May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Stimulation of GTPase Accelerating Activity by R9AP
Author Affiliations & Notes
  • T.G. Wensel
    Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, United States
  • G. Hu
    Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, United States
  • Footnotes
    Commercial Relationships  T.G. Wensel, None; G. Hu, None.
  • Footnotes
    Support  NIH Grant EY11900, Welch Foundation
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 3177. doi:
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      T.G. Wensel, G. Hu; Stimulation of GTPase Accelerating Activity by R9AP . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3177.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: To find conditions for preparing membrane vesicles reconstituted with the transmembrane anchor protein, R9AP, and to test its effects on the GAP activity of RGS9-1. Methods: Full-length R9AP was expressed in E. coli,purified in non-denaturing detergent, and reconstituted into lipid vesicles, with or without purified rhodopsin, by dialysis. Vesicles were characterized by electron cryo-microscopy and biochemical assays. Vesicles containing rhodopsin with or without R9AP were reconstituted with G protein, transducin, and recombinant RGS9-1 in complex with Gß5. Single turnover GTPase assays were used to measure GTPase accelerating protein (GAP) activity. Results: Unilamellar vesicles with a narrow size distribution were readily obtained. R9AP had no effect on R*-catalyzed GTP uptake kinetics. R9AP enhanced the GAP activity of RGS9-1 by a factor of four. The Kd for binding of RGS9-1-Gß5 to membrane-embedded R9AP was < 10 nM. Conclusions: Because some RGS9-1 is bound to R9AP in photoreceptors, and some is not, regulation of binding to R9AP could serve as a mechanism for regulation of GAP activity and recovery kinetics.

Keywords: photoreceptors • protein structure/function 
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