Evidence for the utilization of extracellular [gamma-32P]ATP for the phosphorylation of intracellular proteins in the squid giant axon

Abstract

Proteins in the squid giant axon were labeled with 32P by in vitro incubation of isolated axoplasm with radioactive [gamma-32P]adenosine triphosphate (ATP) and separated by polyacrylamide sodium dodecyl sulfate gel electrophoresis. The two major phosphorylated regions on the gel had molecular weights of 400,000 and 200,000. These two peaks appear to be neurofilament proteins of squid axoplasm. The same set of proteins was phosphorylated in the axoplasm regardless of whether the [gamma-32P]ATP was applied in situ intracellularly or extracellarly. These results suggest that ATP in the extracellular space is, by some ATP-translocation mechanism, utilized in the process of intracellular phosphorylation. Measurements of the apparent influx of ATP across the squid axon membrane yielded results consistent with the view that ATP in the extracellular fluid could be transported into the axoplasm.