Specific binding of dexamethasone to plasma membranes from skeletal muscle.
A procedure was developed to isolate plasma membranes from rabbit skeletal muscle. K+-dependent phosphatase activity was used as marker enzyme for plasma membranes and was determined in the presence of CHAPS (3-( (3-cholamidopropyl)dimethylammonio)-1-propanesulfonate), a zwitterionic detergent. Ca2+-ATPase and succinate dehydrogenase activities were used as marker enzymes for sarcoplasmic reticulum and mitochondria, respectively. Electron-microscopy revealed that plasma membranes were in the form of vesicles. Significant proteolysis of membrane proteins was observed during extraction, which was inhibited by EGTA and 20 mM molybdate. SDS-polyacrylamide gel electrophoresis revealed the disappearance of an intense 96 kDa protein band when membranes were purified in the absence of EGTA and molybdate. Specific binding sites for [3H]dexamethasone were identified in plasma membranes after freezing and incubation with CHAPS. Dithiothreitol was essential for steroid binding and ATP increased it. Under standardized assay conditions, binding was complete with 50 min a 37 degrees C. No binding occurred at 0 degrees C, nor if EGTA and molybdate were absent from the extraction medium.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D002793:Cholic Acids; D003907:Dexamethasone / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D009132:Muscles / Q000648:ultrastructure*; D011188:Potassium / Q000378:metabolism; D011817:Rabbits; D013237:Stereoisomerism
D002793:Cholic Acids; D004533:Egtazic Acid; D003907:Dexamethasone; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D011188:Potassium