Hydrodynamic properties of porcine bestrophin-1 in Triton X-100.
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 (nu) 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 approximately 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.
Stanton
J Brett
JB
Department of Ophthalmology and Vision Science, University of Arizona, Tucson, AZ 85711, and Cole Eye Institute, Cleveland Clinic Foundation, OH 44195, USA.
Goldberg
Andrew F X
AF
Hoppe
George
G
Marmorstein
Lihua Y
LY
Marmorstein
Alan D
AD
D000818:Animals; D002460:Cell Line; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003317:Corneal Dystrophies, Hereditary / Q000235:genetics; D003902:Detergents; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008970:Molecular Weight; D017830:Octoxynol; D010857:Pigment Epithelium of Eye / Q000737:chemistry; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D034421:Sus scrofa; D014018:Tissue Distribution; D014162:Transfection
D018118:Chloride Channels; D003902:Detergents; D005136:Eye Proteins; D011994:Recombinant Proteins; D017830:Octoxynol