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. 1979 Dec;76(12):6071–6075. doi: 10.1073/pnas.76.12.6071

Phosphorylation of specific, distinct proteins in synaptosomes and axons from squid nervous system

Harish C Pant *,†, Harvey B Pollard †,‡, George D Pappas †,§, Harold Gainer †,
PMCID: PMC411804  PMID: 293702

Abstract

Synaptosomes and axons from squid were incubated with [γ-32P]ATP or [32P]orthophosphate and specific, distinct proteins were found to be labeled in each preparation. In axoplasm, only the major 200,000 Mr neurofilament protein and a specific protein of ≈400,000 Mr were labeled, as reported previously [Pant, H. C., Shecket, G., Gainer, H. & Lasek, R. J. (1978) J. Cell Biol. 78, R23-R27]. These results were independent of whether the cosubstrates were 32PO42- or [γ-32P]ATP. However, synaptosomes lacked the 200,000 Mr neurofilament protein and several lower molecular weight proteins were labeled instead, the most prominent being a 47,000 Mr species. [γ-32P]ATP was much more effective in labeling the 47,000 Mr species than 32PO42-. Synaptosomes also contained a distinct 250,000 Mr protein species which, however, was not labeled.

The protein kinase activity in synaptosomes was sensitive to various pharmacological agents, depending on whether the labeled phosphate came directly from ATP or orthophosphate. Carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, a mitochondrial H+ uncoupler, almost completely inhibited incorporation of 32P into protein with 32PO42- as cosubstrate, as expected, but produced only 32% inhibition with [γ-32P]ATP as cosubstrate. The activity could be augmented by incubating synaptosomes in a calcium-free medium and could be suppressed by increasing intrasynaptosomal Ca2+ with A23187, a Ca2+ ionophore. The latter effect was more prominent with 32PO42- than with [γ-32P]ATP as cosubstrate. Depolarizing agents such as veratridine and high K+ also suppressed activity, and the veratridine effect was completely reversed by tetrodotoxin or by omission of Ca2+ when [γ-32P]ATP was used, and partially reversed when 32PO42- was used. We conclude that the morphological transformation of an axon into a terminal is accompanied by significant changes in protein and phospho-protein composition that may be related to synaptic transmission.

Keywords: protein kinase, synaptic transmission

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Selected References

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