Selective solubilization of proteins from red blood cell membranes by protein perturbants

Abstract

Isolated human erythrocyte membranes were exposed to a series of reagents known to modify or perturb proteins; these included sodium hydroxide, lithium diiodosalicylate, acid anhydrides, and organic mercurials. Each reagent liberated the same set of relatively polar polypeptides from the membrane, while the other, more hydrophobic species invariably remained associated with the membrane residue. The selective elution pattern was precisely that seen previously with 6 M guanidine hydrocloride. The released polypeptides, comprising half of the membrane protein mass, contained no carbohydrate; current evidence indicates that all of these components are confined to the cytoplasmic surface of the membrane. The residue contained all the lipids and all the glycoproteins. The latter are accessible to the outer membrane surface and, in at least two cases, seem to extend asymmetrically across the thickness of the membrane. Thus, the distinctive elution behavior which defines these two groups of polypeptides relates both to their chemical composition and their organizational disposition in the membrane.

The lateral distribution of intramembrane particles in the erythrocyte membrane and recombinant vesicles

“…This result did not depend on the detergent concentration, indicating that spectrin-actin is not precipitated by the detergent under these conditions. Treatment of ghosts with 0.01 M NaOH removes virtually all extrinsic proteins [12,26]. After 30 min centrifugation at 50 000 X g 52% of the total prorein and about 4% of both phospholipids and cholesterol were recovered in the supernatant.…”

“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out as described by Steck and Yu [26], using 4.0% acrylamide gels and bromophenol blue as tracking dye.…”

“…Since we have previously found that Triton X-100 can aggregate the particles [12], the effect of this detergent was investigated more systematically. To evaluate the role of spectrin, the aggregation of particles was also studied in membranes from which essentially all extrinsic proteins had been removed by an alkali treatment [12,26]. The role of phospholipids in the aggregation of the particles was examined using vesicles which were reconstituted from a crude Triton X-100 extract and various phospholipids from the erythrocyte membrane.…”

Erythrocyte membrane abnormalities in glucose‐6‐phosphate dehydrogenase deficiency of the mediterranean and A‐types

“…Polyacrylamide gel electrophoresis was performed following the standard procedures [16,17] using 5% acrylamide gels. Gels were run at 5 mA/gel for 2 h, stained with Coomassie brilliant blue and scanned using a densitometer.…”

A 19F‐NMR study of the membrane‐binding region of D‐lactate dehydrogenase of escherichia coli

“…However, D-LDH can be eluted from E. coli membranes by washing with 0.6 M guanidine hydrochloride (Reeves et al, 1973). This suggests that D-LDH, like other primary dehydrogenases involved in electron transfer in E. coli (Cronan et al, 1987), is not an integral membrane protein (Steck & Yu, 1973). Furthermore, the amino acid composition of D-LDH is not particularly hydrophobic, and the molecule does not appear to contain any of the transmembrane hydrophobic structures that are typical of integral membrane proteins (Ho et al, 1988;Fasman, 1989).…”