Use of a small TSK GSW high-performance liquid chromatographic column for large-zone chromatographic studies of monomer-oligomer equilibria of membrane protein.
A TSK GSWP size-exclusion high-performance liquid chromatography "pre-column" (75 mm x 7.5 mm) was used for large-zone chromatographic studies of the effect of ligands on the monomer-oligomer equilibrium in sarcoplasmic reticulum Ca-ATPase solubilized in a non-ionic detergent. The monomer-dimer association constants determined for the Ca2+-occluded E1 P[Ca2] and vanadate-reacted E2V forms were 2-3 fold lower than the association constant of the E1Ca2 form (bound Ca2+ is freely exchangeable). With the TSK GSWP column, large-zone chromatography was rapid and required only small quantities of protein. This column was found to be useful also for studies in the presence of phospholipid, provided that the influence of lipid binding, per se, on the partition coefficient of the protein in the column was taken into consideration. This opens new possibilities for studies of membrane proteins under non-denaturing conditions.
Vilsen
B
B
Institute of Physiology, University of Aarhus, Denmark.
Andersen
J P
JP
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010743:Phospholipids / Q000032:analysis; D011817:Rabbits; D012822:Silicon Dioxide; D014461:Ultracentrifugation
D008565:Membrane Proteins; D010743:Phospholipids; D012822:Silicon Dioxide; C049273:TSK-Gel G 3000SW; D000252:Calcium-Transporting ATPases