Chromatography, delipidation and formation of rhodopsin phospholipid recombinants of wall-eyed pollock
Shukoliukov, S.A.; Kalishevich, O.O.; Tiurin, V.A.; Dikarev, V.P.; Koruagin, V.P.
Biokhimiia 45(3): 398-407
1980
ISSN/ISBN: 0320-9725 PMID: 7378481 Document Number: 164447
Dodecyltrimethylammonium bromide (100 mM) used to solubilize walleyed pollock [Theragra chalcogramma] rhodopsin caused a rapid spontaneous bleaching of the original preparation. Chromatography of the rhodopsin solubilized by 100 mM of N',N'-dimethyldodecylamine N-oxide gave a 30-50% yield of unbleached dark preparations. The rest of the preparation was delipidated down to 5-40 mol phospholipid/mol rhodopsin and was almost completely bleached and aggregated. Rhodopsin was irreversibly adsorbed on hydroxyapatite, but was eluted from a column with agarose A before the unbleached dark preparation. Rhodopsin solubilization in 1-2% sodium cholate was promising. Chromatography of the protein with this detergent on agarose A resulted in 80-90% yield of the unbleached dark preparation, considerable removal of lipids (up to 1-5 mol phospholipids/mol enzyme) and slight bleaching. The regeneration capacity and thermal stability of delipidated preparations decreased almost 2-fold over the original preparation containing up to 100 mol phospholipids/mol rhodopsin. Removal of the main bulk of sodium cholate by a dialysis at 0-2.degree. and a simultaneous administration of natural and synthetic phospholipids gave detergent-free recombinants (proteoliposomes) of the protein. Administration of lipids after a removal of the detergent increased the thermal stability of rhodopsin in the recombinants up to values typical for the enzyme from the native membranes of rod outer segments.