Specific Inhibition of the Phosphorylation of Protein I, a Synaptic Protein, by Affinity-Purified Anti-Protein I Antibody

Abstract

Affinity-purified anti-protein I IgG, either the intact molecule or its monovalent Fab fragment, inhibits specifically the phosphorylation of protein I without affecting the phosphorylation of other proteins, including those that are substrates for the CAMP-dependent protein kinase or calcium/calmodulin-dependent protein kinases. This indicates that the specific inhibition by the anti-protein I IgG probably has resulted from its blocking action on the phosphorylation sites of protein I rather than from its blocking action on the catalytic sites of the protein kinases. This substrate-directed specific inhibitor of the phosphorylation of protein I may provide a unique probe in the investigation of the precise synaptic function of protein I phosphorylation. In particular, the monovalent Fab(t) fragment, which has a substantially lower molecular weight than the intact IgG, and yet is capable of causing the specific inhibition, will be amenable to certain experimentations. To obtain a clue to the presynaptic function of protein I, synaptosomes can be fused with liposomes into which the Fab(t) fragment is incorporated, and it can be examined whether it has any effects on the neurotransmitter release from or uptake into the synaptosomes. It is also possible to test the Fab(t) fragment directly for an effect on neurotransmitter uptake into isolated synaptic vesicles.