Abstract

We have investigated the interaction between concanavalin A-agarose (Con A-agarose) and thyroid peroxidase, an integral membrane protein found in the 105,000 X g, 1-h particulate fraction of thyroid tissue. An intact form of porcine thyroid peroxidase was obtained by solubilization with the nonionic detergent Triton X-100 and two fragmented, hydrophilic forms of the enzyme were prepared by trypsin treatment of the membrane. The three types of thyroid peroxidase bind to Con A-agarose and can be eluted with alpha-methyl-D-mannoside. The alpha-methyl-D-mannoside eluate of the most purified thyroid peroxidase preparation has been analyzed by polyacrylamide gel electrophoresis. Peroxidase activity corresponds with a glycoprotein band. The binding of thyroid peroxidase to Con A-agarose can be inhibited by sugars in the following order: alpha-methyl-D-mannoside greater than D-mannose greater than alpha-methyl-D-glucoside greater than D-glucose greater than D-galactose. This order of specificity is typical of Con A-sugar interactions. Furthermore, inactivation of the carbohydrate binding site of Con A by demetallization greatly reduces the extent of thyroid peroxidase binding. Reactivation of the carbohydrate binding site by the addition of Ca2+ and Mn2+ to demetallized Con A-agarose restores thyroid peroxidase binding. These and other experiments suggest that htyroid peroxidase is, like several other peroxidases, a glycoprotein. In addition, the interaction between thyroid peroxidase and Con A-agarose may provide a new purification tool for thyroid peroxidase.

Highlights

  • From the $ Thyroid Unit, Department of Medicine, Massachusetts General Hospital, Medical School, Boston, Massachusetts 02114 and the Ti Department of Biochemistry, University, Waltham, Massachusetts 02154 and Harvard Brandeis

  • Peroxidase to Con A-Agarose-As shown in Table I, detergent-solubilized, intact thyroid peroxidase binds to Con A-agarose

  • (100 mg/ml) and thyroid peroxidase are mixed and added to Con A-agarose, none of the thyroid peroxidase binds to Con A-agarose (Table I), Znteraction of Thyroid Peroxidase with Concanaualin A

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Summary

Introduction

We have investigated the interaction between concanavalin A-agarose (Con A-agarose) and thyroid peroxidase, an integral membrane protein found in the 105,000 x g, l-h particulate fraction of thyroid tissue. An intact form of porcine thyroid peroxidase was obtained by solubilization with the nonionic detergent Triton X-100 and two fragmented, hydrophilic forms of the enzyme were prepared by trypsin treatment of the membrane. The three types of thyroid peroxidase bind to Con A-agarose and can be eluted with cu-methyl-D-mannoside. The a-methyl-D-mannoside eluate of the most purified thyroid peroxidase preparation has been analyzed by polyacrylamide gel electrophoresis. The binding of thyroid peroxidase to Con A-agarose can be inhibited by sugars in the following order: a-methyl-D-mannoside > D-mannose > cu-methyl-D-glucoside > D-glucose >. Inactivation of the carbohydrate binding site of Con A by demetallization greatly reduces the extent of thyroid peroxidase binding.

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