palmitoylation

[Sean Patterson]

>In article <3A768700.BE5D1F43 at slac.stanford.edu>, Mark Bowen
><mbo at slac.stanford.edu> wrote:
>:Does anyone have experience with in vitro palimoylation?  I've tried the
>:reaction by adding palmitoyl CoA in DMSO to protein (25 kDa with 4 free
>:cys) in the presence of 1% octylglucoside.  This doesn't seems to be
>:working based on SDS-PAGE.
>:

Lot's of experience. I mix 3H-palm-CoA (readily  soluble in water) 
with my protein(s) of interest and incubate at 55C for an hour with 
some detergent so there isn't a solubility problem after 
palmitoylation. What is your assay for successful palmitoylation of 
your protein?

>Just out of curiosity - SNAP-25?
>
>In any case, if this reaction proceeds with any reasonable rate
>spontaneously, I am surprised, I did not know it. I was always
>under impression that you need to have some source of palmitoyl
>transferase activity. This certainly is the case for other lipid
>modifications like myristoylation and geranyl geranylation.

Yup. But with the activated donor - acyl-CoA - you can get chemical 
transfer to protein thiols. This has been documented extensively in 
the literature, to the point that some people are now arguing that 
cellular protein palmitoylation is a non-enzymatic process.

>If you express your protein as a fusion with something, reisolation
>of it after in vitro palmitoylation in the presence of crude
>extract should be relatively straightforward thing to do.
>
>         - Dima

Ditto that.  What is your protein and why do you need to palmitoylate in vitro?

Sean


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