TY - JOUR
T1 - Hydrodynamic properties of porcine bestrophin-1 in Triton X-100
AU - Stanton, J. Brett
AU - Goldberg, Andrew F.X.
AU - Hoppe, George
AU - Marmorstein, Lihua Y.
AU - Marmorstein, Alan D.
N1 - Funding Information:
Work in the author's laboratories is funded by NIH grants EY13160 and EY14898 to ADM, EY13847 to LYM, funds from Phillip Morris USA and Phillip Morris International, and the Macular Degeneration Foundation to ADM, a Career Development Award from Research to Prevent blindness to LYM, and an unrestricted grant from Research to Prevent Blindness to the Department of Ophthalmology at the University of Arizona.
PY - 2006/2
Y1 - 2006/2
N2 - Bestrophin-1 (Best-1) is an integral membrane protein, defects in which cause Best vitelliform macular dystrophy. Best-1 is proposed to function as a Cl- channel and/or a regulator of Ca++ channels. A tetrameric (or pentameric) stoichiometry has been reported for recombinant best-1. Using a combination of gel exclusion chromatography and velocity sedimentation we examined the quaternary structure of native best-1 and found that it migrates as a single species with a Stokes radius of 7.3 nm, sedimentation coefficient (S20,w) of 4.9, and partial specific volume (ν) of 0.80 ml/g. The mass of the protein-detergent complex is calculated to be 206 kDa, with the protein component estimated to be ∼138 kDa. Given a monomeric mass of 68 kDa, we conclude that native best-1 solubilized with Triton X-100 is a homodimer. The differences between this observation and a prior report were examined by comparing recombinant best-1 with tissue derived best-1 using gel exclusion chromatography. Much of the recombinant best-1 eluted in the column void (Vo) fraction, unlike that extracted from RPE cells. We conclude that the minimal functional unit of best-1 is dimeric. This stoichiometry differs from that previously measured for recombinant best-1, suggesting that further studies are necessary to determine the stoichiometry of functional best-1 in RPE membranes.
AB - Bestrophin-1 (Best-1) is an integral membrane protein, defects in which cause Best vitelliform macular dystrophy. Best-1 is proposed to function as a Cl- channel and/or a regulator of Ca++ channels. A tetrameric (or pentameric) stoichiometry has been reported for recombinant best-1. Using a combination of gel exclusion chromatography and velocity sedimentation we examined the quaternary structure of native best-1 and found that it migrates as a single species with a Stokes radius of 7.3 nm, sedimentation coefficient (S20,w) of 4.9, and partial specific volume (ν) of 0.80 ml/g. The mass of the protein-detergent complex is calculated to be 206 kDa, with the protein component estimated to be ∼138 kDa. Given a monomeric mass of 68 kDa, we conclude that native best-1 solubilized with Triton X-100 is a homodimer. The differences between this observation and a prior report were examined by comparing recombinant best-1 with tissue derived best-1 using gel exclusion chromatography. Much of the recombinant best-1 eluted in the column void (Vo) fraction, unlike that extracted from RPE cells. We conclude that the minimal functional unit of best-1 is dimeric. This stoichiometry differs from that previously measured for recombinant best-1, suggesting that further studies are necessary to determine the stoichiometry of functional best-1 in RPE membranes.
KW - Calcium
KW - Centrifugation
KW - Chloride
KW - Gel exclusion chromatography
KW - Ion channel
KW - Oligomer
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U2 - 10.1016/j.bbamem.2006.01.024
DO - 10.1016/j.bbamem.2006.01.024
M3 - Article
C2 - 16600174
AN - SCOPUS:33646267187
SN - 0005-2736
VL - 1758
SP - 241
EP - 247
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
IS - 2
ER -