Purification and characterization of transducin from capybara Hydrochoerus hydrochaeris.
Polypeptides of approximately 39, 36 and <or= 14.4 kDa remained tightly bound to illuminated Hydrochoerus hydrochaeris retinal rod outer segment (ROS) membranes following extensive isotonic and hypotonic washes, and were specifically released in the presence of GTP. These results identified them as the alpha-, beta-and gamma-subunits of transducin (T). Once purified to homogeneity by anion-exchange chromatography, capybara T showed light-dependent beta,gamma-imido-guanosine 5'-triphosphate (GMPPNP) binding and GTPase activities in the presence of bovine rhodopsin, was recognized by anti-bovine T polyclonal antibodies, and was ADP-ribosylated by pertussis toxin. Capybara T bound GMPPNP with an apparent Kd of 18 nM, and the Scatchard and Hill plots revealed positive cooperativity for binding to photoactivated rhodopsin. Using a coupled enzymatic spectrophotometric assay, the initial velocity of its intrinsic light-dependent GTP hydrolytic capacity was calculated to be 0.1 mol of GTP hydrolyzed/min/mol of capybara T. Additionally, chromatography on omega-amino octylagarose of GTPgammaS-extracted capybara T demonstrated that this protein is composed by two functional units, the alpha-subunit and the betagamma-complex. All these results showed that capybara T possesses similar characteristics to other reported transducins. Interestingly, T is the first protein involved in phototransduction that is purified and characterized from H. hydrochaeris.
Ortiz
Julio O
JO
Departamento de Biología Celular, Universidad Simón Bolívar, Caracas, Venezuela.
Rodríguez-Lanetty
Mauricio
M
Bubis
José
J
D000818:Animals; D002417:Cattle; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000737:chemistry / Q000378:metabolism; D008027:Light; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000737:chemistry* / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D012377:Rodentia / Q000378:metabolism; D015293:Transducin / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D021122:Protein Subunits; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D015293:Transducin