis was found to resolve the active papain-purified enzyme into at least 18 components. Seven components with apparent molecular masses of 23,000-26,000 and isoelectric point range of 5.4-7.0 constitute the light subunit, and 11 components with apparent molecular mass of 51,000-53,000 and isoelectric point range of 5.8-7.1 constitute the heavy subunit. Immunoblot analysis of two-dimensional gels showed that all of these components are immunoreactive with a mixture of the two antibodies generated separately against the light and heavy subunits. Preparative subunit separation was achieved using reverse-phase HPLC under acidic but nonreducing conditions. N-Terminal amino acid sequencing of the separated subunits of the papain-purified enzyme yielded sequence information for the first 32 residues of the heavy chain with the N-terminal starting sequence Gly-Lys-Pro-Asp-His-Val-Tyr-Ser-Arg-Ala, and for the first 36 residues of the light subunit with the N-terminal starting sequence Thr-Ala-His-Leu-Ser-Val-Val-Ser-Glu-Asp.