Characterization of cytochalasin B photoincorporation into human erythrocyte D-glucose transporter and F-actin.
The photoincorporation of cytochalasin B into the human erythrocyte glucose transporter and purified G-actin previously reported by this laboratory [Shanahan, M.F. (1982) J. Biol. Chem. 257, 7290-7293] was investigated. [3H]Cytochalasin B photolabeled polypeptides of Mr approximately 43,000-73,000, as determined by polyacrylamide gel electrophoresis, in a concentration-dependent manner with maximum incorporation occurring at 5 microM [3H]cytochalasin B and a half-maximum value of 0.63 microM. This incorporation, previously shown to be partially blocked in the presence of D- but not L-glucose, did not occur in the absence of photolysis and increased linearly with a photolysis time up to 30 s. The reaction was relatively insensitive to pH in the range of pH 6-9, but apparent non-specific labeling significantly increased at pH 5. The effect of cytochalasin B photoincorporation on D-glucose uptake in intact erythrocytes was also examined. Purified chicken muscle F-actin was also photolabeled with this ligand, but at a specific activity of incorporation (pmol/mg of protein) approximately 50 times lower than that of the erythrocyte transporter polypeptides. D-Glucose had no effect on this incorporation while 10(-4) M cytochalasin E completely blocked actin photolabeling. The efficiency of photoincorporation for both the transporter and F-actin was around 1%. Extraction of [3H]cytochalasin B labeled membranes with Triton X-100 resulted in the selective elution of labeled polypeptides from the transporter region while cytochalasin B labeled polypeptides in the region of red cell actin remained in the extracted pellet.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active; D001786:Blood Glucose / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002645:Chickens; D003571:Cytochalasin B / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D009004:Monosaccharide Transport Proteins; D009132:Muscles / Q000378:metabolism; D010777:Photochemistry; D014316:Tritium
D000199:Actins; D001786:Blood Glucose; D002352:Carrier Proteins; D009004:Monosaccharide Transport Proteins; D014316:Tritium; D003571:Cytochalasin B