Thiophosphoproteins in Chromaffin Cells

Brooks, Jack

Abstract

We have used the adrenal chromaffin cell as a model catecholaminergic neuron to study the biochemical events which occur during neurosecretion. Considerable evidence suggests that protein phosphorylation plays a role in catecholamine secretion by chromaffin cells. Permeabilized cells were treated with an ATP analog, ATPgammaS (adenosine-5'-(3-thiotriphosphate) in order to """"""""lock"""""""" phosphorylation reactions in the (thio) phosphorylated state. Our premise has been that this approach would then permit us to relate the various events of secretion to the phosphorylation reaction. ATPgammaS treatment of the cells irreversibly inhibits secretion and results in thiophosphorylation of several proteins. One of these, a 47 kilodalton (kDa) protein may be either a nucleotide translocator in chromaffin vesicles or be thiophosphorylated as a result of nucleotide translocation. The current proposal has two major goals. The first is to determine the intracellular location and function of this protein. An antibody to the 47 kDa protein will be used to help determine its location in subcellular fractions prepared from cultured cells. The function of the protein will be identified by determining if its thiophosphorylation is causally related to nucleotide transport in chromaffin vesicles isolated from cultured cells. The results of this work will provide the identify for a major phosphoprotein in chromaffin cells. The second goal is to extend our observations with thiophosphorylation of permeabilized cells to intact cells. These studies will determine if thiophosphorylation of intact cells produces the same effect on secretion and protein thio phosphorylation as it does in permeabilized cells. They will also provide evidence that the permeabilized cell model faithfully represents the role of phosphorylation in the function of intact cells.