Improved purification of transducin subunits from bovine retinal rod outer segments.
Transducin serves as a mediator between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase, in the visual process. Transducin is a protein composed of three polypeptides: T alpha, T beta, and T gamma, and acts as two functional units, the alpha-subunit and the beta gamma-complex. In the present study, I describe an efficient and fast method of purifying T alpha and T beta gamma using chromatography on a blue agarose column connected in tandem with an omega-amino octylagarose column. The recombination of T alpha and T beta gamma reconstitutes the functional heterotrimeric holoprotein, as demonstrated by the recovery of three native properties of transducin: 1) its capacity to exchange guanine nucleotide, 2) its GTP hydrolytic activity, and 3) the ADP-ribosylation of T alpha catalysed by pertussis toxin.
Bubis
J
J
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela. jbubis@usb.ve
D000818:Animals; D002417:Cattle; D002847:Chromatography, Agarose / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012243:Rhodopsin / Q000737:chemistry*; D012374:Rod Cell Outer Segment / Q000737:chemistry*; D012685:Sepharose / Q000031:analogs & derivatives / Q000494:pharmacology; D015293:Transducin / Q000302:isolation & purification*; D014227:Triazines / Q000494:pharmacology
D011500:Protein Synthesis Inhibitors; D014227:Triazines; C012842:Cibacron Blue F 3GA; C027176:octyl-sepharose CL-4B; D012243:Rhodopsin; D012685:Sepharose; D015293:Transducin