Escherichia coli hemolysin is secreted as a water-soluble polypeptide of Mr 107,000. After binding to target erythrocytes, the membrane-bound toxin resembled an integral membrane protein in that it was refractory towards extraction with salt solutions of low ionic strength. Toxin-induced hemolysis could be totally inhibited by addition of 30 mM dextran 4 (mean Mr, 4,000; molecular diameter approximately 3 nm) to the extracellular medium. Uncharged molecules of smaller size (e.g., sucrose, with a molecular diameter of 0.9 nm, or raffinose, with a molecular diameter of 1.2 to 1.3 nm) did not afford such protection. Treatment of erythrocytes suspended in dextran-containing buffer with the toxin induced rapid efflux of cellular K+ and influx of 45Ca2+, as well as influx of [14C]mannitol and [3H]sucrose. [3H]inulin only slowly permeated into toxin-treated cells, and [3H]dextran uptake was virtually nil. Membranes lysed with high doses of E. coli hemolysin exhibited no recognizable ultrastructural lesions when examined by negative-staining electron microscopy. Sucrose density gradient centrifugation of deoxycholate-solubilized target membranes led to recovery of the toxin exclusively in monomer form. Incubation of toxin-treated cells with trypsin caused limited proteolysis with the generation of membrane-bound, toxin-derived polypeptides of Mr approximately 80,000 without destroying the functional pore. We suggest that E. coli hemolysin may damage cell membranes by partial insertion into the lipid bilayer and generation of a discrete, hydrophilic transmembrane pore with an effective diameter of approximately 3 nm. In contrast to the structured pores generated by cytolysins of gram-positive bacteria such as staphylococcal alpha-toxin and streptolysin O, pore formation by E. coli hemolysin may be caused by the insertion of toxin monomers into the target lipid bilayers.

https://iai.asm.org/content/iai/52/1/63.full.pdf

Escherichia coli hemolysin may damage target cell membranes by generating transmembrane pores.

Widespread occurrence of avian spectrin in nonerythroid cells

High resolution two‐dimensional polyacrylamide gel electrophoresis. I. Methodological procedures

We investigated the erythrocyte membrane proteins of two patients with congenital hemolytic anemia due to increased permeability of the erythrocyte membrane to Na and K (hereditary stomatocytosis and cryohydrocytosis). One-dimensional sodium dodecyl sulfate (SDS) gel electrophoresis resolved the band 7 erythrocyte membrane proteins into three components with approximate molecular weights of 30,000, 28,000, and 26,000. The 28,000-dalton component was decreased in both patients with permeability disorders. Two-dimensional electrophoresis (nonequilibrium pH gradient electrophoresis in the first dimension combined with SDS gel electrophoresis in the first dimension combined with SDS gel electrophoresis in the second dimension) resolved the 28,000-dalton component from normal erythrocyte membranes into two proteins with different isoelectric points, designated 22 x 8 and 60 x 8. In the patients with hereditary stomatocytosis and cryohydrocytosis, 22 x 8 was completely absent, whereas 60 x 8 was detected as usual. In contrast, all the band 7 proteins (including 22 x 8) were invariably present in a survey of normal subjects and reticulocytosis controls. The unique finding of a missing band 7 protein in the patients with hereditary stomatocytosis and cryohydrocytosis raises the possibility that the absence of this protein is responsible for the increased Na and K permeability in these disorders.

/pdf/missing-band-7-membrane-protein-in-two-patients-with-high-na-435s34fplx.pdf

Missing band 7 membrane protein in two patients with high Na, low K erythrocytes

Membrane proteins and cytosol proteins from organs (liver, brain) of inbred mice were separated by two-dimensional electrophoresis (2DE). The 2DE technique revealed high resolution of complex protein solutions by staining the proteins. The 2DE patterns of membrane proteins showed about 660 protein spots (polypeptides) for both liver and brain. The protein patterns of the cytosols revealed about 800 polypeptides for the liver and about 680 for the brain. The membrane proteins and cytosol proteins of the 2DE patterns represented two protein populations specific for these two cell components. They matched in 7 % (liver) or 23 % (brain) of the total number of membrane protein spots. The matched spots represented dissociable membrane proteins rather than contamination of the isolated membrane fractions by cytosol proteins. Protein patterns of the same cell fraction but from the two different organs investigated contained more than 50 % organ-unspecific polypeptides.



Several conditions of our 2DE standard procedure were reinvestigated to test the usefulness for our technique of some methodical steps employed in 2DE by other authors. We found that many steps often used in sample preparations impaired rather than improved the 2DE pattern. In particular, addition of SDS to the protein sample resulted in a considerable loss of proteins during isoelectric foucing. The 2DE itself could be simplified by omitting several steps such as prefocusing, equilibration of focusing gels, forming gel gradients, autoradiography. This has some implications for the development of 2DE to a routine method.

Daniella Harell

Papers

Two-dimensional separation of erythrocyte membrane proteins