Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter.
Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of [3H]forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited [3H]forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP gamma S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP gamma S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.
Morris
D I
DI
Division of Biochemistry and Biophysics, Food and Drug Administration, Bethesda, Maryland 20892.
Robbins
J D
JD
Ruoho
A E
AE
Sutkowski
E M
EM
Seamon
K B
KB
D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D004224:Diterpenes; D004591:Electrophoresis, Polyacrylamide Gel; D007457:Iodine Radioisotopes / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding
D000345:Affinity Labels; D001386:Azides; D004224:Diterpenes; D007457:Iodine Radioisotopes; D009004:Monosaccharide Transport Proteins; C069863:N-(3-(4-azido-3-iodophenyl)propionamide)-6-aminoethylcarbamylforskolin; C069864:N-(3-(4-azido-3-iodophenyl)-propionamide)-7-aminoethylcarbamyl-7-desacetylforskolin; D005576:Colforsin; D000262:Adenylyl Cyclases