Binding of rhodopsin and rhodopsin analogues to transducin, rhodopsin kinase and arrestin-1

Figure 4 Purification of transducin and arrestin-1, and preparation of an enriched fraction of rhodopsin kinase. A: Transducin was purified to homogeneity on a DEAE-cellulose column. The elution profile was monitored at 280 nm (■). Fractions were analyzed for [8-³H] GMPpNp binding activity (CPM) in the absence (○, continuous line) or presence (○, dashed line) of light-activated rhodopsin (as dark-depleted ROS membranes). Fractions were also examined by SDS-PAGE (Inset, top) and Western blot using anti-transducin polyclonal antibodies (Inset, bottom). Lanes a, b, c, d, e, f, and g correspond to column fractions Nº 155, 160, 165, 170, 175, 180 and 185, respectively. Arrows indicate the migration of α-, β- and γ-subunits of transducin (Tα, Tβ and Tγ); B: Autoradiography showing the light-induced in vitro phosphorylation of rhodopsin by rhodopsin kinase (Rho-K). Left, Coomassie blue staining; Right, Autoradiography. Samples of intact ROS membranes, a partially purified fraction of Rho-K, or a mixture of dark-depleted ROS membranes together with the enriched fraction of Rho-K were incubated with [γ-³²P] ATP under light conditions as described in Materials and Methods. Arrows indicate the migration of phosphorylated rhodopsin (Rho), rhodopsin dimers (Rho2), rhodopsin trimers (Rho3) and rhodopsin tetramers (Rho4). A polypeptide band of 80 kDa was phosphorylated in the Rho-K enriched fraction. M: Molecular weight markers; C: Arrestin-1 was purified to homogeneity after three consecutive chromatography steps, a DEAE-cellulose column, a heparin-sepharose column eluted with a gradient of phytic acid, and a second heparin-sepharose column eluted by increasing the salt concentration in the buffer. Shown is the elution profile of the last heparin-sepharose column, which was monitored at 280 nm (•). Fractions were inspected by SDS-PAGE (Inset, top) and Western blot using anti-arrestin-1 polyclonal antibodies (Inset, bottom). Lanes a, b, c, d, e, f, g, h, i, and j correspond to column fractions Nº 17, 21, 23, 25, 27, 29, 31, 33, 35, and 36. Arrows indicate the migration of arrestin-1 (Arr). ROS: Rod outer segments.