39] Iodination of rhodopsin and transmembrane topology

Abstract

This chapter discusses the iodination of rhodopsin and transmembrane topology. In the presence of H 2 O 2 and NaI, lactoperoxidase catalyzes the iodination of accessible tyrosyl residues of proteins. The reaction at neutral pH and low iodide concentration proceeds through an enzyme-substrate complex that transfers the iodine atom directly onto the protein. As most intact cells or sealed membrane vesicles are impermeable to lactoperoxidase, the enzymatic iodination technique is ideally suited for probing the spatial arrangement of proteins in membranes. Several approaches have been developed to identify transmembrane proteins in membranes using this technique. These include iodination of both the exterior and interior surfaces of large sealed vesicles, proteolytic digestion and iodination of “inside-out” and “right-side-out” vesicles, and sequential proteolysis and iodination of reconstituted membrane vesicles containing symmetrically oriented proteins. Rhodopsin is asymmetrically oriented in the native rod outer segment disk membrane. Several sites on the portion of the rhodopsin polypeptide chain exposed to the cytoplasm are accessible to cleavage by papain. As the vectorial orientation of rhodopsin is not preserved after the reconstitution, the percent of rhodopsin molecules in the reconstituted membrane vesicles that is susceptible to papain proteolysis is expected to be different.