非変性2次元電気泳動法によって分離・検出されたカルボキシルエステラーゼとトランスフェリン複合体の電気泳動的抽出

Abstract

Cytosolic proteins from mouse liver were separated and detected using non-denaturing two-dimensional electrophoresis (2DE) and imidazole-zinc reversible staining. A protein spot at pI 5.5/Mr 120,000 in the 2DE exhibited esterase activity, as observed through esterase activity staining and spectrofluorometry. When this spot was eluted and transferred to anti-carboxylesterase (ES) antibody or anti-transferrin (TF) antibody-conjugated protein A beads, a complex of ES and TF was obtained. To investigate the physiological role of this complex, Fe 2+ was added to ES, and a mixture of ES and TF was prepared. We examined the changes in esterase activity in these mixtures. The esterase activity of ES was inhibited by Fe 2+ , however, the inhibition was suppressed in the presence of the ES and TF complex. Therefore, this method is suitable for the separation and elution of the ES and TF complex. The inhibition of esterase activity by Fe 2+ could be suppressed by TF within the ES and TF complex.