Bacterial anion exchange. Use of osmolytes during solubilization and reconstitution of phosphate-linked antiport from Streptococcus lactis

S. V. Ambudkar, P. C. Maloney

Research output: Contribution to journalArticle

Abstract

Membranes of Streptococcus lactis were solubilized with 1.1% octyl-β-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 KP(i) (pH 7). Assays of 32P(i):P(i) exchange showed that antiport was subject to an inactivation which varied in severity according to the lipid present at solubilization. Recovery of P(i)-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 32P(i):P(i) 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 K(t) values for P(i) or 2-deoxyglucose 6-phosphate transport (275 and 25 μM, respectively) were the same as in native membranes. Maximal rates of P(i) and 2-deoxyglucose 6-phosphate transport (200 and 42 nmol/min/mg of protein, respectively) and the turnover number for P(i) 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.

Original languageEnglish (US)
Pages (from-to)10079-10086
Number of pages8
JournalJournal of Biological Chemistry
Volume261
Issue number22
StatePublished - 1986

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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