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

Abstract

The heterodimeric enzyme γ-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 reversephase 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.