Digitonin and its activity

ChenHA Hzhen at freeuk.com
Fri Apr 5 14:58:23 EST 2002


ChenHA wrote:
> 
> Jayakumar R wrote:
> >
> > hi...
> >    I use Digitonin a lot for my mitochondrial fractionation procedures.  I
> > use it to study localization of proteins in mitochondrial compartments.
> > Proteins from different compartments of the mitochondria become selectively
> > released on an increasing gradient of digitonin of around 0.1% to 0.3%.
> > Even though I know that digitonin permeabilizes membranes, forms pores etc.
> > I am not able to understand how exactly Digitonin accomplishes this.
> >     Does Digitonin completely shear and break up the outer membrane of the
> > mitochondria or does it preserve the essential structure of the mitochondria
> > and just permeabilises the membrane without breaking it up?  Or does this
> > break up happen only at high levels of digitonin.
> >     Can anyone advice me  on this.  I need to understand the exact activity
> > of digitonin before formulating a protocol.  Any references also would be
> > very helpful.
> >     By the way, please do not suggest Google?  I have been going through it
> > for the past several hours and did not come up with anything useful.
> >    Any help will be appreciated.
> 
> Found on Google after searching a few minutes -
> 
> 
> Digitonin is a detergent which replaces cholesterol in membranes, and
> creates pores. The plasma membrane has much more cholesterol that the
> nuclear membrane. The net result is that the plasma membrane is
> permeabilized while the nuclear envelope is not. In the process the
> soluble components of the cytoplasm leak out of the cells.

Just adding something vaguely remembered from a project I did as an
undergraduate - I was told that the outer membrane of the mitochondria
was completely removed after treatment with digitonin. Can't remember
what concentration of digitonin was used, so it might not be useful to
you (the post-doc who supervised me was a complete bitch, so I won't
trust what she said either).  

I supposed a membrane shot through with holes wouldn't hold its
integrity easily, therefore whether the membrane can hold its shape
depends very much on how many pores there are which in turn depends on
the concentration of digitonin used and the amount of cholesterol on the
membrane.




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