Thiophosphorylated proteins in chromaffin cells are chromaffin vesicle matrix proteins.
Bovine adrenal chromaffin cells were incubated with inorganic thiophosphate, using a protocol similar to experiments with inorganic phosphate, in order to determine the source of previously observed thiophosphoproteins. Incubation of cultured cells with [35S]thiophosphate resulted in its incorporation into cell constituents within 2 min. SDS-PAGE of the treated cells showed incorporation of label into a broad 97-121 kDa band that was evident after 5 min of treatment and increased progressively to the 40 min exposure limit. Monolayers of chronically treated cells were fractionated into subcellular constituents. The only particulate fraction containing radiolabelled proteins was the chromaffin vesicle fraction. Two-dimensional electrophoresis of the treated cells and isolated chromaffin vesicles showed a majority of proteins in the acidic region of the first dimension gel. A fluorogram of the gel revealed two regions of radiolabelled proteins at acidic and neutral regions of the 2-D gel. These were within the boundaries of the 97-121 kDa band. The thiophosphorylated proteins were released as soluble proteins upon osmotic or freeze-thaw lysis of the vesicles. Chromaffin vesicles isolated from either cultured cells or adrenal medulla tissue were energized by 2 mM ATP but not by the analog adenosine 5'-O-(3-thiotriphosphate). The 97-121 kDa proteins in intact or lysed vesicles prepared from adrenal medulla tissue were not thiophosphorylated by either inorganic thiophosphate or adenosine 5'-O-(3-thiotriphosphate) in the presence or absence of energization by ATP. Nearly complete loss of radiolabel from matrix proteins treated with chondroitinase ABC suggests that it is a component of vesicle proteolgycans.(ABSTRACT TRUNCATED AT 250 WORDS)
Brooks
J C
JC
Marquette University School of Dentistry, Department of Basic Sciences, Milwaukee, WI 53233.
Brooks
M H
MH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000311:Adrenal Glands / Q000166:cytology / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002838:Chromaffin System / Q000166:cytology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*
D010710:Phosphates; D011506:Proteins; D000255:Adenosine Triphosphate; C035638:thiophosphoric acid