Membrane-impermeant reagents

The conformationally sensitive sulphydryl group modified by membrane-impermeant reagents is the only exofacial sulphydryl, and as described in section 4.2.1, has been tentatively identified as Cys429 (Fig. 3). It is the most reactive sulphydryl in the... 

Sretcher, M.S., 1972b, Phosphatidyl-ethanolamine differential labelling in intact ceUs and cell ghosts of human erythrocytes by a membrane-impermeable reagent J. Mol. Biol., 71 523-528. 

Answer Construct and analyze a hydropathy plot for the protein. You can assume that any hydrophobic regions of more than 20 consecutive residues are transmembrane segments of an integral protein. To determine whether the external domain is carboxyl- or amino-terminal, treat intact erythrocytes with a membrane-impermeant reagent known to react with primary amines and determine whether the protein reacts. If it does, the amino terminus is on the external surface of the erythrocyte membrane and this is a type I protein (see Fig. 11-8). If it does not, a type II protein is indicated. 

Staros, J.V. (1988) Membrane-impermeant cross-linking reagents Probes of the structure and dynamics of membrane proteins. Acc. Chem. Res. 21, 435-441. 

Staros, J.V., Lee, W.T., and Conrad, D.H. (1987) Membrane-impermeant cross-linking reagents Application to the study of the cell surface receptor for IgE. In Methods in Enzymology, (G. Di Sabato, ed.), Vol. 150, pp. 503-512. Academic Press, Orlando, FL. 

NMR assays are also useful for characterizing ion transport. Na, Cl, and NMR spectroscopy have been used. A membrane impermeable shift reagent is used to induce a difference in the NMR chemical shift or line width of the ion of interest either inside or outside of the vesicle, thus characterizing translocation. 

Gas electrodes are similar to ISEs and usually work on the same basic principles. They look much like a standard ISE except that they have a gas-permeable, water-impermeable membrane at the tip. Gas present in the environment passes through the membrane, reacts with reagents in the interior, and produces a voltage. This voltage is related to the partial pressure of the gas being measured. 

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