Immunoaffinity purification and reconstitution of the human bilirubin/phenol UDP-glucuronosyltransferase family.
When membrane proteins are solubilized and subjected to purification procedures, the loss of lipids surrounding the protein often results in irreversible inactivation. We describe a procedure for the immunoaffinity purification of the membrane protein UDP-glucuronosyltransferase from human liver. This procedure reduces exposure of the protein to detergent, thereby reducing lipid loss. Triton X-100 was used to solubilize human liver microsomes. The solubilized proteins were applied to a sephacryl 300 (S-300) gel filtration column equilibrated with detergent-free buffer. UDP-glucuronosyltransferase activity eluted in turbid fractions in the void volume. During passage through the column Triton X-100 can partition in the buffer, while lipids are reconstituted into vesicular structures. Proteins with a high affinity for phospholipids remain associated with the lipid and elute in the void volume. The active fractions from the S-300 column were resolubilized with Triton X-100 and applied to a column with immobilized antibody. Washing this column with buffer containing phosphatidylcholine liposomes and no detergent removed unbound proteins and minimized loss of lipid from bound UDP-glucuronosyltransferase. Raising the pH of the washing buffer to 11.5 in the presence of liposomes resulted in elution and simultaneous reconstitution of active UDP-glucuronosyltransferase. Antibodies against membrane proteins are often available but immunoaffinitypurification of active enzyme is difficult. The approach described here could be useful for the isolation of other membrane proteins.
Seppen
J
J
Department of Gastrointestinal and Liver Diseases, Academic Medical Centre, Amsterdam, The Netherlands.
Jansen
P L
PL
Oude Elferink
R P
RP
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001663:Bilirubin / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004789:Enzyme Activation; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C; D008862:Microsomes, Liver / Q000201:enzymology*; D017830:Octoxynol; D019800:Phenol; D010636:Phenols / Q000378:metabolism; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D010636:Phenols; D010713:Phosphatidylcholines; D010743:Phospholipids; D019800:Phenol; D017830:Octoxynol; D014453:Glucuronosyltransferase; C017251:1,2-oleoylphosphatidylcholine; D001663:Bilirubin