Purification of integral plasma membrane proteins by reverse-phase high performance liquid chromatography.
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 Mr 100,000 catalytic polypeptide of each 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 [gamma-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.
Sussman
M R
MR
Department of Horticulture, University of Wisconsin, Madison 53706.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification; D000818:Animals; D002462:Cell Membrane / Q000032:analysis* / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D007668:Kidney / Q000201:enzymology; D008565:Membrane Proteins / Q000302:isolation & purification*; D009492:Neurospora crassa / Q000201:enzymology; D010455:Peptides / Q000302:isolation & purification; D010766:Phosphorylation; D012394:Rosaniline Dyes; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification; D013194:Staining and Labeling; D013552:Swine
D008565:Membrane Proteins; D010455:Peptides; D012394:Rosaniline Dyes; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; C004692:coomassie Brilliant Blue