Abstract
The bovine opsin apoprotein activates transducin, although at a much reduced level than light-activated rhodopsin (Surya, A., Foster, K., and Knox, B. (1995) J. Biol. Chem. 270, 5024-5031). The ability of retinal to modulate opsin apoprotein activity was investigated using a guanyl nucleotide exchange assay on transducin. 11-cis-Retinal reacted with opsin at 22 degrees C to (a) reform pigment having maximal absorbance at 500 nm and (b) reduce opsin activity by >80%. Pigment formation also occurred at 0 degrees C with a t1/2 of 260 min. However, unlike rhodopsin formed at 22 degrees C (R22), the rhodopsin formed at 0 degrees C (R0) activated transducin with the same half-saturating concentration as opsin in an exhaustive binding assay. Thus, the formation of a protonated Schiff base associated with 500 nm absorbance does not by itself lead to the inactivation of opsin. The R0 conformation was partially inactivated by incubation at 22 degrees C (t1/2 = 61 +/- 9 min), suggesting that it may be an intermediate conformation in the regeneration of rhodopsin.
Highlights
The bovine opsin apoprotein activates transducin, at a much reduced level than light-activated rhodopsin (Surya, A., Foster, K., and Knox, B. (1995) J
Unlike rhodopsin formed at 22 °C (R22), the rhodopsin formed at 0 °C (R0) activated transducin with the same half-saturating concentration as opsin in an exhaustive binding assay
In contrast to the high turnover number and rapid kinetics, of transducin activation by metarhodopsin(II), opsin activity is characterized by a low turnover number and slower kinetics, which results in a second-order rate constant for transducin activation by opsin that is approximately 30-fold lower than that for metarhodopsin(II) [5]
Summary
Protein Preparations and Assays—Rhodopsin- and opsin-containing rod outer segment membranes were prepared from frozen dark-adapted bovine retinae as described earlier [5]. Bovine transducin was purified according to Ref. 18, and transducin activation was measured by a guanyl nucleotide exchange assay using a non-hydrolyzable GTP analog, [35S]GTP␥S1 [5]. The reactions were carried out at 22 °C in dim red light for 2 h in 10 mM Tris acetate (pH 7.0), containing 100 mM NaCl, 5 mM MgCl2, and 5 mM 2-mercaptoethanol
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