A third form of the G protein beta subunit. 2. Purification and biochemical properties.
Visual excitation in cones is thought to involve a cone-specific G protein (cone transducin) that transduces the light signal detected by the cone visual pigment into an increase in the enzymatic activity of a cGMP phosphodiesterase. In the preceding paper, we have shown that the G beta 3 isoform of G proteins is specifically localized in bovine cone photoreceptors and proposed that it might be a component of cone transducin. We reported here the purification from bovine retinal extract of a cone-specific T beta 3 gamma complex (where T is transducin), which is composed of a G beta 3 subunit and an immunochemically distinct G gamma subunit. Our purification of this complex is based on a two-stage procedure; the first stage consists of a series of column chromatographies that yield a mixture of purified T beta gamma substantially enriched in T beta 3 gamma, and the second stage involves the removal of all of the rod-specific T beta 1 gamma from the mixture using an affinity column of immobilized monoclonal antibodies directed against the rod T gamma subunit of transducin. Using this procedure, we were able to obtain sufficient amounts of T beta 1 gamma and T beta 3 gamma to begin a comparative study of their properties. We showed that T beta 3 gamma is distinguishable from T beta 1 gamma by isoelectric focusing under nondenaturing conditions. The G beta 3 polypeptide of T beta 3 gamma also migrates slightly slower than the G beta 1 polypeptide of T beta 1 gamma on denaturing polyacrylamide gels. Analysis of the interactions of T beta 3 gamma with other retinal proteins indicated that it has a lower affinity for the T alpha subunit of rod transducin but appears to complex with a phosducin-like protein. The differences in the intrinsic biochemical properties of T beta 3 gamma as compared to T beta 1 gamma may partially account for the lower light sensitivity of cones.
Fung
B K
BK
Jules Stein Eye Institute, UCLA School of Medicine 90024.
Lieberman
B S
BS
Lee
R H
RH
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002417:Cattle; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000276:immunology / Q000302:isolation & purification*; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D012160:Retina / Q000166:cytology / Q000502:physiology; D015293:Transducin / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D010446:Peptide Fragments; D017886:Durapatite; D019204:GTP-Binding Proteins; D015293:Transducin