Resolution of transducin subunits by chromatography on blue sepharose.
The retinal guanine nucleotide-binding protein, transducin (TD), was subjected to chromatography on Blue Sepharose (BLS). A simple two-step protocol was developed, allowing the resolution of the alpha-subunit and the beta gamma-complex of the protein extracted from bovine retina by the use of a poorly hydrolysable GTP analogue. If TD was applied to BLS in a divalent cation-containing buffer, the beta gamma-complex did not bind to the resin, whereas the alpha-subunit was retained; elution of the latter was achieved by removing the divalent cation from the buffer. Binding of the alpha-subunit to BLS was not affected by nucleotides or by ADP ribosylation catalysed by bacterial toxins. However, adsorption of the alpha-subunit by BLS or by a strong cation exchanger (Mono S) depended strictly on divalent cations. In contrast to previous reports, the data suggest the formation of a complex between a sulphonyl residue of Cibacron Blue, a divalent metal ion, and the alpha-subunit as the relevant binding mechanism causing adsorption of the alpha-subunit to BLS.
D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives; D008565:Membrane Proteins / Q000302:isolation & purification*; D012160:Retina / Q000032:analysis; D012685:Sepharose / Q000031:analogs & derivatives; D013462:Sulfur Radioisotopes; D013873:Thionucleotides; D015293:Transducin
D008565:Membrane Proteins; D013462:Sulfur Radioisotopes; D013873:Thionucleotides; C034882:guanosine 5'-O-(1-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); C024908:blue dextran-sepharose; D006160:Guanosine Triphosphate; D012685:Sepharose; D015293:Transducin