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J. Biol. Chem., Vol. 261, Issue 22, 10079-10086, Aug, 1986
SV Ambudkar and PC Maloney
Membranes of Streptococcus lactis were solubilized with 1.1% octyl-beta-
D-glucopyranoside in the presence of 0.37% acetone/ether-washed
phospholipid from several sources. After adding excess Escherichia coli
phospholipid as bath-sonicated liposomes, phosphate:sugar phosphate
antiport was reconstituted in proteoliposomes by a 25-fold dilution in 0.1
M KPi (pH 7). Assays of 32Pi:Pi exchange showed that antiport was subject
to an inactivation which varied in severity according to the lipid present
at solubilization. Recovery of Pi-linked exchange was improved by the
presence of 10-20% glycerol or other osmolyte during extraction. The
osmolytes tested in this regard have included polyols (glycerol,
erythritol, xylitol, sorbitol), sugars (glucose, trehalose), and two amino
acids (glycine, proline). Each gave 10--20-fold increased recoveries of
32Pi:Pi antiport compared to controls using only detergent and lipid; these
precautions were not required for the efficient reconstitution of
F0F1-ATPase. Antiport in the artificial system was studied most carefully
when glycerol was the stabilizing additive. For that case, the Kt values
for Pi or 2-deoxyglucose 6- phosphate transport (275 and 25 microM,
respectively) were the same as in native membranes. Maximal rates of Pi and
2-deoxyglucose 6-phosphate transport (200 and 42 nmol/min/mg of protein,
respectively) and the turnover number for Pi exchange (25--50/s) suggested
that antiporters were recovered without loss of activity. We conclude that
the quantitative aspects of bacterial anion exchange are amenable to study
in an artificial system, and that the use of osmolytes as general
stabilants can be a valuable adjunct to current techniques for
reconstitution of integral membrane transport proteins.
Bacterial anion exchange. Use of osmolytes during solubilization and reconstitution of phosphate-linked antiport from Streptococcus lactis
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