Purification and characterization of a thiol:protein disulfide oxidoreductase from bovine liver

Abstract

A thiol:protein disulfide oxidoreductase which degrades insulin into its A and B chains has been purified to homogeneity from bovine liver. The initial extraction procedure used 1% Triton X-100, which increased the soluble insulin degrading activity 100 to 800% as a result of solubilizing a membrane-associated enzyme. The criteria for homogeneity of the isolated protein include sodium dodecyl sulfate and disc gel electrophoresis. Characterization of the homogeneous protein by sedimentation equilibrium centrifugation indicates that a single protein is present which has a mass of 60,000 daltons. A single amino-terminal end group and molecular weight estimation by sodium dodecyl sulfate gel electrophoresis indicated that the thiol:protein disulfide oxidoreductase consists of a single polypeptide chain. The pure enzyme was also examined by gel filtration chromatography and has an apparent molecular weight of 92,000. The amino acid composition and carbohydrate content were also determined. The carbohydrate analysis demonstrated the presence of D-mannose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and N-acetylneuraminic acid which comprise a total of 12% by weight of the isolated protein.