Ts. The phosphate transporter in the plasma membrane of Saccharomyces cerevisiae was effectively produced in Pichia pastoris and purified in DPC detergent. Its activity was totally recovered right after reconstitution in proteoliposomes having a comparable substrate specificity as observed in an intact cell technique.117 Conversely, opposite results were obtained with mitochondrial uncoupling proteins. The Chou laboratory reported protontransport activity for each UCP1 and UCP2 proteins in DPC,118,119 while Zoonens and co-workers found that DPC fully inactivates both transporters.120 Asmar-Rovira and colleagues investigated how nine detergents influence the function in the nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays.121 Below 45 mol of phospholipids per mole of nAChR, the receptor was swiftly inactivated. By carefully measuring the volume of residual lipids soon after solubilization of enriched Torpedo membranes, they could show that most detergents degraded the receptor throughout purification below the essential threshold to preserve its activity. For example, Cymal-6, DDM, LDAO, and OG showed decreased stability and considerable reduction or loss of ion-channel function. In contrast, CHAPS, DPC, and sodium cholate maintained stability and supported ion-channel function. Asmar-Rovira and colleagues concluded that within the case of nAChR, CHAPS, DPC, and sodium cholate mimic the lipids in the sense of becoming capable to sustain lipiddependent activity and stability. The predicament is much more complicated together with the human ABCG2 multidrug pump. MacDevitt et al. had been capable to solubilize the recombinant 477-47-4 medchemexpress protein from sf9 insect cell membranes only with hexadecyl phosphocholine.122 Right after 3 purification steps in hexadecyl phosphocholine, the protein was nevertheless able to bind the substrate, but its ATPase activity in detergent was low, as well as the authors did not test ATPase activity immediately after reconstitution in the protein in liposomes. They have been nevertheless capable to analyze single particles by cryoEM and obtained a low-resolution threedimensional projection map displaying a tetrameric structure, which was interpreted as 4 homodimers of ABCG2. A second study appeared a handful of years later, showing that the ABCG2 receptor purified in hexadecyl Thiodicarb Technical Information phosphocholine was irreversibly inactivated, even though precisely the same protein purified in DDM was active when reconstituted in liposomes containing an excess of cholesterol (40 ).123 The authors concluded that the homodimers of ABCG2 have been disrupted by hexadecyl phosphocholine, resulting within a total inactivation from the receptor.124 Related benefits had been obtained for BmrA, a multidrug resistance efflux pump. The protein was inactivated by DPC, but inside a reversible manner. Exchanging the alkyl phosphocholine detergent with DDM or anionic calix[4]arene-based detergents restored its activity. Reversible activation of pumps has also been observed with the human bile salt export pump, BSEP, developed in Pichia pastoris membranes and purified in phosphocholine detergents with linear or cyclic alkyl chains.125 Its activity was restored by exchanging the detergent with DDM.125 Within the case in the multidrug resistance pump MDR3, addition of lipids towards the alkyl phosphocholine-MDR3 complex resulted inside a partial restoration of its activity.126 Aside from these examples of partial tolerance to DPC, you’ll find quite a few examples of membrane proteins that happen to be totally inactivated by this detergent (see Table S2). For example, diacylglycecol-kinase activity within the pres.
