Isolation and purification of bacterial membrane proteins by the use of organic solvents: The lactose permease and the carbodiimide-reactive protein of the adenosinetriphosphatase complex of escherichia coli

✍ Scribed by Altendorf, Karlheinz ;Lukas, Margot ;Kohl, Brigitte ;Müuller, Clemens R. ;Sandermann, Heinrich

• Publisher: Wiley (John Wiley & Sons)
• Year: 1977
• Tongue: English
• Weight: 545 KB
• Volume: 6
• Category: Article
• ISSN: 0091-7419
• DOI: 10.1002/jss.400060208

✦ Synopsis

Abstract

Techniques for the solubilization and fractionation of integral membrane proteins have been developed in recent years. A small portion of membrane protein (about 2%, proteolipid fraction) will partition into chloroform or 1‐butanol, and, in several cases, these proteins retain functional activity. A virtually complete solubilization can be achieved at neutral pH by use of aprotic solvents, like hexamethylphosphoric triamide or N‐methylpyrrolidone.

At relatively low concentrations (< 3 M) aprotic solvents inhibited β‐D‐galactoside transport by whole cells and the derivative membrane vesicles of Escherichia coli, but this inhibition could be largely reversed by a simple washing procedure. At higher concentrations of aprotic solvent (5–6 M), 50–80% of the total protein of lactose transport‐positive membrane vesicles was solubilized. When these extracts were added to intact lactose transport‐negative membrane vesicles, lactose transport was reconstituted, the required energy being provided by either respiration (e.g., addition of D‐lactate) or by a K^+^ diffusion potential established with the aid of valinomycin.

The dicyclohexylcarbodiimide (DCCD)‐reactive subunit of the E. coli ATPase complex was found to partition into chloroform, and to be amenable to further purification in organic solvent. Ether precipitation and chromatography on DEAE‐cellulose and hydroxypropyl‐Sephadex G‐50 yielded an homogeneous polypeptide of an apparent molecular weight of 9,000.

The purified and unlabeled DCCD‐reactive protein was incorporated into K^+^‐loaded liposomes, and a membrane potential was generated by the addition of valinomycin. There are indications that the DCCD‐reactive protein alone made the membrane specifically permeable for protons.