title: Thiophosphorylation and phosphorylation of saponin-permeabilized cultured chromaffin cells.
abstract: There is increasing evidence that phosphorylation of cellular proteins plays a role in the control of events surrounding secretion in neurons and chromaffin cells. In previous studies, we have used thiophosphorylation of cell proteins as a means of fixing cellular phosphorylation reactions in the phosphorylated state. Thiophosphorylation of permeabilized chromaffin cells with adenosine-5?-O-(3-thiotriphosphate) results in irreversible inhibition of secretion. Thiophosphate is incorporated primarily by two cellular proteins of 58 and 47 kDa. Calcium enhanced thiophosphorylation of the 47 kDa protein but not the 54 kDa protein. This pattern of thiophosphorylation differed markedly from that for phosphorylation under similar treatment conditions. The phosphoprotein composition of the cells depended upon the medium calcium and ATP concentration. In the absence of exogenous ATP, fewer phosphoproteins were seen in calcium stimulated cells than in unstimulated cells. Proteins labelled with (32)P or (35)S migrated to the same position on polyacrylamide gels containing sodium dodecyl sulfate. In the presence of exogenous ATP, (32)P incorporation was similar for both control and calcium-stimulated cells and was found primarily in a 64 kDa protein. Incorporation of [(32)P]phosphate by calcium-stimulated cells was reduced to the same extent by pretreatment of the cells with either adenosine-5?-O-(3-thiotriphosphate) or ATP. The different electrophoretic banding patterns for thiophosphorylation and phosphorylation are likely due to the irreversibility of the thiophosphorylation reaction and reversibility of the phosphorylation reaction. The inability to turn over thiophosphate groups, in association with changes in secretion, may permit identification of those phosphoproteins that are putatively involved in secretion.
authors.affiliation: Marquette University School of Dentistry, Milwaukee, WI 53233, U.S.A.
authors.forename: J C
authors.initials: JC
authors.lastname: Brooks
authors.forename: M H
authors.initials: MH
authors.lastname: Brooks