107 Biochimica et Biophysica Acta, 582 (1979) 107--114 © Elsevier/North-Holland Biomedical Press BBA 28780 EVIDENCE FOR THE UTILIZATION OF EXTRACELLULAR [7-32p]ATP FOR THE PHOSPHORYLATION OF INTRACELLULAR PROTEINS IN THE SQUID GIANT AXON HARISH C. PANT, SUSUMU TERAKAWA,TOHRU YOSHIOKA, ICHIJI TASAKI and HAROLD GAINER Laboratory of Neurobiology, National Institute of Mental Health, Bethesda, and Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, Bethesda, MD 20014 (U.S.A.) (Received April 10th, 1978) Key words: ATP; Phosphorylation; Membrane translocation; Neurofilament protein; Axon; Axoplasm Summary Proteins in the squid giant axon were labeled with 32p by in vitro incubation of isolated axoplasm with radioactive [~-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 regard- less of whether the [~/-32p]ATP was applied in situ intracellularly or extracel- larly. These results suggest that ATP in the extracellular space is, by some ATP- translocation mechanism, utilized in the process of intracellular phosphoryla- tion. Measurements of the apparent influx of ATP across the squid axon mem- brane yielded results consistent with the view that ATP in the extracellular fluid could be transported into the axoplasm. Introduction Many studies on the phosphorylation of proteins in various eukaryotic tis- sues have been carried out [1--6]. The squid giant axon provides a unique op- portunity for studying phosphorylation of intracellular proteins. Some of the special features of this experimental preparation for biochemists is that the sheath, which consists of the plasma membrane and surrounding sattelite cells, can easily be separated from the axoplasm by extrusion. With this prepara- tion, the radioactive substrate, [~/-32p]ATP, can be applied directly to extruded axoplasm in vitro under rigorously controlled conditions, or alternatively it can be injected into the axoplasm in situ, namely, in the excitable axon. In an