May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Hydrodynamic Properties of Porcine Bestrophin in Triton X-100
Author Affiliations & Notes
  • B. Stanton
    Ophthalmic Research, i31, Cleveland Clinic Foundation, Cleveland, OH, United States
  • A.F. Goldberg
    Eye Research Institute, Oakland University, Rochester, MI, United States
  • A.D. Marmorstein
    Eye Research Institute, Oakland University, Rochester, MI, United States
  • Footnotes
    Commercial Relationships  B. Stanton, None; A.F.X. Goldberg, None; A.D. Marmorstein, None.
  • Footnotes
    Support  NIH grants EY 13160 and EY 13246
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 5099. doi:
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      B. Stanton, A.F. Goldberg, A.D. Marmorstein; Hydrodynamic Properties of Porcine Bestrophin in Triton X-100 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5099.

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

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Abstract

Abstract: : Purpose: To determine the hydrodynamic properties of bestrophin and the stoichiometry of the detergent solubilized bestrophin complex under non-denaturing conditions. Methods: Using porcine RPE detergent lysates or a solubilized membrane fraction derived from porcine RPE, the Stokes radius of bestrophin was determined using size exclusion chromatography on a Sephacryl S-300HR column. The Svedberg coefficient (S20,w) and partial specific volume (ν) of the bestrophin-detergent complex were determined by velocity sedimentation through 5% - 20% (W/V) sucrose gradients prepared in either H2O or D2O containing 0.2% Triton X-100 (TX-100). An estimation of molecular mass of the protein portion of the bestrophin / TX-100 complex was made assuming additivity of ν for TX-100 (0.94 ml/g) and protein (0.74 ml/g). Results: The Stokes radius of bestrophin was determined to be ~7.25nm. Velocity sedimentation experiments indicate a S20,w for bestrophin of 4.9 ± 0.4 and ν of the bestrophin / TX-100 complex of 0.80 ± .02 (ml/g). Based on these data we estimate a mass of ~206,000 kDa for the bestrophin / TX-100 complex. Assuming ν = 0.74 ml/g for the protein portion of the complex, we calculate that bestrophin binds~0.31 g of Triton X-100 / g protein and is present as a single major species with an estimated mass of ~ 138 kDa. Conclusions: We have determined the hydrodynamic properties of porcine bestrophin solubilized in TX-100. The combined data indicate a protein mass, measured under non-denaturing conditions, of approximately twofold that of monomeric bestrophin (138 kDa / 68 kDa = 2.03). We consider it likely that the dimeric form is the minimal unit of function for bestrophin in the porcine RPE plasma membrane.

Keywords: protein structure/function • proteins encoded by disease genes • electroretinography: non-clinical 
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