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Unformatted text preview: nding between peptides that are in the bilayer, which is an
energetically favorable outcome since there is no water present in the interior of the
(d) You attempt to culture keratinocytes from these mutant mice and find that, unlike
wild type keratinocytes, they don’t form a compact monolayer and detach from the cell 7.06 Spring 2004 PS 1 key 9 of 11 culture dish very easily. In an attempt to demonstrate that Epidermo-1 is involved in cellcell interactions at desmosomes you treat wild type keratinocytes with EGTA, a calcium
chelator, and find that they detach from the dish just like the mutant cultures. Why does
Desmosomes, adherens junctions, and tight junctions all need high concentrations of
calcium to stay intact. EGTA is a calcium chelator and so removing the calcium
from the culture media prevents the formation of these junctions and so the cells
lose their adhesive properties and detach.
5. Below are five of the ways by which proteins can associate with the plasma membrane:
as a single-pass transmembrane protein
as a multi-pass transmembrane protein
(iii) via a covalent fatty acid chain
via an oligosaccharide to phosphatidylinositol
as a peripheral protein via electrostatic interactions or a lipid binding
(a) Give an example of each and detail the conditions by which you could purify each of
these proteins from the cell membrane.
Single-pass transmembrane protein: e.g. glycophorin A on erythrocytes or EGFR
(epidermal growth factor receptor).
Multi-pass transmembrane protein: e.g. bacteriorhodopsin, G-protein coupled
receptors such as b-adrenergic receptor. Both single and multi-pass transmembrane
receptors can be purified by adding detergent to cells to disrupt the membrane.
Ionic detergents, such as SDS, will denature the protein and so prevent any
functional assays being carried out. Non-ionic detergents, such as Triton X-100, can
be used to purify these proteins in a non-denatured form.
Covalent fatty acid chain: e.g...
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- Spring '11