Reconstitution of intestinal Na(+)-phosphate cotransporter.
The rabbit intestinal brush-border membrane Na(+)-phosphate cotransporter was purified from sodium dodecyl sulfate (SDS)-brush-border membrane vesicles (BBMV) protein (SDS-treated Ca(2+)-precipitated BBMV) by a three-column chromatography protocol. The purification included a preparative scale chromatofocusing chromatography column over the pH range from 7.4 to 4 after solubilization in 3-[(3-cholamidopropyl)-diamethylammonia]-1-propanesulfonate (CHAPS), a chromatofocusing column over the pH range from 5.6 to 4 after solubilization in n-octyl glucoside, and gel filtration chromatography on a Sephacryl S-200 column. Verification of Na(+)-phosphate cotransporter purification involved substrate affinities, substrate stoichiometry, and inhibitor sensitivity after proteoliposome reconstitution and SDS-polyacrylamide gel electrophoresis (PAGE). After gel filtration Na(+)-dependent phosphate uptake was 3,300-fold enriched compared with the cell homogenate. A single 130-kDa polypeptide was visualized by SDS-PAGE under reducing conditions using silver stain. The coenrichment of this 130-kDa polypeptide and proteoliposome reconstituted Na(+)-dependent phosphate uptake suggest that the intestinal brush-border membrane Na(+)-phosphate cotransporter has been purified and proteoliposome reconstituted.
Peerce
B E
BE
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77555-06.
Cedilote
M
M
Seifert
S
S
Levine
R
R
Kiesling
C
C
Clarke
R D
RD
D000818:Animals; D052160:Benzothiazoles; D002232:Carbocyanines; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002793:Cholic Acids; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008564:Membrane Potentials; D008871:Microvilli / Q000378:metabolism*; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters
D052160:Benzothiazoles; D002232:Carbocyanines; D002352:Carrier Proteins; D002793:Cholic Acids; D005456:Fluorescent Dyes; D008081:Liposomes; D010710:Phosphates; D011510:Proteolipids; D050608:Sodium-Phosphate Cotransporter Proteins; D027981:Symporters; C020485:proteoliposomes; C012944:3,3'-dipropyl-2,2'-thiadicarbocyanine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate