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Binding proteins to cytoplasmic surface of cellular membrane

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Binding proteins to cytoplasmic surface of cellular membrane

Postby havery01 » Mon Nov 22, 2010 9:00 pm

When you create vesicles made from the plasma membrane that are both normal in orientation and that are inside out and you want to remove the normal vesicles, and obtain a more pure harvest of inside-out vesicles, its suggested that you pass the preparation of mixed vesicles over an affinity column. There is an affinity column made of the lectin pisum sativum from the common pea attached to solid beads for support. Will using this approach to effectively remove the normal vesicles from the inside out ones actually work?

me: ok i know that pisum sativum is the scientific name for the common pea. And that by using lectin in the experiment that they have binding sites for specific mono or oligosaccharides in cell walls or membranes. They thereby change the physiology of the membrane and can cause agglutination, or clumping together of cells or particles. I also know that inside out vesicles re a mechanical disruption of cell membranes that give rise to small closed vesicles surrounded by a bilayer membrane. They can become inside out by freezing and thawing. what i cant find is any information on how the common pea affinity column can have any sort of affect on sorting the vesicles except for the lectin found in it that causes agglutination.
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Postby canalon » Mon Nov 22, 2010 11:00 pm

The question is whether the agglutination wil be specific to one type of vesicle or the other. I.e. are there lectin binding site on the cytoplasm site of your membrane.
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Postby JackBean » Tue Nov 23, 2010 6:03 am

I think only extrecellular side should contain oligosaccharides, rigth? So, it won't bind the inside-out vesicles, but binds the normal one ;)
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Re: Binding proteins to cytoplasmic surface of cellular membrane

Postby brantozeke » Sat Nov 27, 2010 2:56 pm

Look in the lab book. There's a table on lectins on page 55 that says that Lectin Pisium Sativum binds specifically to Sialic Acid. SO! The question is, does sialic acid only present itself on the outside of the cell membrane, or both inside and out? Also... if a vesicle is made of the cell membrane, does the outside of the cell membrane NORMALLY become the inside of the vesicle? I'm not sure. I'm still hunting that one down. Maybe somebody can shed some light.
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Postby havery01 » Tue Nov 30, 2010 8:13 pm

this is what i read. From 61 to 92% of the total sialic acid is present on the cell SURFACE as measured by its susceptibility to cleavage. But its amount and density varies considerably throughout the cell cycle. If most of the sialic acid is found on the surface and the vesicles are inside out with the cytoplasmic surface now being on the outside, does that mean that sialic acid would now have no capability of binding to anything since it is on the inside on the cell?
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Postby JackBean » Fri Dec 02, 2011 4:29 pm

yes
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