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Title: Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

Abstract

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.

Authors:
; ; ; ;  [1]
  1. (Division of Biochemistry and Biophysics, Food and Drug Administration, Bethesda, MD (USA))
Publication Date:
OSTI Identifier:
5464015
Alternate Identifier(s):
OSTI ID: 5464015
Resource Type:
Journal Article
Journal Name:
Journal of Biological Chemistry; (United States)
Additional Journal Information:
Journal Volume: 266:20; Journal ID: ISSN 0021-9258
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CYCLASES; LABELLING; GLUCOSE; MEMBRANE TRANSPORT; PHOSPHOLIPIDS; BIOCHEMICAL REACTION KINETICS; AFFINITY; AZIDES; BRAIN; CATTLE; CELL MEMBRANES; ERYTHROCYTES; IODINE 125; MAN; MOLECULAR STRUCTURE; MOLECULAR WEIGHT; SPECIFICITY; TRACER TECHNIQUES; ALDEHYDES; ANIMALS; BETA DECAY RADIOISOTOPES; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY; BODY FLUIDS; CARBOHYDRATES; CELL CONSTITUENTS; CENTRAL NERVOUS SYSTEM; DAYS LIVING RADIOISOTOPES; DOMESTIC ANIMALS; ELECTRON CAPTURE RADIOISOTOPES; ENZYMES; ESTERS; HEXOSES; INTERMEDIATE MASS NUCLEI; INTERNAL CONVERSION RADIOISOTOPES; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; LIPIDS; LYASES; MAMMALS; MATERIALS; MEMBRANES; MONOSACCHARIDES; NERVOUS SYSTEM; NITROGEN COMPOUNDS; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; ORGANIC PHOSPHORUS COMPOUNDS; ORGANS; PRIMATES; RADIOISOTOPES; REACTION KINETICS; RUMINANTS; SACCHARIDES; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Morris, D.I., Robbins, J.D., Ruoho, A.E., Sutkowski, E.M., and Seamon, K.B. Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter. United States: N. p., 1991. Web.
Morris, D.I., Robbins, J.D., Ruoho, A.E., Sutkowski, E.M., & Seamon, K.B. Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter. United States.
Morris, D.I., Robbins, J.D., Ruoho, A.E., Sutkowski, E.M., and Seamon, K.B. Mon . "Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter". United States.
@article{osti_5464015,
title = {Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter},
author = {Morris, D.I. and Robbins, J.D. and Ruoho, A.E. and Sutkowski, E.M. and Seamon, K.B.},
abstractNote = {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.},
doi = {},
journal = {Journal of Biological Chemistry; (United States)},
issn = {0021-9258},
number = ,
volume = 266:20,
place = {United States},
year = {1991},
month = {7}
}