Purification of integral plasma membrane proteins by reverse-phase high performance liquid chromatography

Abstract

Following dissolution in anhydrous trifluoroacetic acid, plasma membrane isolated from two eukaryotic species was directly injected onto a reverse-phase high performance liquid chromatograph column. Upon development with a 60 to 100% ( v v ) linear gradient of ethanol containing 0.1% trifluoroacetic acid, most of the polypeptides eluted without retention. Only the lipids and very hydrophobic proteins were retained and resolved. Most noticeable among retained proteins was the M r 100,000 catalytic polypeptide of cach species' primary plasma membrane cation pump, the Na +,K +-ATPase of pig kidney and the H +-ATPase of Neurospora crassa hyphae. This simple 60-min procedure yielded nearly pure ATPase starting from crude membranes and in a completely volatile solvent, without detergent. When fungal plasma membranes were phosphorylated in vitro with [γ- 32P]ATP prior to injection, protein kinase activity was observed and this resulted in the phosphorylation of the H +-ATPase as well as of several other less-abundant hydrophobic membrane proteins. This procedure is useful as an alternative method for the rapid characterization of those membrane-associated polypeptides that contain several hydrophobic, transmembrane sequences.