In vivo protein phosphorylation and labeling of ATP in sea urchin eggs loaded with 32PO4 via electroporation.
Protein phosphorylation was examined in sea urchin eggs in which the ATP was labeled with 32P over a brief period of time using reversible electrical poration to gain access to the cytoplasm. Unfertilized eggs from two species, Lytechinus pictus and Strongylocentrotus purpuratus, were electrically permeabilized and incubated in the presence of [32P]H3PO4, under conditions allowing label uptake. After a 5-min loading period the eggs were resealed and the fate of the label was monitored. The label had equilibrated with the cellular ATP pool within the 13-min period required for loading and resealing the eggs. Furthermore, this equilibrium was maintained for at least 2 hr beyond the loading period in either unfertilized or fertilized eggs (i.e., the specific activity of ATP was the same for fertilized and unfertilized eggs). We also examined the position of the label within the ATP and found that 40-45% of the label isolated with the ATP was in the gamma phosphate of ATP and hence was immediately available for protein phosphorylation. The label was maintained in this position in the ATP for at least 2 hr following the loading period and was not affected by fertilization (determined for L. pictus only). The phosphoprotein banding pattern was determined by gel electrophoresis and autoradiography at various time points following the loading period. There was a continuous increase of label incorporated into protein over time; however, the banding pattern did not change. This pattern was not affected by fertilization. Furthermore, inhibition of protein synthesis (with emetine) had no effect on this phosphoprotein banding pattern. Although the loading period was brief there was sufficient incorporation of label into protein during this time to obscure potential regulatory phosphorylation events.
Larochelle
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Stanford University, Hopkins Marine Station, Pacific Grove, California 93950.
Epel
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002463:Cell Membrane Permeability; D002851:Chromatography, High Pressure Liquid; D004560:Electricity; D004591:Electrophoresis, Polyacrylamide Gel; D004626:Embryology / Q000379:methods*; D007700:Kinetics; D009865:Oocytes / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D012617:Sea Urchins / Q000196:embryology / Q000378:metabolism
D010761:Phosphorus Radioisotopes; D011506:Proteins; D000255:Adenosine Triphosphate