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J. Biol. Chem., Vol. 279, Issue 38, 39565-39573, September 17, 2004

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The Hydrodynamic Properties of Dark- and Light-activated States of n-Dodecyl -D-Maltoside-solubilized Bovine Rhodopsin Support the Dimeric Structure of Both Conformations^*

Rafael Medina, Deisy Perdomo, and José Bubis

From the Departamento de Biología Celular, Universidad Simón Bolívar, Apartado 89.000, Valle de Sartenejas, Caracas 1081-A, Venezuela

Rhodopsin (Rho) has been extracted in n-dodecyl -D-maltoside (DM) from bovine retinal rod outer segments and purified to homogeneity by affinity chromatography on concanavalin A-Sepharose. Because chemical cross-linking of Rho and photoactivated Rho (Rho*) provided initial evidence for the oligomeric nature of the photoreceptor protein, we carried out a hydrodynamic characterization of the native and activated conformations of detergent-solubilized Rho. The molecular weights of the complexes between dark and photoexcited states of Rho and DM were determined by gel filtration chromatography on Sephacryl S-300, in the presence of 0.1% DM. Subtracting the size of the corresponding detergent micelles resulted in molecular masses of 78 kDa for native Rho and 76 kDa for Rho*. The measured content of 0.97 g of detergent/g of protein resulted in a calculated partial specific volume of 0.765 cm³/g for the protein-detergent complex and a molar mass of 64–65 kDa for the protein moiety. The sizes of Rho·DM and Rho*·DM complexes were also evaluated by sedimentation on 10–30% sucrose gradients, in the presence of 0.1% DM, and molecular masses of about 60 kDa were estimated for both the dark- and light-activated states of the photoreceptor protein. The size of Rho was determined to be 65,300 and 69,800 Da, respectively, when the purified Rho·DM complex was either chromatographed on Sephacryl S-300 or ultracentrifuged on sucrose gradients in the absence of DM. All these results were consistent with a dimeric quaternary structure for both conformations of Rho. Additionally, the functional integrity of the purified photoreceptor protein following gel filtration chromatography and ultracentrifugation was demonstrated by three criteria as follows: (i) its characteristic UV-visible absorption spectra, (ii) its capability to photoactivate transducin, and (iii) its ability to serve as a substrate for rhodopsin kinase.

Received for publication, March 4, 2004 , and in revised form, July 6, 2004.

^* This work was supported in part by FONACIT Grant S1-2000000514, Caracas, Venezuela. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a graduate student research assistantship from Decanato de Investigación y Desarrollo, Universidad Simón Bolívar, Caracas, Venezuela.

To whom correspondence should be addressed. Tel.: 58-212-9064219; Fax: 58-212-9063064; E-mail: jbubis{at}usb.ve.

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