Hexamethylphosphoric triamide as a solubilizing agent: Purification and reactivation of diglyceride kinase

Abstract

The tightly bound proteins of biological membranes are usually solubilized with the aid of detergents. Experimental problems arising are binding and inactivation by the detergent and difficulty of detergent removal in lipid/protein reconstitution. An alternative way to purify membrane proteins is based on the observation that a small amount of membrane protein can usually be solubilized with organic solvents such as chloroform or butanol-1 [ 1,2]. Two hydrophobic bacterial membrane enzymes, isoprenoid alcohol kinase and diglyceride kinase, have been highly purified and reactivated using butanol-1 [3,4]. The aprotic organic solvents, HMPT and MP, have subsequently been found to be much more effective in solubilizing membrane proteins than chloroform or butanol-1 [5]. Precipitation with ethanol and gel permeation chromatography on Sephacryl were developed as methods for protein purification in HMPT. The following defined membrane proteins have so far been solubilized and purified in HMPT, the Escherichia coli phage lambda receptor protein [6], bacterioopsin [7], the pig kidney Na+,K+-ATPase subunits [S. Maier, H. S. unpublished] and certain erythrocyte and brain membrane proteins [R. Schmitt, S. Maier, H. S., unpublished]. The functional reconstitution from HMPT has remained a largely unsolved problem although certain soluble enzymes regained activity from HMPI’-solution [5]. Reconstitution of lactose transport has been achieved in membrane vesicles prepared from a transport-negative strain of E. coli by addition of an