[28] Depalmitoylation of rhodopsin with hydroxylamine

This chapter describes the procedures for the in vivo labeling of rhodopsin with [3H]palmitate and the removal of palmitate from rhodopsin in vitro using hydroxylamine (NH2OH). The properties of hydroxylamine-depalmitoylated rhodopsin exhibited in assays of regeneration and transducin activation, as well as information obtained by others using the complementary approach of site-directed mutagenesis to eliminate the palmitate groups from opsin is summarized in the chapter. Rhodopsin plays a critical role in visual transduction, the process that links the absorption of light with generation of an electrical response within the rod. Proteins containing palmitate include p21ras, viral glycoproteins, and mammalian transferrin receptor. Palmitoylation occurs posttranslationally, either in the Golgi apparatus or in the endoplasmic reticulum. The palmitoylation is a cotranslational or early posttranslational modification of rat rhodopsin. However, the nonenzymatic exchange of palmitate can be demonstrated in isolated rod outer segment (ROS13).