Phosphorylation of purified thyroid plasma membranes incubated with [32-P]ATP.
Plasma membranes have been prepared from porcine thyroid glands using sucrose gradients. The fractions having a density in sucrose of 1.18 g/ml mainly contained plasma membranes and were moderately contaminated with other subcellular components as shown by marker enzyme data. Purified plasma membranes incubated in the presence of [32-P]gamma ATP incorporated 32-P. Kinetics of incorporation of 32-P into endogenous substrates studied in various buffers and with increasing ATP concentration suggest a phosphodephosphorylating system related to cAMP-dependent protein kinase and phosphoprotein phosphatase activities. The two enzymatic activities associated with plasma membranes have been demonstrated using exogenous substrates. cAMP increases and fluoride ions decrease the extent of membrane phosphorylation. The specific activity of protein kinase was 10-12 times higher than in the initial homogenate and was only slightly enhanced in the presence of 0.5% Nonidet as compared to microsomal fraction. cAMP binding to membrane proteins was 3 times higher than to the other particulate fractions. TSH present in the incubating medium or added after 5 min of 32-P labelling induced a rapid stimulation of endogenous phosphorylation followed by a rapid decrease. Phosphorylated membrane substrates were analyzed: high voltage paper electrophoresis after partial hydrolysis indicated that [32-P]phosphate is incorporated into serine and threonine residues as o-phosphate derivatives. SDS-polyacrylamide gel electrophoresis showed several 32--labelled fractions. When enhanced by cAMP, no specific phosphorylation of protein components was observed.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D003579:Cytochrome Reductases / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D009708:Nucleotidases / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011479:Protamines / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D013552:Swine; D013961:Thyroid Gland / Q000378:metabolism*; D013972:Thyrotropin / Q000494:pharmacology*
D010761:Phosphorus Radioisotopes; D011479:Protamines; D000255:Adenosine Triphosphate; D013972:Thyrotropin; D000242:Cyclic AMP; D003579:Cytochrome Reductases; D003576:Electron Transport Complex IV; D011494:Protein Kinases; D009708:Nucleotidases; D000469:Alkaline Phosphatase