Abstract
The properties of detergents required to substitute the lipid environment of sarcoplasmic reticulum Ca2+-ATPase with retention of good functional properties were determined by the use of a large number of diverse detergents and delipidated enzyme. Detergents having an intermediate chain length (approximately equal to C12) and a polyoxyethylene glycol or carbohydrate polar group were optimal for Ca2+-ATPase function and stabilization, while detergents with short alkyl chain (C8) or bulky head groups and many zwitterionic detergents led to rapid inactivation. Under optimal conditions (including solubilization in the E1 state), stability of delipidated Ca2+-ATPase approximated that obtained by solubilization of Ca2+-ATPase with a layer of bound lipid. Some detergents (in particular long chain members of the Tween family) were characterized by an inadequate interaction with delipidated Ca2+-ATPase, resulting in biphasic inactivation. According to analytical ultracentrifugation and high performance liquid chromatography experiments, the rapid and slow components of biphasic inactivation were due to the formation of monomeric and oligomeric Ca2+-ATPase, respectively. It is concluded that both hydrophobic and polar interactions are important for the detergent effect and that solubilizing detergents of intermediate and short chain length may be bound as a monolayer, differently than the membrane lipid. Long chain detergents cause protein aggregation and, despite their resemblance to natural lipids, are inferior in their activity-retaining properties. The previous use of such detergents to prepare oligomeric Ca2+-ATPase with long term retention of activity (cf. Møller, J. V., Anderson, J. P., and le Maire, M. (1988) Methods Enzymol. 157, 261-270) is shown to depend on the presence of residual lipid in these preparations.
Highlights
The properties of detergents required to substitute of membrane proteins [1,2,3,4,5,6,7]
Irrespective of the detergent used, it is quite often found chromatography experimentst,he rapid ansdlow com- that solubilized membrane proteins are inactivated at high ponents of biphasic inactivation were due to the for- detergent to protein ratios [8, 31] which either suggests dismation of monomeric and oligomeric Ca2+-ATPase,re- sociation of solubilized membrane protein to smaller entities spectively
It is concluded that both hydrophobic and polar in- moval of residual lipid attached to the solubilized detergent teractions are important for the detergent effect and membrane protein complexes
Summary
Pended at 0.05 mg of protein/ml and containing about 0.025 mg/ml endogenousphospholipid, i.e. about 30 p M lipid) C12E8. Cient of 5 S, corresponding to monomeric Ca2+-ATPase[16], while the remainder of the boundary produced a drawn out Oligomers of Ca2+-ATPasePrepared by Gel Chromatography and more rapidly moving boundary, representing various oli- The poor performance demonstrated here for detergents gomers of the protein This occurred at a concentration of with bulky head groups as protecting agents contrawstith the free C12E8of 0.02 mg/ml, significantly below the cmc, which previous use of Tween 80 to prepare active and stable oligois 0.05 mg/ml at 20 “C [31,46]T. he formation of a monomeric mers of CaZ+-ATPase[14, 15], and theuse of Lubrol W X to fraction of denaturedand water-soluble Ca2+-ATPasewas purify active Na+,K+-ATPasein an oligomeric form [58].
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