translocation of PKC?

Maikel van der Velden biochemphysiol at pobox.ruu.nl
Wed Jun 5 18:53:17 EST 1996


Hi,

I am trying to investigate the translocation of PKC from the cytosol of the 
cell to the membrane in the fatbody of locust Locusta migratoria.
Therefore 10 fatbodies are incubated with PMA (6.5 microM) in a shaking 
water-bath for 1 h at 30 degrees Celcius. Following incubation, the 
fatbodies are homogenised in 5 ml ice-cold 25 mM Tris-HCl (pH 7.5)/0.25 mM 
sucrose/2.5 mM MgCl2/2.5 mM EGTA/50 mM 2-mercaptoethanol. The homogenate is 
then centrifuged at 1500g with the resulting supernatant centrifuged for 1 
h at 100000g to separate cytosolic and particulate fractions. The 100000g 
pellet is washed and resuspended in 5 ml homogenisation buffer with 0.3% 
Triton X-100, incubated for 1 h at 4 degrees Celcius and centrifuged at 
100000g for 1 h to separate detergent-solubilised material. PKC activity 
present in both cytosolic and detergent-solubilised particulate fractions is 
measured by using a radio-active PKC-assay. Thereby PKC is assayed by 
measuring the incorporation of 32P into neurogranin from [gamma-32P]ATP.
The reaction mixture (60 microliter) contains 20 mM Tris-HCl (pH 7.5)/7.5 mM 
MgAc/30 microM neurogranin/10 microM ATP/2.2 nM [gamma-32P]ATP/0.5 mM NaF/2 
mM Ca2+/83.33 microgram/ml phosphatidylserine/2 microgram/ml diacylglycerol
and 6 microliter cytosolic or particulate fraction. The basal activity is 
measured with 100 microM EGTA instead of Ca2+, phosphatidylserine and 
diacylglycerol. 

I don't find any (extra) activation of PKC with Ca2+, phosphatidylserine and 
diacylglycerol in contrast to the basal activity level.
I found out that the presence of EGTA in the homogenisationmedium causes 
this phenomenon.
EGTA, however, is necessary as a chelator to dissociate the reversible 
Ca2+ induced membrane binding of PKC so only the phorbol ester induced 
membrane binding of PKC (which is stable to chelator and high salt, but 
can be dissociated with detergents) is investigated.

Can anyone help me and tell me how to continue my research?
Does anyone have an explanation for this phenomenon?

Thanks, 

        Maikel van der Velden  



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