Isolation of human platelet and red blood cell plasma membrane proteins by preparative detergent electrophoresis

High resolution polyacrylamide gel electrophoretic techniques have been applied to the preparative isolation and analysis of plasma membrane proteins and glycoproteins from human platelets and red blood cells. The techniques presented allow relatively simple, direct, rapid and quantitative purification of a broad molecular weight range of membrane proteins, by means of continuous elution preparative gel electrophoresis of proteins solubilized with sodium dodecyl sulfate. Spectrophotometric and fluorophotometric (fluorescamine) profiling, and high resolution gel electrophoretic analysis (SDS-acrylamide gradient slab gels, and gel electrofocusing) of eluted protein species indicate that purified membrane proteins of a broad molecular weight range may be obtained in a one step procedure, and in quantities and concentrations sufficient for further analytical or experimental procedures.     

The structure of mixed cholesterol-phospholipid monolayers spread at the air-water interface as probed by interactions with band 3 protein from erythrocyte membranes

Solubilized band 3 protein from human erythrocyte membranes (the anion transport protein) interacts strongly and specifically with monolayers of cholesterol spread at the air-water interface whereas, at pH 7–10, it shows only moderate interactions with phospholipid monolayers (Klappauf, E. and Schubert, D. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1225–1235). When band 3 protein, at pH 7 and an ionic strength of approx. 100 mM, is added to the subphase of mixed cholesterol-glycerophospholipid monolayers, the changes Δπ in monolayer surface pressure induced by the protein depend on the mole fraction X of sterol in the mixture. However, Δπ(X) only increases with increasing X towards the high values of Δπ that are characteristic of cholesterol monolayers if X>0.67±0.04; at lower cholesterol content, Δπ(X) is practically identical to the value obtained with the pure glycerophospholipid. With mixtures of coprostanol and glycerophospholipids, the break in the Δπ(X) curves occurs when X=0.33±0.03. Cholesterol-sphingomyelin and epicoprostanol-phosphatidylethanolamine mixtures show an increase of Δπ(X) when X>0. The data seem to support earlier claims that cholesterol can form stoichiometric complexes with glycerophospholipids, the stoichiometries revealed by the band 3-monolayer interactions being 2:1 and 1:2. They also show that cholesterol-sphingomyelin complexes, if they should exist, must be structurally different from the cholesterol-glycerophospholipid complexes.     

A study on the renaturation of membrane proteins after solubilization in SDS or following polyacrylamide gel electrophoresis in SDS,with special reference to a phosphatase from acholeplasma laidlawii

It may be easier to renature SDS-denatured hydrophobic proteins than to renature SDS-denatured water-soluble proteins. This paper presents some support for this hypothesis in the form of literature reports and an experiment of our own with an intrinsic membrane protein (a phosphatase from Acholeplasma laidlawii), that could be completely renatured, to judge from the restored activity, which was equal to (or higher than) that of the untreated enzyme. If this hypothesis is correct it might be possible to devise general methods to reverse the SDS denaturation of hydrophobic membrane proteins. This would be a breakthrough in the purification of at least some membrane proteins, because the high-resolving polyacrylamide gel electrophoresis in SDS could then be used to prepare membrane proteins in a native state. The method used for the renaturation of the SDS-denatured, entirely inactive, phosphatase comprised removal of SDS with the aid of conventional dialysis against a buffer containing the neutral, very efficient and non ultraviolet light-absorbing detergent G3707. For renaturation of the enzyme following an SDS-electrophoresis in polyacrylamide the gel was immersed in the same buffer for several hours; by staining for phosphatase the enzyme could easily be localized in the gel in the form of a yellow band, coinciding with a protein zone.     

A study on the separation of reconstituted proteoliposomes and unincorporated membrane proteins by use of hydrophobic affinity gels, with special reference to band 3 from bovine erythrocyte membranes

Alkyl-Sepharose 4B with octyl, decyl, or dodecyl groups as an alkyl chain was a good adsorbent for any type of detergents and a variety of proteins, but not for phospholipids in a vesicle form. When these gels were added to the mixtures of reconstituted proteoliposomes prepared by using bovine band 3 and the protein unincorporated into liposomes, free band 3 in solution was adsorbed onto the gels and the proteoliposomes could be recovered by filtration, suggesting that this procedure, when applicable, permits a rapid isolation of proteoliposomes without loss and dilution of the sample. In addition, the results indicated that Bio-Beads SM-2 resin, which is virtually nonadsorbing for most proteins, can be used in removing any kind of detergents from those protein-detergent mixtures.