Electrotransfer of [32P]NAD allows labeling of ADP-ribosylated proteins in intact Chinese hamster ovary cells.
CHO-K1D cells electroporated in buffers containing [32P]NAD incorporated the label in a voltage-dependent manner. Electroporation with 650 V/cm at 1460 microF in Ham's F12 medium supplemented with 10 mM Hepes, pH 7.1, resulted in a greater than 20-fold increase in [32P]NAD uptake, while decreasing relative cellular survival by only 6%. Exposure of cells to gamma irradiation (20 Gy) prior to electroporation increased the steady-state level of poly(ADP-ribosylated) nuclear proteins two- to four-fold over that of unirradiated control cells. These data indicate that electrotransfer of [32P]NAD is a simple and rapid means of labeling the cellular NAD pool and should prove useful in the analysis of the relationship between poly(ADP-ribosylation) of nuclear proteins and DNA repair.
Elia
M C
MC
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Motyka
L E
LE
Stamato
T D
TD
D000244:Adenosine Diphosphate / Q000032:analysis* / Q000378:metabolism / Q000528:radiation effects; D000818:Animals; D002021:Buffers; D002478:Cells, Cultured; D006224:Cricetinae; D004560:Electricity; D004591:Electrophoresis, Polyacrylamide Gel; D005720:Gamma Rays; D009243:NAD / Q000378:metabolism; D009687:Nuclear Proteins / Q000032:analysis* / Q000378:metabolism / Q000528:radiation effects; D012266:Ribose / Q000032:analysis* / Q000378:metabolism / Q000528:radiation effects
D002021:Buffers; D009687:Nuclear Proteins; D009243:NAD; D000244:Adenosine Diphosphate; D012266:Ribose